Evidence Report/Technology Assessment

Number 196

Assessment of Thiopurine Methyltransferase Activity 

in Patients Prescribed Azathioprine or Other 

Thiopurine-based Drugs

Prepared for: 

Agency for Healthcare Research and Quality 

U.S. Department of Health and Human Services 

540 Gaither Road 

Rockville, MD 20850 

www.ahrq.gov 

Contract No. 290-2007-10059-I (EPCIII)

Prepared by: 

University of Ottawa Evidence-based Practice Centre, Ottawa, ON, Canada

EPC Director

David Moher, Ph.D. 

Investigators

Ronald A. Booth, Ph.D. 

Mohammed T. Ansari, M.B.B.S, 

M.Med.Sc., M.Phil.

Andrea C. Tricco, Ph.D. 

Evelin Loit, Ph.D. 

Laura Weeks, Ph.D.

Steve Doucette, M.Sc. 

Becky Skidmore, M.L.S.

Jeffrey S. Hoch, Ph.D. 

Sophia Tsouros, B.H.Kin. 

Margaret Sears, Ph.D.

Richmond Sy, M.D.

Jacob Karsh, M.D.

Suja Mani, B.Sc, D.M.M.

James Galipeau, Ph.D.

Alexander Yurkiewich, B.Sc. 

Raymond Daniel, B.A. 

Alexander Tsertsvadze, M.D., M.Sc.

Fatemeh Yazdi, M.Sc. 

AHRQ Publication No. 11-E002 

December 2010
This report is based on research conducted by the University of Ottawa Evidence-based 

Practice Center (EPC) under contract to the Agency for Healthcare Research and Quality 

(AHRQ), Rockville, MD (Contract No. 290-2007-10059-I (EPCIII)). The findings and 

conclusions in this document are those of the author(s), who are responsible for its content, 

and do not necessarily represent the views of AHRQ. No statement in this report should be 

construed as an official position of AHRQ or of the U.S. Department of Health and Human 

Services.

The information in this report is intended to help clinicians, employers, policymakers, and 

others make informed decisions about the provision of health care services. This report is

intended as a reference and not as a substitute for clinical judgment.

This report may be used, in whole or in part, as the basis for the development of clinical 

practice guidelines and other quality enhancement tools, or as a basis for reimbursement and 

coverage policies. AHRQ or U.S. Department of Health and Human Services endorsement of 

such derivative products may not be stated or implied. 

 
ii

This document is in the public domain and may be used and reprinted without permission except 

those copyrighted materials noted for which further reproduction is prohibited without the 

specific permission of copyright holders.

Suggested Citation

Booth RA, Ansari MT, Tricco AC, Loit E, Weeks L, Doucette S, Skidmore B, Hoch JS, Tsouros 

S, Sears M, Sy R, Karsh J, Mani S, Galipeau J, Yurkiewich A, Daniel R, Tsertsvadze A, Yazdi F. 

Assessment of thiopurine methyltransferase activity in patients prescribed azathioprine or other 

thiopurine-based drugs. Evidence Report/Technology Assessment No. 196. (Prepared by the 

University of Ottawa Evidence-based Practice Center under Contract No. 290-2007-10059-I 

AHRQ Publication No. 11-E002. Rockville, MD: Agency for Healthcare Research and Quality. 

December 2010.

No investigators have any affiliations or financial involvement (e.g., employment, 

consultancies, honoraria, stock options, expert testimony, grants or patents received or 

pending, or royalties) that conflict with material presented in this report. 
iii

Preface

The Agency for Healthcare Research and Quality (AHRQ), through its Evidence-Based 

Practice Centers (EPCs), sponsors the development of evidence reports and technology 

assessments to assist public- and private-sector organizations in their efforts to improve the 

quality of health care in the United States. The reports and assessments provide organizations 

with comprehensive, science-based information on common, costly medical conditions and new 

health care technologies. The EPCs systematically review the relevant scientific literature on 

topics assigned to them by AHRQ and conduct additional analyses when appropriate prior to 

developing their reports and assessments.

To bring the broadest range of experts into the development of evidence reports and health 

technology assessments, AHRQ encourages the EPCs to form partnerships and enter into 

collaborations with other medical and research organizations. The EPCs work with these partner 

organizations to ensure that the evidence reports and technology assessments they produce will 

become building blocks for health care quality improvement projects throughout the Nation. The 

reports undergo peer review prior to their release. 

AHRQ expects that the EPC evidence reports and technology assessments will inform 

individual health plans, providers, and purchasers as well as the health care system as a whole by 

providing important information to help improve health care quality.

We welcome comments on this evidence report. They may be sent by mail to the Task Order 

Officer named below at: Agency for Healthcare Research and Quality, 540 Gaither Road, 

Rockville, MD 20850, or by e-mail to epc@ahrq.gov. 

Carolyn M. Clancy, M.D.

Director

Agency for Healthcare Research and Quality

Jean Slutsky, P.A., M.S.P.H.

Director, Center for Outcomes and Evidence

Agency for Healthcare Research and Quality

Stephanie Chang, M.D. 

Director, EPC Program

Agency for Healthcare Research and Quality

Ernestine W. Murray R. N., B.S.N, M.A.S

EPC Program Task Order Officer

Agency for Healthcare Research and Quality
iv

Acknowledgments

The University of Ottawa Evidence-based Practice Center thanks Dr. David Moher (Director 

EPC), Dr. Timothy Ramsay (Director Methods Centre, Ottawa Hospital Research Institute) and 

Ms. Chantelle Marie Garritty (Senior Program Manager, Ottawa Hospital Research Institute) for 

their intellectual contribution and administrative support. We also give our sincere thanks to Ms. 

Mary P. Nix and Dr. Gurvaneet Randhawa at the Agency for Healthcare Research and Quality 

for their tremendous help in thinking though the early phase of topic refinement and analytic 

framework. 
v

Structured Abstract

Objectives: To examine whether pretreatment determination of thiopurine methyltransferase 

(TPMT) enzymatic activity (phenotyping) or TPMT genotype, to guide thiopurine therapy in 

chronic autoimmune disease patients, reduces treatment harms. Other objectives included 

assessing: preanalytic, analytic, and postanalytic requirements for TPMT testing; diagnostic 

accuracy of TPMT genotyping versus phenotyping; association of thiopurine toxicity with 

TPMT genotypic or phenotypic status; and costs of testing, care, and treating drug-associated

complications.

Data Sources: MEDLINE®, EMBASE®, and Healthstar were searched from inception to May 

2010; the Cochrane Library® to October 2009; and BIOSIS®, Genetics Abstracts, and 

EconLitTM to May 2009, for English language records.

Review Methods: A reviewer screened records, and a second reviewer verified exclusions and 

subsequent selection of relevant studies. Studies in patients with leukemia and organ transplant 

were excluded. Additionally, laboratories that provide TPMT analytical services were surveyed 

to assess means of TPMT testing in practice. Where possible, risk of bias was assessed using 

standard criteria. Meta-analyses estimated diagnostic sensitivity, and specificity; and odds ratios 

of associations.

Results: 1790 titles or abstracts, and 538 full text records were screened. 114 observational 

studies and one RCT were included. Majority of studies were rated fair quality, except for 

diagnostic studies with 37 percent of studies rated poor. In general, there were few patients who 

were homozygous (or compound heterozygous) for TPMT variant alleles in the included studies 

limiting applicability. 

There is insufficient evidence examining effectiveness of pretesting in terms of reduction in 

clinical adverse events.

Sufficient preanalytical data were available regarding preferred specimen collection, stability 

and storage conditions for TPMT testing. There was no clinically significant effect of age, 

gender, various coadministered drugs, or most morbidities (with the exception of renal failure 

and dialysis). TPMT phenotyping methods had coefficients of variation generally below 10 

percent. TPMT genotyping reproducibility is generally between 95-100 percent.

The sensitivity of genotyping to identify patients with low or intermediate TPMT enzymatic 

activity is imprecise, ranging from 70.70 to 82.10 percent (95 percent CI, lower bound range 

37.90 to 54.00 percent; upper bound range 84.60 to 96.90 percent). Sensitivity of homozygous 

TPMT genotype to correctly identify patients with low to absent enzymatic activity was 87.10 

percent (95 percent CI 44.30 to 98.30 percent). Genotyping specificity approached 100 percent. 

Leukopenia was significantly associated with low and intermediate enzymatic activity (low 

activity OR 80.00, 95 percent CI 11.5 to 559; and intermediate activity OR 2.96, 95 percent CI 

1.18 to 7.42), and homozygous and heterozygous TPMT variant allele genotype (OR 18.60, 95 

percent CI 4.12 to 83.60; and 4.62, 95 percent CI 2.34 to 9.16, respectively). In general, TPMT 

phenotyping costs less than genotyping, although estimates across studies are quite 

heterogeneous. 
vi

Conclusions: There is insufficient direct evidence regarding the effectiveness of pretesting of 

TPMT status in patients with chronic autoimmune diseases. Indirect evidence confirms strong 

association of leukopenia with lower levels of TPMT activity and carrier genotype already 

established in the literature. 
vii

Contents

Executive Summary........................................................................................................................ 1

Evidence Report.......................................................................................................................... 11

Chapter 1. Introduction ................................................................................................................. 13

Background............................................................................................................................... 13

Biochemistry of Thiopurines .................................................................................................... 13

Thiopurine Toxicity .............................................................................................................. 14

Scope, Topic Development, and the Key Questions................................................................. 17

Key Questions Addressed in This Report................................................................................. 20

Chapter 2. Methods....................................................................................................................... 21

Literature Search Strategies and Peer Review of Electronic Search Strategies........................ 21

Study Selection ......................................................................................................................... 21

Data Extraction ......................................................................................................................... 24

Assessment of Risk of Bias....................................................................................................... 25

Evidence Synthesis and Analysis.............................................................................................. 26

Pooling of Evidence of Diagnostic Accuracy (Key Question 1c) ........................................ 27

Pooling of Evidence of Association of Thiopurine Toxicity With TPMT Genotypic or 

Phenotypic Status (Key Question 3c) ................................................................................... 29

Rating the Strength of Evidence and Assessing Applicability ............................................. 29

Survey of Laboratories Conducting TPMT Analyses............................................................... 29

Chapter 3. Results......................................................................................................................... 31

Screening and Inclusion of Records for Key Questions........................................................... 31

KQ 1a: In terms of the analytical performance characteristics of enzymatic measurement 

of TPMT activity and determination of TPMT allelic polymorphisms, what are the 

preanalytical requirements for enzymatic measurement of TPMT and determination of 

TPMT allelic polymorphisms? ............................................................................................. 35

KQ 1b: In terms of the analytical performance characteristics of enzymatic measurement 

of TPMT activity and determination of TPMT allelic polymorphisms, what are the within 

and between laboratory precision and reproducibility of the available methods of 

enzymatic measurement of TPMT and determination of TPMT allelic polymorphisms? ... 43

KQ 1c: What is the diagnostic sensitivity and specificity of TPMT allelic polymorphism 

measurement compared to the measurement of TPMT enzymatic activity in correctly 

identifying chronic autoimmune disease patients eligible for thiopurine therapy with low 

or absent TPMT enzymatic activity? How do effect modifiers explain any observed 

heterogeneity in sensitivity and specificity? ......................................................................... 45

KQ 2: Does the measurement of TPMT enzymatic activity or determination of TPMT 

allelic polymorphisms change the management of patients with chronic autoimmune 

disease when compared with no determination of TPMT status? ........................................ 58
viii

KQ 3a: In chronic autoimmune disease patients prescribed thiopurine-based drugs, does 

the assessment of TPMT status to guide therapy, when compared with no 

pretreatmentassessment, lead to reduction in rates of mortality, infection, hospitalization, 

withdrawal due to adverse events, serious adverse events and improvement in 

health-related quality of life? ................................................................................................ 60

KQ 3b: In chronic autoimmune disease patients prescribed thiopurine-based drugs, does

the assessment of TPMT status to guide therapy, when compared with no 

pretreatmentassessment, lead to reduction in rates of myelotoxicity, liver toxicity, and 

pancreatitis? .......................................................................................................................... 62

KQ 3c: In the absence or inconclusiveness of evidence answering key question 3a 

and/or 3b above, is there an association between TPMT status (as determined by TPMT 

enzymatic activity and/or TPMT allelic determination) and/or the following amongst 

chronic autoimmune disease patients treated with thiopurines: ........................................... 65

TPMT Enzymatic Activity Determination............................................................................ 65

TPMT Allelic Determination................................................................................................ 74

KQ 4: What are the costs of determining TPMT enzyme activity and/or genotyping for 

patients with chronic autoimmune disease being considered for thiopurine-based 

therapy?................................................................................................................................. 88

Survey of Laboratories Conducting Analyses of TPMT Enzymatic Activity and 

Genotyping............................................................................................................................ 92

Chapter 4. Discussion ................................................................................................................... 97

TPMT Status Determination..................................................................................................... 97

Preanalytic Requirements and Sample Stability....................................................................... 97

TPMT Variation Amongst Patient Populations.................................................................... 98

Analytic Performance ............................................................................................................... 99

Diagnostic Sensitivity and Specificity................................................................................ 100

Postanalytic Requirements.................................................................................................. 101

Clinical Laboratory Survey................................................................................................. 102

Knowledge of TPMT Status to Guide Therapy ...................................................................... 102

Association of TPMT Status With Thiopurine Toxicity .................................................... 104

Costs of Determining TPMT Status Versus Costs of Treating Drug-Associated 

Complications..................................................................................................................... 106

Strengths and Limitations....................................................................................................... 106

Recommendations and Future Research................................................................................. 106

Conclusions............................................................................................................................. 107

References and Included Studies............................................................................................ 109

List of Acronyms/Abbreviations............................................................................................. 121

Figures

Figure 1. Metabolic pathways of thiopurine drugs....................................................................... 14

Figure 2. Analytic framework....................................................................................................... 19

Figure 3. PRISMA diagram of record identification, screening and inclusion ............................ 32
ix

Figure 4. Risk of bias of studies of diagnostic accuracy of TPMT genetic testing compared 

with TPMT enzymatic activity ..................................................................................................... 47

Figure 5. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, *3B

and *3C, to diagnose TPMT activity ........................................................................................... 49

Figure 6. Meta-analysis 2 of specificity of genotyping TPMT *2, *3A, *3B and *3C, to 

diagnose TPMT activity................................................................................................................ 50

Figure 7. Additional meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, 

*3A, *3B and *3C, or TPMT *3A, *3B and *3C, to diagnose TPMT activity............................ 51

Figure 8. Additional meta-analysis 2 of specificity of genotyping TPMT *2, *3A, *3B and 

*3C, or TPMT *3A, *3B and *3C, to diagnose TPMT activity................................................... 52

Figure 9. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, and 

*3C, to diagnose TPMT activity................................................................................................... 53

Figure 10. Meta-analysis 2 of specificity of genotyping TPMT *2, *3A, and *3C, to diagnose 

TPMT activity............................................................................................................................... 53

Figure 11. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *3A, *3B and 

*3C, to diagnose TPMT activity................................................................................................... 54

Figure 12. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, *3B, 

*3C, *3D, *4, *5, *6, *7, *8, *10, *14, and *15, to diagnose TPMT activity ............................. 55

Figure 13. Meta-analysis 2 of specificity of genotyping TPMT *2, *3A, *3B, *3C, *3D, *4, 

*5, *6, *7, *8, *10, *14, and *15, to diagnose TPMT activity..................................................... 56

Figure 14: Risk of bias of the randomized controlled trial answering KQ 2................................ 59

Figure 15: Risk of bias of the randomized controlled trial answering KQ 3a .............................. 60

Figure 16: Risk of bias of the single randomized controlled trial answering KQ 3b ................... 63

Figure 17: Risk of bias of the single retrospective cohort study answering KQ 3b ..................... 63

Figure 18. Risk of bias of TPMT enzymatic activity, and thiopurine toxicity association 

studies ........................................................................................................................................... 67

Figure 19. Odds ratio of withdrawal due to adverse events during thiopurine treatment of 

chronic autoimmune disease; intermediate versus normal enzymatic activities .......................... 68

Figure 20. Odds ratios of myelotoxicity during thiopurine treatment of chronic autoimmune 

disease; intermediate/normal, low/intermediate and low/normal comparisons............................ 69

Figure 21. Odds ratios of leukopenia during thiopurine treatment of chronic autoimmune 

disease; intermediate/normal, low/intermediate and low/normal comparisons............................ 70

Figure 22. Odds ratio of anemia during thiopurine treatment of chronic autoimmune disease; 

intermediate versus normal TPMT enzymatic activities .............................................................. 71

Figure 23. Odds ratios of hepatitis or elevated hepatic transaminases during thiopurine 

treatment of chronic autoimmune disease: intermediate/normal, low/intermediate and 

low/normal comparisons............................................................................................................... 72

Figure 24. Odds ratios of pancreatitis during thiopurine treatment of chronic autoimmune 

disease: intermediate/normal, and low/intermediate comparisons ............................................... 73

Figure 25. Risk of bias of TPMT allelic determination and thiopurine toxicity association 

studies ........................................................................................................................................... 77

Figure 26. Meta-analysis of odds ratios of infections during thiopurine treatment of chronic 

autoimmune disease, heterozygotes versus noncarriers................................................................ 78

Figure 27. Odds ratio of withdrawal due to adverse events during thiopurine treatment of 

chronic autoimmune disease, heterozygotes versus noncarriers................................................... 78
x

Figure 28. Odds ratio of myelotoxicity during thiopurine treatment of chronic autoimmune 

disease, heterozygotes versus noncarriers..................................................................................... 79

Figure 29. Odds ratio of myelotoxicity during thiopurine treatment of chronic autoimmune 

disease, homozygotes versus noncarriers ..................................................................................... 79

Figure 30. Odds ratio of leukopenia during thiopurine treatment of chronic autoimmune 

disease, heterozygotes versus noncarriers..................................................................................... 80

Figure 31. Odds ratio of leukopenia during thiopurine treatment of chronic autoimmune 

disease, homozygotes versus noncarriers ..................................................................................... 81

Figure 32. Odds ratio of leukopenia during thiopurine treatment of chronic autoimmune 

disease, homozygotes versus heterozygotes................................................................................. 81

Figure 33. Odds ratio of anemia during thiopurine treatment of chronic autoimmune disease, 

heterozygotes versus noncarriers.................................................................................................. 82

Figure 34. Odds ratio of thrombocytopenia during thiopurine treatment of chronic 

autoimmune disease, heterozygotes versus noncarriers................................................................ 82

Figure 35. Odds ratio of hepatotoxicity during thiopurine treatment of chronic autoimmune 

disease, heterozygotes versus noncarriers..................................................................................... 83

Figure 36. Odds ratio of hepatotoxicity during thiopurine treatment of chronic autoimmune 

disease, homozygotes versus noncarriers ..................................................................................... 83

Figure 37. Odds ratio of pancreatitis during thiopurine treatment of chronic autoimmune 

disease, heterozygotes versus noncarriers..................................................................................... 84

Figure 38. Odds ratio of pancreatitis during thiopurine treatment of chronic autoimmune 

disease, homozygotes versus noncarriers ..................................................................................... 85

Figure 39. Costs of genotype test per patient from public laboratory data in USD (2008), in 

chronological order....................................................................................................................... 90

Figure 40. Costs of phenotype test per patient from public laboratory data in USD (2009), in 

chronological order....................................................................................................................... 90

Figure 41. One-time cost estimates of adverse events associated with AZA in USD (2009), in 

chronological order....................................................................................................................... 91

Tables

Table 1. Rating the strength of evidence ........................................................................................ 7

Table 2. TPMT polymorphisms.................................................................................................... 16

Table 3. Study selection criteria.................................................................................................... 22

Table 4. Two by two tables for analysis of diagnostic accuracy .................................................. 25

Table 5. Categorization of overall risk of bias of individual studies............................................ 26

Table 6. Meta-analyses 1 and 2, for Key Question 1c ................................................................. 28

Table 7. Primary records with companion reports passing level III screen.................................. 33

Table 8. Distribution of records by key questions....................................................................... 34

Table 9. Stability of TPMT enzymatic activity ........................................................................... 36

Table 10. Gender related differences in TPMT enzymatic activity.............................................. 37

Table 11. Effect of age on TPMT enzymatic activity................................................................... 38

Table 12. Effects of drugs on TPMT enzymatic activity.............................................................. 39

Table 13. Variation of TPMT activity with race........................................................................... 41

Table 14. TPMT activity in various disease groups ..................................................................... 41
xi

Table 15. Characteristics of studies of diagnostic accuracy of TPMT genetic testing compared 

with TPMT enzymatic activity ..................................................................................................... 45

Table 16. Meta-analyses 1 and 2, for sensitivity and specificity of variant allelic determination 

to diagnose TPMT enzymatic activity .......................................................................................... 48

Table 17. Rating strength of evidence-key question 3a................................................................ 61

Table 18. Rating strength of evidence-key question 3b................................................................ 64

Table 19. Summary characteristics of TPMT enzymatic activity and thiopurine toxicity 

association studies......................................................................................................................... 65

Table 20. Summary characteristics of TPMT allelic determination and thiopurine toxicity 

association studies......................................................................................................................... 76

Table 21. Pooled odds ratios of outcomes on thiopurine treatment between TPMT variant 

allele carriers and noncarriers, as well as between the two carrier states (allele specific 

subgroups)..................................................................................................................................... 85

Table 22. Characteristics of TPMT testing, care and drug-associated complications costs 

studies ........................................................................................................................................... 88

Table 23. Laboratory questionnaire responses.............................................................................. 94

Appendixes

Appendix A: Exact Search Strings

Appendix B: Sample Data Abstraction Forms

Appendix C: Evidence Tables

Appendix D: List of Excluded Studies

Appendix E: Peer Reviewers & Technical Expert Panel

Appendixes and Evidence Tables for this report are provided electronically at 

http://www.ahrq.gov/downloads/pub/evidence/pdf/tpmt/tpmt.pdf.
1

Executive Summary

Background

Thiopurine drugs are used to treat chronic autoimmune inflammatory conditions and 

hematological malignancies, and to prevent organ transplant rejection. The present study focuses 

on populations with autoimmune disease.

Thiopurine drugs are associated with various toxic adverse effects, including 

myelosuppression, hepatotoxicity, pancreatitis, and flu-like symptoms. One of the most serious 

dose-dependent reactions is severe myelosuppression that is thought to be caused by the active 

metabolite, deoxy-6-thioguanosine 5’ triphosphate (6-tGN). Excessive levels of 6-tGN may arise 

not only due to overdosing, but also because of decreased inactivation of the drug. 

The most extensively characterized enzyme in the metabolism of thiopurines is thiopurine 

methyltransferase (TPMT). TPMT inactivates the active forms of two commonly used thiopurine 

drugs, azathioprine (AZA) and 6-mercaptopurine (6-MP), by methylation. Multiple studies have 

shown that lower TPMT enzymatic activity is correlated with higher levels of the active drug 

metabolites and increased thiopurine toxicity. Genetic polymorphisms associated with lower 

TPMT enzymatic activity are similarly correlated. 

Approximately 0.3 percent of the population with chronic autoimmune disease that could 

potentially benefit from thiopurine treatment is homozygous for a variant TPMT allele expressed 

as low or even absent TPMT activity. These patients are at greatest risk of myelosuppression. 

Approximately 15 percent of the patient population is heterozygous for variant alleles; they are 

likely to have intermediate TPMT enzymatic activity, with moderate risk of myelosuppression 

with thiopurine therapy. Four common variant alleles (TPMT*2, *3A, *3B, and *3C) account for 

80 percent to 95 percent of individuals with below normal TPMT activity; however the 

frequency of these alleles varies among Caucasians, Asians, and Africans. 

Until recently, the recommended starting dose of either AZA or 6-MP did not take into 

account patients with very low or absent TPMT activity. The initial doses range from 1.0 to 2.5 

mg/kg/day for AZA, and 0.75 to 1.25 mg/kg/day for 6-MP. It has been proposed that patients 

with either intermediate or low to absent TPMT activity may benefit from lower initial doses. 

Various clinical guidelines recommend measuring TPMT enzymatic activity or screening for 

TPMT alleles before starting patients on thiopurine drugs. However, the evidence base for these 

recommendations is unclear. It is also unclear whether one or both of the tests, TPMT 

genotyping or enzymatic activity (phenotyping) should be used to determine TPMT status before 

thiopurine treatment initiation. As such, there is a need to review the current literature regarding 

the assessment of TPMT status prior to administration of thiopurine drugs, to determine if 

pretreatment TPMT testing reduces drug-related toxicity. The population of interest was 

restricted to those with chronic autoimmune disease, as patients with malignancy or organ 

transplant frequently require concomitant treatments of similar toxicity profile. 

This evidence report was commissioned by the Agency for Healthcare Research and Quality 

(AHRQ) to address the following of questions about TPMT genotypic and phenotypic testing 

methodology, their comparative diagnostic accuracy, effectiveness of pretreatment testing, 

association with drug toxicity, and costs involved.
2

Key Questions

KQ1. In terms of the analytical performance characteristics of enzymatic measurement of 

TPMT activity and determination of TPMT allelic polymorphisms:

a) What are the preanalytical requirements for enzymatic measurement of TPMT and 

determination of TPMT allelic polymorphisms? (e.g. specimen types and collection 

procedures, lab transportation, interference of coadministered drugs, patient preparation 

and identification etc.)

b) What are the within and between laboratory precision and reproducibility of the available 

methods of enzymatic measurement of TPMT and determination of TPMT allelic 

polymorphisms (proficiency testing)?

c) What is the diagnostic sensitivity and specificity of TPMT allelic polymorphism 

measurement compared to the measurement of TPMT enzymatic activity in correctly 

identifying chronic autoimmune disease patients eligible for thiopurine therapy with low 

or absent TPMT enzymatic activity? How do effect modifiers (e.g. underlying disease 

prevalence and severity, different activity thresholds, Hardy-Weinberg equilibrium, 

number and types of alleles tested) explain any observed heterogeneity in sensitivity and 

specificity?

d) Are there any postanalytical requirements specific to measurement of TPMT enzymatic 

activity or TPMT allelic polymorphism measurement? (e.g. timely reporting of data, 

reference intervals, immediate or reporting within a time-frame, highlighting of extreme 

results)

KQ2. Does the measurement of TPMT enzymatic activity or determination of TPMT allelic 

polymorphisms change the management of patients with chronic autoimmune disease when 

compared with no determination of TPMT status? 

KQ3. In chronic autoimmune disease patients prescribed thiopurine-based drugs (AZA or 6-

MP), does the assessment of TPMT status to guide therapy, when compared with no pretreatment

assessment, lead to:

a) reduction in rates of mortality, infection, hospitalization, withdrawal due to adverse 

events (WDAE), serious adverse events (SAE) and improvement in health-related 

quality of life?

b) reduction in rates of myelotoxicity, liver toxicity, and pancreatitis? 

c) In the absence or inconclusiveness of evidence answering key question 3a and/or 3b 

above, is there an association between TPMT status (as determined by TPMT 

enzymatic activity and/or TPMT allelic determination) and/or the following amongst 

chronic autoimmune disease patients treated with thiopurines?

i. the clinical outcomes of mortality, infections, hospitalization, WDAE, SAE 

and health-related quality of life? 

ii. surrogate outcomes of myelotoxicity, liver toxicity, and pancreatitis? 

KQ4. What are the costs of determining TPMT enzyme activity and/or genotyping for 

patients with chronic autoimmune disease being considered for thiopurine-based therapy (e.g., 

costs of testing, costs of care, and costs of treating drug-associated complications)?
3

Methods 

Search Strategy 

The following databases were searched: Ovid MEDLINE® 1950 to May Week 3 2010; The 

Cochrane Library® (CLIB 2009 3) including CENTRAL, CDSR, DARE, HTA, and NHSEED; 

BIOSIS® May 6 2009; EMBASE® 1980 to 2010 Week 21; Genetics Abstracts: May 7 2009; 

and Ovid Healthstar 1966 to April 2010. EconLitTM was searched May 7 2009 for the economic 

question (Key Question 4).

Study Selection

English language records of any study design in chronic autoimmune disease populations 

were included. Effectiveness studies of testing prior to treatment were restricted to comparative 

experimental or observational designs.

Outcomes included determinants of preanalytic variability and proficiency of TPMT 

genotypic and phenotypic (enzymatic activity) testing; diagnostic accuracy of genotypic testing 

compared with the enzymatic assay; clinical and laboratory measures of drug toxicity; and costs 

of both testing and drug-associated complications. 

One reviewer screened abstracts to include studies, and a second reviewer independently 

verified exclusions. Two reviewers independently screened full-text reports, with conflicts 

resolved by consensus or third party adjudication. Data were extracted in standardized forms.

Risk of Bias Assessment 

Standard criteria were used to assess risk of bias of individual studies, except for studies 

eligible for questions pertaining to TPMT testing methods (KQ 1a and 1b) and costs (KQ4), for 

which no assessment scales exist. Studies were assessed as good, fair or poor. 

Evidence Synthesis

Evidence was synthesized qualitatively for key questions 1a, 1b and 4. Data synthesis was 

not possible for key questions 1d, 2, 3a and 3b due to scarcity of evidence. We therefore 

examined associations between thiopurine toxicity and TPMT genotype and phenotype (KQ3c). 

For key question 4, costing data were converted to U.S. dollars (2009) using purchasing power 

parities, inflated to reflect 2009 values using the consumer price index for U.S. medical care for 

all urban consumers. 

Quantitative syntheses were undertaken with the underlying assumption that given similar 

doses of the drugs, differences in outcomes of thiopurine toxicity arise from differences in 

TPMT enzymatic activities. Because enzymatic activity or genotype are the main determinant of 

thiopurine toxicity, we assumed that the underlying autoimmune disease, method of genotyping 

or phenotyping (enzymatic activity testing), population demographics, and study design did not 

give rise to substantial diversity in effect estimates. We, therefore, pooled studies across these 

covariates to estimate diagnostic accuracy and strength of association with adverse events related 

to TPMT testing and status, respectively. Individual study estimates (odds ratios, or sensitivity 

and specificity) were pooled using DerSimonian and Laird’s random-effects model, with 
4

weighting by individual study variance and the estimated between-study heterogeneity. Data 

were pooled when two or more studies were in a given analysis for an outcome. Pooled estimates 

of diagnostic sensitivity and specificity, and odds ratios and their 95 percent confidence intervals 

(CIs) were calculated using CMA software (version 2.2.046). With small numbers of studies in 

most analyses, we could explore clinical and/or methodological diversity for very few of the 

preidentified covariates. When feasible, statistical heterogeneity was tested using Cochran’s Q, 

and reported when found to be substantial (p value for chi-squared test of heterogeneity below 

0.10, and I2 above 50 percent).

For quantitative syntheses of evidence of genetic association studies for drug toxicity 

outcomes, a codominant model was used to pool estimates associated with noncarrier, 

heterozygous carrier and homozygous carrier states. Noncarrier state indicated absence of tested 

TPMT polymorphisms. Heterozygous carrier state indicated presence of one variant TPMT allele 

on one of the paired chromosomes; homozygous carrier state implied presence of one of the 

identical TPMT variant alleles on each one of the paired chromosomes, or presence of two 

different variant alleles each on one of the two paired chromosomes (the latter is also called a 

compound heterozygous state). Similarly, three categories of enzymatic activities were defined 

(high/normal, intermediate and low/absent). We compared each state with the other two 

genotypic or phenotypic states. The TPMT enzymatic activity assay was considered to be the 

reference, for the index test of genotyping of the different single nucleotide polymorphisms 

(SNPs). With a dichotomous index test, i.e. the presence or absence of variant alleles, 

investigation of implicit or explicit cut-off threshold effects was ruled out by design. Therefore, 

we pooled for the outcomes of test sensitivity and specificity for each set of variant TPMT alleles 

tested. 

Rating the Strength of Evidence

Evidence of comparative effectiveness of TPMT pretesting versus no testing for the critical 

and important outcomes of mortality (critical), serious adverse events (critical), myelotoxicity 

(important), and health-related quality of life (important) was rated across the domains of risk of 

bias, consistency, directness and precision as high, moderate, low or insufficient. 

Laboratory Survey

To augment the limited published literature to answer key questions 1a, 1b, 1c, and 1d, 

further data regarding the preanalytical and postanalytical requirements and performance 

characteristics of TPMT laboratory analyses were collected. With advice from the Technical

Expert Panel, the review team decided to survey English speaking laboratories that provide 

TPMT analytical services. Seven laboratories were contacted. An 11-item questionnaire 

addressing TPMT analytical methods (e.g., sample type and handling), preanalytical 

requirements (e.g., specimen stability), quality control procedures, and reporting of results was 

administered via Survey MonkeyTM.

Results 

We screened 1790 titles or abstracts and 538 full text records. One hundred and fifteen 

unique studies and their 21 companion reports were included. One randomized controlled trial
5

was included; all other studies were of observational design. The majority (greater than 75 

percent) of studies were rated as fair, while a substantial (37 percent) percentage of diagnostic 

studies were of poor design. No evidence was found to answer Key Question 1d. Sparse 

evidence answered Key Questions 2, 3a and 3b. In general, there were few patients who were 

homozygous (or compound heterozygous) for TPMT variant alleles in the included populations. 

Six of the seven laboratories invited to participate in the survey returned responses; three 

from Canada and three from the United Kingdom. Among the responses, yearly TPMT testing 

volumes ranged from 50 to 1500 allelic determinations and 600 to 19,000 enzymatic 

determinations.

KQ 1a: Preanalytical Requirements for TPMT Enzymatic Activity and 

allelic Polymorphisms Measurements

Storage conditions and study designs varied widely across 13 studies assessing the influence 

of storage on TPMT activity. Temperatures ranging from -85°C to room temperature, and time 

periods ranging from a few hours to 16 months were studied. TPMT is a stable enzyme and its 

activity remained constant during storage at room temperature for five days or at -20°C for three 

months. Storage at -80°C resulted in 15 percent of TPMT activity decrease after 16 months. All 

surveyed laboratories analyzed specimens of blood with EDTA anticoagulant, stored for up to 

eight days, at 4o

C or room temperature before analysis. Other factors noted prior to testing, such 

as gender, age, and race did not significantly affect the TPMT enzyme activity. 

Nineteen different drugs studied to date in patients being treated for autoimmune conditions 

had no clinically relevant inhibiting effect on TPMT activity (see the main Results section for a 

list of drugs). Studies showing potentially clinically significant effects were conducted in vitro, 

and therefore their in vivo influence on TPMT activity remains unknown.

Research suggests that younger red blood cells (RBCs) have higher TPMT activity than older 

RBCs, but these differences are not clinically relevant and can be avoided if the TPMT activity is 

expressed per grams of hemoglobin or per milliliter of packed RBCs. However, these two 

reporting units are not identical and results are not directly comparable.

Two studies investigated the effect of comorbid conditions on TPMT activity, including 

inflammatory bowel disease, autoimmune hepatitis, multiple sclerosis, myasthenia gravis, 

pemphigus and chronic renal failure. They reported clinically insignificant differences in TPMT 

activity for all diseases, except for patients with the chronic renal failure. These patients’ TPMT 

activity predialysis was 50 percent higher than healthy controls, but postdialysis levels dropped 

to levels comparable with the controls’.

No evidence was reported for patient preparation or identification. Also, no evidence was 

found regarding preanalytical factors influencing TPMT genotyping. However, since 

preanalytical requirements are commonly understood for genetic testing, previously published 

guidelines can be used. The Clinical and Laboratory Standards Institute (CLSI) has published 

guidelines covering all preanalytical requirements for collection, transportation, preparation and 

storage of specimens for genetic testing. 
6

KQ 1b: Within and Between Laboratory Precision and Reproducibility 

of Enzymatic Measurement of TPMT and Determination of TPMT 

Allelic Polymorphisms

Enzymatic assays measure the S-methylation of 6-MP by TPMT to form 6-

methylmercaptopurine. Initially, 6-methylmercaptopurine was measured using a radiolabel 

method, which was later replaced by high performance liquid chromatography (HPLC). 

Alternatively, 6-thioguanine (6-TG) may be used as a substrate in HPLC-based methods, which 

measure 6-methyl-TG. All methods used to measure the TPMT activity are highly precise and 

accurate. The radiolabel method reported by eight studies had interassay and intra-assay

variation coefficients from 0.51 to 8.4 percent and from 0.72 to 6.8 percent, respectively. The 

HPLC based methods produced inter-assay and intra-assay coefficients of variation ranging from 

0.2 to nine percent and from zero to 9.5 percent respectively, in 16 studies. Among surveyed 

laboratories, enzymatic analysis repeatability ranged from three to 10 percent within runs, and 

from five to 20 percent between runs. 

We found only three studies that investigated TPMT genotyping test performance. Matrixassisted laser desorption/ionization, with a time-of-flight (MALDI-TOF) mass spectrometry 

multiplex assay and TaqMan® 5' nuclease assays were compared with denaturating HPLC, and 

100 percent concordance was observed for 586 and 50 genotypes, respectively. The MALDITOF study also measured reproducibility to be in 100 percent agreement, when 10 percent of 

randomly selected samples of the study population were genotyped in duplicate. A novel 

microchip platform was compared with TaqMan® and with a conventional restriction fragment 

length polymorphism (RFLP) assay, resulting in 100 percent concordance. 

KQ 1c: Diagnostic Sensitivity and Specificity of TPMT Allelic 

Polymorphism Measurement, Compared to the Measurement of TPMT 

Enzymatic Activity 

A total of 16 studies, mostly of cross-sectional and prospective observational design, 

contributed to quantitative syntheses. Studies did not specifically examine diagnostic accuracy of 

genetic testing with the TPMT enzymatic activity test as the reference standard, so we designated 

the activity test to be the reference standard and genotyping to be the index test. The pooled 

sensitivity of the carrier genotype (i.e. homozygous plus heterozygous patients) to correctly 

identify all those patients with subnormal (intermediate, or low to absent) enzymatic activity was 

imprecise and ranged from 70.70 to 82.10 percent across the different subgroups of alleles tested 

(95 percent CI, lower bound range 37.90 to 54.00 percent; upper bound range 84.60 to 96.90 

percent). The pooled sensitivity of a homozygous TPMT genotype to correctly identify patients 

with low to absent enzymatic activity was based on two studies with few homozygotes (87.10, 

95 percent CI 44.30 to 98.30 percent). Meta-regression analysis did not identify any significant 

effect modifiers. Compared with the reference standard of TPMT enzymatic activity, the 

specificity of TPMT genotyping to correctly identify patients with normal/high enzymatic 

activities, or normal/high and intermediate enzymatic activities, was very high (greater than 90 

percent) across all combinations of alleles tested.
7

There was insufficient data to determine the optimum combination of TPMT alleles for 

testing. Approximately 80 percent of the studies tested at least the TPMT *3A, *3B, and *3C or 

TPMT *2, *3A, and 3C variant alleles, irrespective of testing additional polymorphisms. 

Among the surveyed laboratories, reported concordance between enzymatic analysis 

genotyping ranged from 60 percent to 100 percent.

KQ 2: Knowledge of TPMT Status and Change in Management 

Evidence from one randomized controlled trial in 333 patients with chronic inflammatory 

conditions of whom only one patient was homozygous for a variant allele suggests that physician 

azathioprine prescribing practice may not entirely be guided by pharmacogenetic testing. 

Cautious prescribing is adopted by physicians regardless of pre treatment genotyping results. No 

significant differences could be observed between the prethiopurine tested and nontested groups 

in azathioprine starting doses or mean doses at the end of the study period, however, 

heterozygotes received lower doses compared with noncarriers in the group pretested before 

therapy.

KQ 3a and 3b: Knowledge of TPMT Status to Guide Therapy 

Table 1. Rating the strength of evidence

Pretreatment genotyping to guide thiopurine treatment vs. thiopurine treatment without pretesting

Outcome N of 

studies

N of 

Subjects Domains pertaining to strength of evidence

OR 

(95% 

CI)

Strength of 

evidence

Risk of 

Bias Consistency Directness Precision 

Mortality 1 RCT1 333 Medium Unknown Direct Imprecise

0.33 

(0.03 

to 

3.18)

Insufficient 

Serious 

adverse 

events 

1 RCT1 333 Medium Unknown Direct Imprecise

0.48 

(0.14 

to 

1.64)

Insufficient

Healthrelated 

quality of life

0 0 - - - - - Insufficient 

Myelotoxicity 0 0 - - - - - Insufficient 

Applicability 

of evidence 

There is limited applicability of evidence for the outcomes of mortality and serious adverse 

events since there was just one homozygous carrier of TPMT variant allele in the entire 

sample of mostly IBD patients observed for just 4 months. Also, patients likelier to 

experience adverse events were excluded during the screening phase

Abbreviations: RCT = randomized controlled trial

Evidence comparing efficacy of prior TPMT status determination with no pretesting from 

one fair quality randomized controlled trial and a poor quality retrospective cohort study 

demonstrated no significant differences in the outcomes of leukopenia, neutropenia and 

pancreatitis, while significantly higher odds were observed for hepatitis in the group randomized 

to prior TPMT genotyping, odds ratio 2.54 (95 percent CI 1.08 to 5.97). Other intermediate 

outcomes were not reported.
8

KQ 3c: Association Between TPMT Status and Thiopurine Toxicity

TPMT enzymatic activity. Among 15 studies, mostly cross-sectional in design, quantitative 

syntheses demonstrated a dose response relationship associating subnormal TPMT enzymatic 

activities with leukopenia and myelotoxicity. In comparison with normal enzymatic activity, 

greater odds of leukopenia were noted with low enzymatic activity (OR 80.00, 95 percent CI 

11.5 to 559), than intermediate activity (OR 2.96, 95 percent CI 1.18 to 7.42). Greater odds of 

myelotoxicity were also noted with low activity when compared with intermediate (OR 10.20, 95 

percent CI 2.23 to 46.60) and normal (OR 13.60, 95 percent CI 3.52 to 52.80) TPMT enzymatic 

activities. 

Pooling of the few small studies with events for the outcomes of withdrawal due to adverse 

events, anemia, hepatitis or elevated hepatic transaminases and pancreatitis, revealed no 

significant associations. 

No evidence was available for the outcomes of mortality, hospitalization, serious adverse 

events, and health related quality of life. The sparse data available for the outcomes of infection, 

neutropenia and thrombocytopenia did not permit a meaningful synthesis. 

TPMT genotype. Thirty studies contributed to quantitative syntheses. A dose response 

relationship was suggested between TPMT genotypic status and leukopenia. In studies testing 

TPMT *2, *3A, *3B, and * 3C, plus/minus additional genetic variants, homozygosity for a 

variant TPMT allele yielded the highest odds ratio for leukopenia (18.60, 95 percent CI 4.12 to 

83.60) compared with noncarrier status, while heterozygous patients experienced lower, but still 

significantly increased odds (4.62, 95 percent CI 2.34 to 9.16) compared with noncarriers. 

However, with only 6 homozygous participants, direct comparison with heterozygous carriers

did not yield statistically significant results.

For all other outcomes of mortality, hospitalization, serious adverse events (SAE), health 

related quality of life (HQOL), neutropenia, infection, withdrawal due to adverse events, 

myelotoxicity, anemia, thrombocytopenia, hepatitis or elevated hepatic transaminases, 

and pancreatitis, evidence was either absent, insufficient or lacked power to demonstrate 

significant differences between heterozygous and homozygous carriers in comparisons with 

noncarriers, and between themselves.

KQ 4: Costs of TPMT Testing, Costs of Care, and Costs of Treating 

Drug-Associated Complications

Eleven studies reported data on the costs of determining TPMT activity and/or genotyping 

for patients with chronic autoimmune disease being considered for thiopurine therapy. The 

studies were conducted in Canada, United States, New Zealand, Europe (Italy, Scotland, United 

Kingdom, Spain), and Korea. All data were converted into U.S. dollars (2009).

Eight studies reported 11 cost estimates for TPMT genotyping, which were obtained from 

public and private laboratories or hospitals. The cost of obtaining a test per patient ranged from 

$29.43 to $617.80, with the highest cost being reported by a private laboratory. Excluding the 

cost from the private laboratory, the average cost for the genotype test per patient was $89.94. 

Four studies reported five cost estimates for the TPMT enzymatic analysis, which were also 

from laboratories or hospitals. The cost of obtaining a test per patient ranged from $46.36 to 

$320.98 and the source for the highest cost was not reported. Excluding the highest costing item, 

the average cost for the TPMT phenotype test per patient was $53.13.
9

Seven studies reported eight cost estimates for treating AZA related complications. The costs 

were obtained from hospitals and governmental agencies. The one-time cost of adverse events 

associated with AZA ranged from $1,366.82 to $7,110.02 USD (2009), with an average of 

$4,019.29. One study reported two cost estimates for the average cost per identified TPMTdeficient individual, with an average of $11,848.51. 

Conclusions

There is currently insufficient evidence regarding effectiveness of determining TPMT status 

prior to thiopurine treatment in terms of improvement in clinical outcomes and incident 

myelotoxicity in comparison with routine monitoring of full blood counts and adverse events. It 

is also unclear whether pretesting guides appropriate prescribing. Indirect evidence confirmed

previously known strong associations between lower levels of TPMT enzymatic activity and the 

presence of TPMT variant alleles with thiopurine related leukopenia. Sufficient preanalytical 

data were available to recommend preferred specimen collection, stability and storage conditions 

for determination of TPMT status. There was no clinically significant effect on TPMT activity of 

age, gender, various coadministered drugs, or most morbidities (with the exception of renal 

failure and dialysis). The available methods for determination of TPMT enzymatic activity 

showed good precision, with coefficients of variation generally below 10 percent. Based upon 

limited evidence, the reproducibility of TPMT allelic polymorphism determination is acceptable. 

However, the sensitivity of genetic testing to identify patients with low or intermediate TPMT 

enzymatic activity is imprecisely known. Thus, if knowledge of TPMT status is desired and there 

has been no recent transfusion of RBCs, with the current evidence enzymatic assay 

(phenotyping) rather than allelic polymorphism determination is preferred. Enzymatic assay will 

capture effects of other polymorphisms that are not detected by genotyping the common alleles; 

laboratories tend to use genotyping as a confirmatory test for low TPMT activity. The average 

cost of TPMT phenotyping was approximately half of the average cost of TPMT genotyping, but 

these costs may not be generalizable to all TPMT tests. More research has been conducted 

examining TPMT genotyping but the cost estimates are heterogeneous, likely due to different 

methodological choices. 

Remaining Issues

There is insufficient evidence examining the effectiveness of TPMT pretreatment enzymatic 

or genetic testing, to minimize thiopurine related toxicity in patients with chronic autoimmune 

diseases. As a priority, well powered, good quality, randomized controlled studies need to be 

conducted, in diverse and representative patient populations, to compare the effectiveness of 

TPMT genotyping and phenotyping with one another, and with no TPMT testing. These studies 

should be large enough to include a sizable number of patients homozygous for the variant 

alleles and should be pragmatic in conduct, mimicking routine clinical practice. Outcomes would 

include both treatment efficacy and harms associated with thiopurine therapy. Another objective 

would be to establish the optimum initial dose adjustment for a given TPMT status. These 

studies should ensure that outcomes are truly assessed without prior knowledge of results of 

TPMT testing and administered drug dose, by employing appropriate blinding procedures. The 

recently concluded pragmatic TARGET study by Newman and associates was under-powered to 

detect differences in clinically important outcomes, largely because it faced recruitment 
10

problems. In future such recruitment problems may be mitigated by educating the public and 

clinicians that the evidence base for pretreatment TPMT testing is lacking and that it is unclear 

whether pretreatment testing does more good (i.e. reduction in thiopurine related toxicity) than 

harm (i.e. reduction in thiopurine efficacy because of overzealous dose reductions based on prior 

testing). 

Until such experimental high quality evidence becomes available, alternative evidence may 

be sought in prospectively designed observational studies that estimate health related quality of 

life, drug prescription patterns, and myelotoxicity related mortality as important outcomes 

associated with and with no pretreatment TPMT testing. With availability of empiric evidence 

from such studies, decision-analytic modeling that comprehensively consider alternative 

strategies such as regular blood cell count and liver enzyme testing, metabolite monitoring, and 

dose adjustments for concomitant medications that impact the TPMT enzymatic pathway can 

help guide practice until evidence becomes available from well powered pragmatic trials. 

Subsequent models might also need to consider new information as technologies develop and 

knowledge evolves. 

TPMT genotyping should test for the most common TPMT polymorphisms in the population 

of interest. There is little direct evidence identifying the optimum set of alleles to be tested, and 

this may need to be established for specific populations if TPMT genotyping turns out to be 

effective in future studies. 

TPMT activity analyses are reported on one of two bases: per milliliter of packed red blood 

cells; or per gram of hemoglobin. These are not readily or exactly comparable. Common 

reporting units are needed, as well as cutoffs for low/absent, intermediate, normal TPMT 

enzymatic activity, and high enzymatic activities. 

Future studies should clearly report numbers of uninterpretable or equivocal test results.
Evidence Report 

13

Chapter 1. Introduction

Background

Thiopurines make up a class of immunosuppressive and chemotherapeutic drugs that is used 

effectively in the treatment of chronic autoimmune inflammatory conditions, hematological 

malignancies, and prevention of organ transplant rejection. Azathioprine (AZA), 6-

mercaptopurine (6-MP) and 6-thioguanine (6-TG) are the thiopurine drugs currently used in 

clinical practice. The clinical response to thiopurines varies according to the nature of disease, 

dose and patient metabolism of the drugs.

AZA and 6-MP are currently widely used as steroid sparing agents in chronic autoimmune 

inflammatory conditions, including pemphigoid, inflammatory bowel disease, and rheumatoid 

arthritis, among others. AZA and 6-MP are effective in inducing remission in 50 percent to 60 

percent of inflammatory bowel disease patients, and permit steroid reduction or withdrawal in up 

to 65 percent of patients.2 Clinical response rates using AZA to treat nonbullous inflammatory 

dermatoses can be as high as 75 percent.3 However, use of AZA or 6-MP in other chronic 

inflammatory disorders including lupus and rheumatoid arthritis has been variable, and they are 

often not the primary drugs of choice.

When used among organ transplant patients, although AZA has been associated with a 5-year 

renal graft survival ranging from 70 percent to 92 percent,4,5 use of AZA and 6-MP in 

transplantation has declined somewhat in favor of other immunosuppressive drugs. 

Both 6-MP and 6-TG have been used effectively in treatment of childhood acute 

lymphoblastic leukemia, with remission rates (5-year relapse free survival) of approximately 80 

percent using 6-MP.6

Patients with cancer or transplanted organs are clinically more complex, so this review 

focuses on thiopurine use in autoimmune conditions. 

Biochemistry of Thiopurines

AZA and 6-MP are pro-drugs that have no intrinsic biological activity, and require extensive 

metabolism for activity (Figure 1). After oral administration of AZA or 6-MP, between 27 

percent and 83 percent is biologically available. AZA is often used clinically, as it is more stable 

and soluble than 6-MP. AZA doses are higher because the molecular weight of 6-MP is 55 

percent of that of AZA. 

In the gut, approximately 90 percent of AZA is converted to 6-MP, a thiopurine analogue of 

the purine base hypoxanthine, by cleavage of the imidazolyl moiety which is thought to be 

catalyzed through the action of glutathione transferase.7 6-MP is then enzymatically converted to 

its active metabolite, deoxy-6-thioguanosine 5’ triphosphate (6-tGN), through successive 

enzymatic conversions by hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and 

inosine monophosphate dehydrogenase (IMPDH). Inactivation of 6-MP (and hence AZA) occurs 

primarily through S-methylation by thiopurine S-methyltransferase (TPMT), and to a minor 

degree by catabolism, to thiouric acid by xanthine oxidase (XO). 

6-TG is converted to its active metabolite (6-tGN) in a single step involving HGPRT, while 

inactivation occurs through two pathways. The major metabolic pathway involves guanine 

deaminase (GD) and aldehyde oxidase (AO) to form inactive 6-thiouric acid. Metabolism by 
14

TPMT, to form inactive 6-methyl-TG, is a minor contributor to drug inactivation. TPMT also 

plays a minor role in directly methylating and inactivating 6-tGN.

Incorporation of 6-tGN into DNA triggers cell-cycle arrest and apoptosis through the 

mismatch repair pathway. Until recently, this was considered the primary mechanism of action.8

However, recent evidence has suggested other mechanisms of immunosuppression not directly 

related to 6-tGN incorporation into DNA. Metabolism by TPMT of 6-thiomercaptopurine (6-

tIMP), an intermediate metabolite, to produce 6-methyl –tIMP has been shown to inhibit de novo 

purine synthesis in lymphocytes, which likely contributes to the immunosuppressive effects of 

thiopurines.9 Furthermore, accumulation of 6-tGN in lymphocytes has been demonstrated to 

decrease the expression of TRAIL, TNFRS7, and α-4 integrin, effectively decreasing 

inflammation. Thiopurine drugs have also been shown to induce apoptosis in T-cells through 

modulation of Rac1 activation upon CD28 costimulation. Rac1 is a GTPase upstream of MEK, 

NF-κB, and bcl-xL. Upon binding of 6-thio-GTP with Rac1, activation of its downstream 

mediators is blocked, inducing apoptosis.10 

Figure 1. Metabolic pathways of thiopurine drugs

Abbreviations: 6-MP = 6-mercaptopurine; 6-tGN = 6-thioguanine nucleotides; 6-tIMP = 6-thiomercaptopurine; 6-TG = 6-

thioguanine monophosphate; 6-tGN = deoxy-6-thioguanosine 5’ triphosphate; 6-tXMP = 6-thiooxanthosine; AO = aldehyde 

oxidase; AZA = azathioprine; GD = guanine deaminase; HGPRT = hypoxanthine guanine phosphoribosyltransferase; IMPDH = 

inosine monophosphate dehydrogenase; TPMT = thiopurine S-methyltransferase; XO = xanthine oxidase

Thiopurine Toxicity

Thiopurine-based drugs have been associated with various toxic adverse events, including 

myelosuppression, hepatotoxicity, pancreatitis, and flu-like symptoms, among others. One of the 

most serious dose-dependent reactions is myelosuppression, which is believed to be caused by 

increased 6-tGN levels (the active metabolite), either due to overdosing or a low rate of 

AZA

TPMT

6-tIMP 6-tXMP

TPMT

6-tGN 6-TG

TPMT

6-MethylMP

(Inactive) 6-MethyltIMP 6-methylTG

(Inactive)

6-thiouric acid

(Inactive)

XO

HGPRT IMPDH

TPMT

Increased

Apoptosis

6-MethyltGMP

(Inactive)

HGPRT

GD or AO

XO

6-MP

6-thiouric acid

(Inactive)

Inhibition of de novo

purine synthesis

Rac1 

inhibition

Incorporation 

into DNA

Decreased expression:

TRAIL

TNFRSF7

α4-integrin

Decreased

Inflammation

 

 

 

 

AZA

TPMT

6-tIMP 6-tXMP

TPMT

6-tGN 6-TG

TPMT

6-MethylMP

(Inactive) 6-MethyltIMP 6-methylTG

(Inactive)

6-thiouric acid

(Inactive)

XO

HGPRT IMPDH

TPMT

Increased

Apoptosis

6-MethyltGMP

(Inactive)

HGPRT

GD or AO

XO

6-MP

6-thiouric acid

(Inactive)

Inhibition of de novo

purine synthesis

Rac1 

inhibition

Incorporation 

into DNA

Decreased expression:

TRAIL

TNFRSF7

α4-integrin

Decreased

Inflammation

 

 

 

 
15

thiopurine metabolism. The most extensively characterized enzyme in the metabolism of 

thiopurines is TPMT. 

TPMT polymorphisms. The TPMT gene is located on chromosome 6 at 6p22.3. It is 

approximately 27 kb in size and contains 9 exons.11,12 A nonfunctional TPMT pseudogene has 

also been identified on chromosome 18 at 18q21.1.13 TPMT is widely expressed in many tissues, 

but TPMT expression in lymphocytes, red blood cells and bone marrow is most relevant 

clinically for immunosuppression by thiopurine drugs. To date, at least 30 variant (or mutant)

alleles of TPMT have been identified, the majority of which have been associated with lower 

TPMT enzymatic activity or protein expression (Table 2).14 Several studies have highlighted the 

importance of thiopurine drug metabolism by TPMT, as lower TPMT may place patients at 

higher risk of developing drug-related toxicity.15,16 The four most common alleles (TPMT*2, 

TPMT*3A, TPMT*3B, and TPMT*3C) seen in Caucasians, Asians, and Africans account for 

approximately 80 percent to 95 percent of individuals with lower TPMT activity.17-22 When 

comparing genotype to phenotype (enzymatic activity), homozygous mutant individuals have 

very low or absent enzymatic activity while those heterozygous for a mutant allele demonstrate 

intermediate enzymatic activity, between those of noncarrier and homozygous individuals. The 

frequency of the common alleles within each ethnic group varies, as does the overall number of 

individuals with lower TPMT activity. Heterozygous individuals with intermediate enzymatic 

activity comprise five percent to 15 percent of patients, while approximately 0.3 percent are 

homozygous, with very low or absent enzymatic activity.17,18,23
16

Table 2. TPMT polymorphisms 

Note: a Originally called TPMT*20,29 another paper by the same group refers to the same allele as TPMT*24.14 Abbreviation: 

TPMT = Thiopurine methyltransferase

Allele Nucleotide Amino acid 

substitution Enzyme activity

TPMT*1 WT Wild-type activity.

TPMT*2 238G→C 80Ala→Pro Low24

TPMT*3A 460G→A 154Ala→Thr Low24

719A→G 240Tyr→Cys

TPMT*3B 460G→A 154 Ala→Thr In vitro assay: significant enzyme activity decrease24

TPMT*3C 719A→G 240Tyr→Cys In vitro assay: enzyme activity decrease25

TPMT*3D 460G→A 154Ala→Thr Intermediate24

719A→G 240Tyr→Cys

292G→T 98Glu→X

TPMT*4 _1G→A 

(intron 9)

Splicing defect Low24

TPMT*5 146T→C 49Leu→Ser In vitro assay: enzyme activity decrease14,25

TPMT*6 539A→T 180Tyr→Phe Low24

TPMT*7 681T→G 227His→Gln Intermediate24

TPMT*8 644G→A 215Arg→His Intermediate24

TPMT*9 356A→C 119Lys→Thr Intermediate/normal24

TPMT*10 430G→C 144Gly→Arg In vitro assay: enzyme activity decrease25,26

TPMT*11 395G→A 132Cys→Tyr Low24

TPMT*12 374C→T 125Ser→Leu In vitro assay: enzyme activity decrease24

TPMT*13 83A→T 28Glu→Val In vitro assay: enzyme activity decrease25,26

TPMT*14 1A→G 1Met→Val Low24

TPMT*15 _1G→A 

(intron 7)

Splicing defect Low24

TPMT*16 488G→A 163Arg→His Intermediate24

TPMT*17 124C→G 42Gln→Glu Intermediate24

TPMT*18 211G→A 71Gly→Arg Intermediate24

TPMT*19 365A→C 122Lys→Thr Normal24

TPMT*20 712A→G 238Lys→Glu Intermediate24

TPMT*20a 106G→A 36Gly→Ser In vitro assay: significant enzyme activity 

decrease24

TPMT*21 205C→G 69Leu→Val Intermediate24

TPMT*22 488G→C 163Arg→Pro Intermediate24

TPMT*23 500C→G 167Ala→Gly Low24

TPMT*24 537G→T 179Gln→His Intermediate24

TPMT*25 634T→C 212Cys→Arg Intermediate24

TPMT*26 117T→ C 208Phe→Leu Intermediate27

TPMT*27 19T→ G 107Tyr→ Asp Intermediate28
17

TPMT analysis. Analysis of TPMT status can be accomplished through either analysis of 

the red blood cell TPMT enzymatic activity, or genotyping. Genetic analysis in routine clinical 

laboratories involves targeting specific TPMT mutations, usually at least three of the four 

common alleles. Depending on the mutant alleles targeted and the ethnic background of the 

patient, genotyping can identify up to 95 percent of affected individuals, but it will not identify 

those patients with rare mutations. Since the frequency of the rare mutations is exceedingly low, 

the probability of missing patients with rare mutations is also low. The enzymatic assay is 

currently considered to be the gold standard measurement, since it should identify all patients 

with reduced enzymatic activity, regardless of mutations. However, the enzymatic assay is 

technically more challenging to perform. In current clinical practice, both enzymatic testing and 

genetic analysis are being performed, depending on the laboratory.

Clinical utility and validity of TPMT analyses. Currently, there is no evidence that the 

presence of one or more mutant TPMT alleles causes disease or places one at increased risk for 

disease. However, the presence of a mutant allele has been suggested to increase the risk of 

thiopurine-related drug toxicity, particularly when using AZA or 6-MP (6-TG is not metabolized 

to as great an extent by TPMT). Therefore, a fraction of patients prescribed thiopurines are at 

greater risk of developing drug-related toxicity. Until recently, all patients were prescribed a 

standard starting dose of either AZA or 6-MP. The current starting dose for AZA ranges from 1.0 

to 2.5 mg/kg/day and 0.75 to 1.25 mg/kg/day for 6-MP. Patients with either intermediate or low 

to absent TPMT activity may benefit from a decreased starting dose. 

Various clinical guidelines suggest measuring TPMT enzymatic activity or screening for 

TPMT alleles associated with reduced enzymatic activity before starting patients on thiopurine 

drugs.30,31 However, measuring TPMT activity may not lead to reduced drug-related toxicity 

since regular monitoring is recommended. Complete blood counts, including platelet counts are 

recommended to be done weekly during the first month, twice monthly for the second and third 

months of treatment, then monthly or more frequently if dosage alterations or other therapy 

changes are necessary.32 As such, there is a need to review the current literature regarding the 

assessment of TPMT status prior to administration of thiopurine drugs, to determine if TPMT 

testing will reduce drug-related toxicity.

Scope, Topic Development, and the Key Questions

This review of the effectiveness of determining thiopurine methyl transferase (TPMT) 

enzymatic activity prior to initiation of thiopurine therapy in patients with chronic autoimmune 

diseases was nominated by the American Association for Clinical Chemistry (AACC), and 

commissioned by the Agency for Healthcare Research and Quality (AHRQ).

TPMT status can be assessed by direct determination of the TPMT enzymatic activity 

(phenotyping), or by genotyping TPMT gene coding for the enzyme for common single 

nucleotide polymorphisms (SNPs), also referred to as variant alleles, coding for the enzyme.

In theory, TPMT status determination before initiating thiopurine therapy may be undertaken 

in order to address two potential clinical scenarios:

• minimize thiopurine toxicity in up to 15 percent of patients with lower TPMT 

enzymatic activity, by thiopurine dose reduction or switching to alternative treatment 

• optimize clinical responsiveness in patients with abnormally elevated TPMT 

enzymatic activity, by dose escalation 
18

In the first scenario, thiopurine dose reduction, in order to minimize drug toxicity associated 

with excessively elevated levels of thioguanine nucleotides, may negatively affect treatment 

efficacy (i.e. overzealous dose reduction to subtherapeutic thiopurine levels). Hence, an 

additional concern in tandem with the first scenario is what an optimally effective dose reduction 

should be, in light of pretreatment knowledge of TPMT status, in patients likely to experience 

increased thiopurine toxicity. Based on a scoping review of the literature, it was anticipated that 

this additional concern had not been adequately investigated in primary research. Therefore, the 

current systematic review of literature does not investigate the relative efficacy or the 

effectiveness of thiopurine dose adjusted treatment of chronic autoimmune diseases with 

pretreatment TPMT testing. As such, this review focuses on the equipoise of whether 

pretreatment determination of the TPMT status (using genotyping and/or phenotyping) mitigates 

harms associated with thiopurine therapy. In addition, the accuracy of TPMT status 

determination by genotyping is also investigated, in reference to the enzymatic activity assay, as 

well as the costs and potential savings associated with TPMT testing.

The second clinical scenario could not be investigated because abnormally high TPMT 

enzymatic activity has not been investigated in any detail in primary research, so its significance 

is not yet appreciated. 

The analytic framework (Figure 2) depicts the causal pathways forming the basis of the key 

questions. Since study of myelosuppression in organ transplant and cancer patients poses several 

potential confounders (namely, concomitant myelosuppressive treatment and the short-term 

complications induced by the procedure or disease), the eligibility criteria were restricted to 

populations with chronic autoimmune diseases. As well, since the metabolism of the drug 6-

thioguanine does not involve the TPMT enzyme, we focused on the two thiopurine drugs, AZA 

and 6-MP.
19

Figure 2. Analytic framework

Abbreviations: KQ = key question; TPMT = thiopurine methyltransferase

Preanalytic 

validity

Analytic 

validity Clinical utility

5-10% normal 

dose of thiopurine 

or alternative 

immunosuppressa

nt 

Testing: 

genotyping 

OR 

phenotyping

Wild type 

genotype or 

normal TPMT

enzymatic activity

Non-transplant, non-cancer 

patients with chronic 

autoimmune disease, of any 

age, potentially benefiting 

from azathioprine or 6-MP 

Heterozygous for 

inactive allele or 

intermediate TPMT 

enzymatic activity 

Homozygous for 

inactive allele or 

very low to absent 

TPMT enzymatic 

activity 

Normal dose of 

thiopurine 

50% reduced dose 

of thiopurine 

• mortality

• infection

• hospitalization / 

ICU admission

• WDAE

• SAE

• HQOL

No prior 

testing 

KQ 1a

pretesting

KQ 1b

precision, reliability

KQ 1d

reporting

KQ 1c 

diagnostic accuracy

Standard initial 

dose of thiopurine 

KQ 2 – patient management

KQ 3a, b – adverse outcomes

KQ 4

costs

• myelotoxicity

• pancreatitis

• hepatitis

KQ 3c

TPMT status 

associated with 

outcomes
20

Key Questions Addressed in This Report

The following key questions were generated by the University of Ottawa Evidence Based 

Practice Centre in consultation with American Association for Clinical Chemistry and the 

Agency for Health Research Quality. Outcomes are considered only in the context of drug 

toxicity/adverse events, and not of efficacy.

KQ1. In terms of the analytical performance characteristics of enzymatic measurement of 

TPMT activity and determination of TPMT allelic polymorphisms:

a) What are the preanalytical requirements for enzymatic measurement of TPMT and 

determination of TPMT allelic polymorphisms? (e.g. specimen types and collection 

procedures, lab transportation, interference of coadministered drugs, patient preparation 

and identification etc.)

b) What are the within and between laboratory precision and reproducibility of the 

available methods of enzymatic measurement of TPMT and determination of TPMT 

allelic polymorphisms (proficiency testing)?

c) What is the diagnostic sensitivity and specificity of TPMT allelic polymorphism 

measurement compared to the measurement of TPMT enzymatic activity in correctly 

identifying chronic autoimmune disease patients eligible for thiopurine therapy with low 

or absent TPMT enzymatic activity? How do effect modifiers (e.g. underlying disease 

prevalence and severity, different activity thresholds, Hardy-Weinberg equilibrium, 

number and types of alleles tested) explain any observed heterogeneity in sensitivity and 

specificity?

d) Are there any postanalytical requirements specific to measurement of TPMT enzymatic 

activity or TPMT allelic polymorphism measurement? (E.g. timely reporting of data, 

reference intervals, immediate or reporting within a time-frame, highlighting of extreme 

results)

KQ2. Does the measurement of TPMT enzymatic activity or determination of TPMT allelic 

polymorphisms change the management of patients with chronic autoimmune disease when 

compared with no determination of TPMT status? 

KQ3. In chronic autoimmune disease patients prescribed thiopurine-based drugs (AZA or 6-

MP), does the assessment of TPMT status to guide therapy, when compared with no pretreatment 

assessment, lead to:

a) reduction in rates of mortality, infection, hospitalization, withdrawal due to adverse 

events (WDAE), serious adverse events (SAE) and improvement in health-related 

quality of life?

b) reduction in rates of myelotoxicity, liver toxicity, and pancreatitis?

c) In the absence or inconclusiveness of evidence answering key question 3a and/or 3b 

above, is there an association between TPMT status (as determined by TPMT enzymatic 

activity and/or TPMT allelic determination) and/or the following amongst chronic 

autoimmune disease patients treated with thiopurines?

i. the clinical outcomes of mortality, infections, hospitalization, WDAE, 

SAE and health-related quality of life? 

ii. surrogate outcomes of myelotoxicity, liver toxicity, and pancreatitis? 

KQ4. What are the costs of determining TPMT enzyme activity and/or genotyping for 

patients with chronic autoimmune disease being considered for thiopurine-based therapy (e.g., 

costs of testing, costs of care, and costs of treating drug-associated complications)?
21

Chapter 2. Methods

Literature Search Strategies and Peer Review of Electronic 

Search Strategies

Electronic search strategies were developed and tested through an iterative process by an 

experienced medical information specialist (BS) in consultation with the team. The search 

strategy was peer reviewed by RD according to the PRESS guideline.33 The following databases 

were searched: Ovid MEDLINE® 1950 to May Week 3 2010; The Cochrane Library® (CLIB 

2009 3) including CENTRAL, CDSR, DARE, HTA, and NHSEED; BIOSIS® May 6 2009; 

EMBASE® 1980 to 2010 Week 21; Genetics Abstracts: May 7 2009; and Ovid Healthstar 1966 

to April 2010. EconLitTM was searched May 7 2009 for the economics section (question 4). 

Strategies utilized a combination of controlled vocabulary and keywords. No language or date 

restrictions were imposed on any of the searches and animal studies were excluded. Additional 

references were identified through scanning reference lists of relevant articles, and by expert 

nomination. Grey (unpublished, unindexed or difficult to locate) literature were sought through 

searching the websites of relevant specialty societies and organizations. The detailed search 

strategies are available in Appendix A.

Study Selection 

We employed three levels of screening of retrieved records. Study selection was based on 

predefined eligibility criteria of interventions (testing and treatment), patient populations, 

outcome measures, and study design (Table ). Studies that included patients reporting to a clinic 

then tested their TPMT genotype or phenotype to correlate with AEs in which adverse events 

were noted from patients charts were considered cross-sectional in design. The electronic 

literature search and expert-nominated records were uploaded to the software program EPPIReviewer 3.0,

34 along with screening questions developed by the review team. Titles and 

abstracts were screened by one reviewer for potential relevance, and exclusions at this level were 

verified by a second reviewer. In case of disagreement or uncertainty about relevance, the record 

was passed through to the next level for full-text review. Two reviewers reviewed full text 

reports independently, applying a priori eligibility criteria. Discrepancies were resolved through 

discussion and consensus or by third party adjudication if consensus could not be reached. 

Reviewers were not masked to the reports’ authors, institution or journal. For key questions 1a 

and 1b, a third level of screening was undertaken by content experts (RB and EL) to confirm 

records marked as relevant at a relatively liberal level II screening. Records that were 

unavailable as abstract or full text reports, or were commentaries, letters, editorials and reviews 

were excluded. Only articles in the English language were eligible.
22

Table 3. Study selection criteria 

Criterion Key 

question Included Excluded Eligible study design

Patient 

population

All key 

questions

Patients with chronic autoimmune diseases, 

of any age

Cancer, organ transplant -

Drug 

treatment(s) 

All key 

questions

Azathioprine (AZA), and 6-mercaptopurine 

(6-MP)

6-thioguanine, excluded 

because it is not metabolized 

by the TPMT enzyme

-

Test(s) All key 

questions

1. TPMT enzymatic activity as determined by 

HPLC, radioassay or mass spectrometry

2. Genotyping using PCR, direct sequencing 

or RFLP

Mass quantitation was 

excluded because it is rarely 

used

-

Outcomes 1a Specimen types and collection procedures, 

lab transportation, interference of 

coadministered drugs, patient preparation and 

identification, enzyme stability, and factors 

affecting preanalytic enzymatic variability (e.g. 

gender, age, comorbid conditions etc.) 

- Any

1b Inter-, and intra-assay coefficients of 

variations and/or test concordance

- Any

1c Testing sensitivity and specificity - Any

2 Patients requiring thiopurine dose reduction, 

patients switching to nonthiopurine treatment, 

and number of specific monitoring tests per 

person time at risk, or per patient per unit 

treatment time 

Differences in efficacy (i.e. 

clinical response to 

treatment)

Randomized controlled 

trials, controlled clinical 

trials, cohort, and casecontrol studies reporting 

numerical data in which 

administration of 

thiopurine treatment

followed thiopurine status 

testing and was adjusted 

or replaced (by another 

disease modifying 

treatment) in at least one 

group

3a Mortality, infection, hospitalization (including 

ICU admissions if available), withdrawal due to 

adverse events (WDAE), serious adverse 

events (SAE), Health-related quality of life 

measures (HQOL)

3b Myelotoxicity (two or more of leukopenia, 

thrombocytopenia, and anemia), leukopenia, 

neutropenia, thrombocytopenia, and anemia, 

hepatoxicity (raised ALT/AST) and pancreatitis 

3c Mortality, hospitalization (including ICU 

admissions if available), withdrawal due to 

adverse events (WDAE), serious adverse 

events (SAE), Health-related quality of life 

measures (HQOL), myelotoxicity (two or more 

Any study design in which 

thiopurine treatment was 

not guided by prior 

knowledge of TPMT status
Table 3. Study selection criteria (continued)

23

Criterion Key 

question Included Excluded Eligible study design

of leukopenia, thrombocytopenia, and 

anemia), leukopenia, neutropenia, 

thrombocytopenia, anemia, hepatoxicity 

(raised ALT/AST) and pancreatitis 

4 Costs of testing, costs of care, costs of 

treating drug-associated complications

Economic analyses (e.g., cost 

analysis, cost-benefit 

analysis, cost-effectiveness 

analysis, cost-utility analysis) 

Studies that provide 

costing data

Publication 

status 

All key 

questions

NonEnglish publication, 

editorial, review, commentary, 

letter, news report or case 

report. Also records 

unavailable as abstracts or 

full text reports

Abbreviations: 6-MP = 6-mercaptopurine; AZA = azathioprine; HQOL = Health-related quality of life; ICU = intensive care unit; PCR = polymerase chain reaction; RFLP = 

restriction fragment length polymorphism; WDAE = withdrawal due to adverse events; SAE = serious adverse events; TPMT = thiopurine methyltransferase
24

Data Extraction 

Sample data extraction forms are presented in Appendix B. Where applicable for a key 

question, we extracted the following data from studies: first author’s name, year of publication, 

country, study design (prospective or retrospective), sample size, eligibility criteria, population 

characteristics, type of genetic testing (e.g. alleles tested, method of testing and source of DNA) 

and enzymatic assay, testing thresholds, percentage of nondiagnostic results, deviation from 

Hardy-Weinberg equilibrium (HWE), reported sensitivity and specificity, drug treatment and 

dose, definitions of outcomes, number of patients with and without events as dichotomous 

outcomes data for a given genotypic or phenotypic enzymatic status, adjusted and crude effect 

estimates with standard error or confidence intervals, validation of health profiles, coefficient of 

variations, preanalytic variables for TPMT activity, costing items (e.g., costs of adverse events, 

cost of testing, costs of care, currency, discount rate, source of cost data) and determinants of 

preanalytic validity as reported in papers. 

For the diagnostic accuracy question, numerical and meta-analyzable data were extracted in 

two 2×2 tables as shown in Table . Patients tested negative for any of the SNPs were considered 

noncarriers (wild type homozygous), while carriers were either heterozygous for the variant 

allele or homozygous for it. For data clarification and missing data we wrote to study authors. 

Heterozygous carrier state implied presence of one variant allele on one of the two paired 

chromosomes; homozygous state implied presence of one of the same variant alleles on each one 

of the two paired chromosomes, or presence of two different mutant alleles on each of the two 

paired chromosomes (the latter is also called a compound heterozygous state). In other words, 

compound heterozygosity was extracted and interpreted as homozygous carrier data. We did not 

differentiate between normal and high TPMT activities, and most studies used the terms 

interchangeably. When investigators reported raw data, we considered TPMT activity of less 

than 5.0 Units per milliliter of packed red blood cells (U/mL RBCs) as low, between 5.0 and 

13.5 U/mL RBCs as intermediate, and greater than 13.5 U/mL RBCs as normal activity. 

Extracted data were verified and corrections made where necessary. For clarity of presentation, 

we standardized and presented TPMT activity data according to two standard units, U/mL RBCs 

or U/gram of hemoglobin (g Hb). Note that 1 Unit is equivalent to 1 nanomole of product per 

hour (nmol/h), and 1 nmol/h/g Hb is equivalent to 1 pmol/h/mg Hb. Unless otherwise stated, the 

product was 6-MMP.

When there were multiple reports of the same study we referenced the most relevant record 

as the primary identifying study, and also extracted additional data as available from companion 

report(s). 
25

Table 4. Two by two tables for analysis of diagnostic accuracy

2×2

Table A

Phenotyping (enzymatic activity assay)

Reference standard

Normal or high 

activity

Intermediate or low activity

Genotyping

Index test

Heterozygous or homozygous 

carriers 

Noncarriers

2×2

Table B

Phenotyping (enzymatic activity assay)

Reference standard

Intermediate, 

normal, or high 

activity

Low activity

Genotyping

Index test

Homozygous carriers

Noncarriers or heterozygous 

carriers only

Assessment of Risk of Bias

For key questions 1c (diagnostic accuracy); 2, 3a, and 3b (effectiveness of testing versus no 

testing;) and 3c (association studies), we assessed risk of bias of studies using standard 

questionnaires. 

For diagnostic studies we used a modified QUADAS tool35 in which we replaced the item 

number 4 about the time period between the two tests with an item questioning whether HardyWeinberg equilibrium (HWE) was tested or not. 

For randomized controlled trials and cohort studies we assessed the following:

• similarity of groups at baseline in terms of baseline characteristics and prognostic 

factors

• extent to which outcomes were described

• blinding of subjects and providers

• blinded assessment of the outcome

• intention-to-treat analysis

• differential loss to follow-up between the compared groups or overall high loss to 

follow-up, 

• Potential for financial conflict of interest

For trials, two additional elements were considered:

• methods used for randomization and

• allocation concealment

• Treatment adherence

For cohort studies, yet another set of elements was considered:

• sample size

• methods for selecting participants (inception cohort, methods to avoid selection bias);

• methods for measuring exposure variables
26

• methods to control confounding (e.g. design features of matching or restriction to 

particular subgroups; and analytic methods of stratification or regression modeling 

with propensity scores or covariates) 

• appropriateness of methods of measuring TPMT status

For key question 3c, risk of bias items included assessment of comparability of groups, 

blinded assessment of outcome and categorization of TPMT genotyping or phenotyping testing 

results, sample selection and survival bias, adequacy of description of TPMT testing 

methodology and its reliability, reporting of ambiguous and uninterpretable results of TPMT 

testing, compliance with thiopurine treatment, sources of TPMT genotyping or phenotyping for 

all patients, loss to followup, clear description of outcomes definitions, adequacy of sample size, 

methods to control for confounding, potential for financial conflict of interest, and finally 

assessment of HWE and gene-gene interaction where applicable. 

Each study was given an overall risk of bias assessment of Good (low risk of bias), Fair or 

Poor (high risk of bias) (Table ). Because of a lack of standards for assessment of risk of bias or 

quality of preanalytic, analytic, postanalytic, and costing data studies, risk of bias was not 

assessed for key questions 1a, 1b, 1d and question 4. To rate the strength of the body of 

evidence, we planned to qualify the corpus of evidence into low, medium and high risk for an 

outcome of interest. Since only one study contributed to the outcomes preplanned for rating the 

strength of evidence, individual risk of bias of this study determined the overall risk of bias of 

the evidence for those outcomes in form of a single study. In other words, fair risk of bias of the 

individual study for the outcomes determined the medium risk of bias of the available evidence. 

Details of items contributing to risk of bias assessment for each study are reported in Appendix C 

(Evidence tables).

Table 5. Categorization of overall risk of bias of individual studies

Good (low risk of bias). These studies have the least bias and results are considered valid. A 

study that adheres mostly to the commonly held concepts of high quality including the 

following: a formal randomized controlled study; clear description of the population, setting, 

interventions, and comparison groups; appropriate measurement of outcomes; appropriate 

statistical and analytic methods and reporting; no reporting errors; low dropout rate; and clear 

reporting of dropouts

Fair. These studies are susceptible to some bias, but it is not sufficient to invalidate the results. 

They do not meet all the criteria required for a rating of good quality because they have some 

deficiencies, but no flaw is likely to cause major bias. The study may be missing information, 

making it difficult to assess limitations and potential problems

Poor (high risk of bias). These studies have significant flaws that imply biases of various types 

that may invalidate the results. They have serious errors in design, analysis, or reporting; large 

amounts of missing information; or discrepancies in reporting.

Evidence Synthesis and Analysis 

For key questions 1a, 1b, and 4, evidence was synthesized qualitatively. 

Data synthesis was not possible for key questions 1d due to lack of evidence, and for 

question 2, 3a and 3b sparse evidence precluded evidence synthesis. 

For key question 4, costing data were converted to United States Dollar 2009 values using 

purchasing power parities

36 and were inflated to reflect 2009 values using the consumer price 

index for US medical care for all urban consumers (series identification # CUUR0000SAM).37
27

For the main quantitative syntheses, we did not consider underlying autoimmune disease, 

method of genotyping or phenotyping (i.e. enzymatic activity testing), population demographics, 

and the different observational study designs, as contributors to any important clinical or 

methodological diversity. In this light, the underlying assumption was that differences in 

outcomes of thiopurine toxicity are explained by differences in TPMT enzymatic activities 

across similar doses of the drugs. We used DerSimonian and Laird’s random-effects model for 

pooling individual study estimates, weighting them by individual study variance and the 

estimated between-study heterogeneity. We pooled whenever there were two studies in a given 

analysis for an outcome. Pooled estimates of diagnostic sensitivity and specificity percentages, 

and odds ratios and their 95 percent confidence intervals were calculated using CMA software 

(version 2.2.046).

Despite the underlying assumption, we planned to explore clinical and methodological 

diversity in a random effects univariate meta-regression based upon preidentified covariates of 

study design, overall risk of study bias, TPMT testing methodology, specific age subgroups, and 

underlying autoimmune disease(s) subgroups. Data on variables such as racial groups were too 

sparse to be considered as covariates. HWE was not considered as a covariate because it has 

been shown that including studies that appear to violate the HWE in the meta-analysis does not 

bias the summary estimates.38Enzymatic activity cutoffs used in the studies differed trivially 

between them. This variability is not unexpected because laboratories employ in-house reference 

intervals based on slight population and assay differences. As such, the TPMT enzymatic activity 

cutoff was also not considered to be an important effect modifier. For key questions other than 

the diagnostic question 1c, an additional covariate considered was blinded assessment of 

outcomes and TPMT status (by genotyping or phenotyping). Meta-regression was done in SAS 

using the NLMIXED procedure whenever six or more studies were involved in the pooled result 

of a binary covariate and 12 or more if the covariate had three distinct levels. When possible, we 

tested for statistical heterogeneity using Cochran’s Q and reported it when found to be 

substantial (p value for chi-squared test of heterogeneity less than 0.10, and I2 greater than or 

equal to 50 percent).

Pooling of Evidence of Diagnostic Accuracy (Key Question 1c)

We considered the TPMT enzymatic activity assay as the reference for the index test of 

genotyping of the different SNPs. Since the index test reported presence or absence of variant 

allele as a dichotomous outcome, implicit or explicit cut-off threshold effects were ruled out. 

We, therefore, pooled for the outcomes of test sensitivity and specificity instead of summary 

receiver-operating characteristics. 

The role of testing for statistical heterogeneity in effect estimates from studies of diagnostic 

accuracy is less well understood. Since the estimates of sensitivity and specificity are interrelated, the significance of heterogeneity in one and not the other is unclear. We, therefore, did 

not formally test for the extent of statistical heterogeneity; however, we did carry out univariate 

meta-regression based upon preidentified covariates stated above when possible.

Since studies identified homozygous and heterozygous carrier states by testing for different 

TPMT variant alleles, we pooled only the studies testing for the same set of SNPs.

When possible, two meta-analyses were considered for each set of variant alleles investigated 

by at least two studies. In meta-analysis 1, genotypes were dichotomized into noncarriers and 

carriers (or homozygotes and/or heterozygotes) and phenotypes were dichotomized into normal 
28

(or high) and subnormal (or intermediate and/or low and/or absent) enzymatic activities. In metaanalysis 2, we dichotomized genotypes into noncarriers and/or heterozygotes and homozygotes, 

and phenotype into normal and/or high and/or intermediate and low/absent activities (Table ).

For studies with 100 percent sensitivity or specificity, 0.5 was added to either both TP (true 

positive) and FN (false negative) or TN (true negative) and FP (false positive), in order to 

estimate pooled proportions.

Table 6. Meta-analyses 1 and 2, for Key Question 1c

Meta-analysis 1

Normal/high activity Intermediate / 

low / absent 

activity

Sensitivity 1 Specificity 1

Homozygotes 

and/or 

heterozygotes

A1 B1 B1/(B1+D1) C1/(C1+A1)

Noncarrier (or Wild 

types)

C1 D1

Meta-analysis 2

Normal/high/intermediate 

activity

Low/absent 

activity

Sensitivity 2 Specificity 2

Homozygotes A2 B2 B2/(B2+D2) C2/(C2+A2)

Noncarrier (or Wild 

types) and/or 

heterozygotes

C2 D2
29

Pooling of Evidence of Association of Thiopurine Toxicity With TPMT 

Genotypic or Phenotypic Status (Key Question 3c)

Outcomes data were number of patients with and without events. With respect to genotype, 

we considered a codominant model in which TPMT enzymatic activities are highest when both 

TPMT alleles are Wild type (i.e. noncarrier state without TPMT polymorphisms), followed by 

somewhat decreased enzymatic activity when the one of the two alleles is mutant or variant (i.e. 

heterozygous carrier of mutation, or heterozygote) and lowest to absent TPMT enzymatic 

activities when both alleles are variants (i.e. homozygous carrier of mutation, or homozygote). 

The possible comparisons were heterozygotes versus noncarriers, homozygotes versus 

noncarriers, and homozygotes versus heterozygotes. For the association with TPMT enzymatic 

activity, the comparisons were intermediate enzymatic activity versus normal/high, low to absent 

activity versus normal/high and low/absent activity versus intermediate activity. 

For estimating association with genotype, the primary analysis included all studies as long as 

they tested for the most common TPMT alleles, irrespective of testing additional polymorphisms. 

Alleles we considered most common were TPMT*2, *3A, *3B, *3C (Table ). Secondary 

analyses were allele specific and restricted to only those subgroups of studies that tested specific 

alleles. Additionally, we assessed deviations of the genotype frequencies from those predicted by 

the Hardy-Weinberg law (chi-square test). A p-value less than 0.05 indicated possible 

genotyping error (other factors, such as inbreeding could also be at play). The Hardy-Weinberg 

law states that if there are 2 alleles at a particular locus, named A and a, with frequency p and q, 

respectively, then after 1 generation of random mating the genotype frequencies of the AA, Aa, 

and aa groups in the population will be p2

, 2pq, and q2, respectively. Given that there are only 2 

alleles possible, A or a, then p + q = 1, and p2 + 2pq + q2 = 1. 

Rating the Strength of Evidence and Assessing Applicability 

For the prespecified outcomes of mortality (critical), serious adverse events (critical), 

myelotoxicity (important), and health-related quality of life (important) in Key Questions 3a, and 

3b, two methodologists rated the strength of the body of evidence across the domains of risk of 

bias, consistency, directness and precision as per published guidance.

39 Disagreements were 

resolved by consensus. Applicability was assessed according to the domains of patient 

population, intervention and dose, comparator, outcome and study duration. We examined 

eligibility criteria, participant demographics, and distribution of genotype to assess 

generalisability of population. To assess applicability of testing we examined the type of TPMT 

tests employed and their validity and reproducibility. We also ascertained dose and type of 

thiopurine. We also examined whether non-pretested group of patients received routine blood 

count monitoring or not, and the study duration. 

Survey of Laboratories Conducting TPMT Analyses

To supplement the limited published literature to answer key questions 1a, 1b, 1c, and 1d, 

further data regarding the preanalytical and postanalytical requirements and performance 

characteristics of TPMT laboratory analyses was collected. With advice from the Technical 

Expert Panel, the review team decided to survey laboratories that provide TPMT analytical 

services.
30

Our target population included all laboratories in which medium of language was English 

and provide TPMT analytical services. Potential participating laboratories were identified by the 

Technical Expert Panel, which also queried two listservs: Clinical Chemistry General Topics 

(American Association for Clinical Chemistry) and CSCC Listserv (Canadian Society of Clinical 

Chemists). Based on these expert recommendations, two organizations and seven laboratories 

were contacted to determine their willingness either to complete a questionnaire or to 

disseminate the questionnaire to other relevant laboratories. Contact was initiated using an 

emailed letter explaining the intent behind the request, as well as background information on the 

questionnaire. The letter and questionnaire are included in Appendix B.

The review team developed a draft questionnaire, which was assessed by the Technical 

Expert Panel for face validity and comprehensiveness and the logical flow of questions and 

responses were ensured for both paper and electronic versions. The questionnaire was also 

revised after some initial responses revealed ambiguity. The final questionnaire included 11 

questions with multiple subquestions relating to TPMT analytical methods (e.g., sample type and 

handling), preanalytical requirements (e.g., specimen stability), quality control procedures, 

reporting of results and cost of TPMT services. The questionnaire took approximately 15-20 

minutes to complete. The full questionnaire is included in Appendix B.

The survey was administered electronically via Survey MonkeyTM to representatives of the 

six laboratory representatives who had agreed to participate, with up to three weeks provided for 

completion. Reminder emails were sent one week after initial contact as well as three days before 

responses were due. Responses were captured in a Survey MonkeyTM database and were 

subsequently downloaded to Microsoft Excel to conduct the analysis. We conducted a 

descriptive analysis, and summarized results using frequencies and ranges as appropriate.

Ethics approval was obtained from the Ottawa Hospital Research Ethics Board before initial 

contact with any potential participating laboratory.
31

Chapter 3. Results

Screening and Inclusion of Records for Key Questions

The PRISMA diagram in Figure 3 depicts the flow of retrieved records through the phases of 

screening and inclusion. Overall, 1783 records were identified by systematic searches of 

databases and seven1,40-45 were nominated by the Technical Expert Panel and reviewers. A third 

level of content expert screening was employed to ensure that laboratory studies identified for 

key questions 1a, 1b and 1d did not include irrelevant records, as a result of relatively liberal 

screening at the two prior levels.

Studies that were reported in more than one included record are listed in Table . We 

identified one of these records as the primary report and others as companion papers, however, 

where applicable, data from both were used in evidence syntheses. 

For 33 of 169 included records, extractable data were not available (Table 8).

We contacted authors of 24 records requesting additional data or seeking data 

clarification,1,46-68 and additional data received were incorporated in evidence syntheses. 

One hundred and thirty six records associated with 115 unique studies addressing the key 

questions were included in this systematic review. Additional individual study data and quality 

assessment are presented in Appendix C. Forty-five studies contributed to meta-analyses and the 

remaining were included in qualitative syntheses of evidence. No record was identified that 

answered Key Questions 1d (Table ).
32

Figure 3. PRISMA diagram of record identification, screening and inclusion

* Two records had two primary reasons for exclusion at level III

Nominated 

Records

(n = 7)

Screening Included Eligibility Identification

Records identified though 

data base searching

(n = 1783)

Records screened by title and abstract

(Level I)

(n= 1783)

Full text articles 

assessed for eligibility 

(level II)

(n = 538)

Records excluded at 

Level I

(n = 1252)

Records included at 

Level II

(n =204)

Full text records excluded at 

level II. Reasons:

• Editorial, review, 

commentary, letter, news, 

report or case report (n = 44)

• Study population was not 

relevant (n = 159)

• Language of publication was 

not English (n = 2)

• Neither abstract nor full text 

could be retrieved (n = 11)

• Other reasons (n = 118) Further full text eligibility 

assessment for key 

questions 1a and 1b by 

content experts (level III)

 

Records included at 

level III (n = 169)

Full text records excluded at 

level III. Reasons:

• No direct preanalytic data of 

relevance (n = 32)

• Non-original data (n = 1)

• No proficiency, 

reproducibility and precision 

data (n = 4*)

Records included in the report

(n= 136 records of 115 unique 

studies)

Studies included in 

quantitative 

syntheses

(n = 45)

Eligible records not 

reporting relevant data 

for any key question

(n = 33)
33

Table 7. Primary records with companion reports passing level III screen 

Primary record Companion record(s)

Palmieri, 200747 Palmieri, 200669

Winter, 200770 Winter, 200771

Gisbert, 200672 Gisbert, 200773

Lindqvist, 200649 Hindorf, 200674, Hindorf, 200575

Heneghan, 200676 Heneghan, 200377

Hindorf, 200650 Hindorf, 200678

Czaja, 200679 Czaja, 200480

Gearry, 200481 Gearry, 200382

Winter, 200483 Winter, 200484

Derijks, 200454 Derijks, 200385

Marinaki, 200486 Ansari, 200387

Campbell, 200258 Campbell, 200288

Black, 199889 Black, 199890

Stolk, 199891 Stolk, 199692

Snow, 199593 Snow, 199494

Kerstens, 199595 Kerstens, 199296

Walsh, 200897 Walsh, 200898

Schmeling, 200799 Schmeling, 2007100

Ansari, 200462 Ansari, 2004101

Colletti, 200945 Colletti, 200660

Xin, 2005102 Xin, 2005103

One companion record was considered primary paper for key question 1b74
34

Table 8. Distribution of records by key questions

Key question Total number 

of studies

Full text 

records Abstracts

Eligible 

studies not 

reporting 

relevant data

Studies for 

which (some 

or all) data 

were received 

from authors

1a 35102,104-137 33102,104-135 2136,137 0 0

1b

3674,106-108,111-

116,118,121,124,128,1

31,135,136,138-156

3374,106-108,111-

116,118,121,124,128,1

31,135,138-154

3136,155,156 3139,141,149 0

1c

241,46,49-

53,56,59,70,93,99,157-

168

201,46,49-

53,56,59,70,93,157-

163,167,168

499,164-166 599,164-166,168 551-53,56,59

1d 0 NA NA NA NA

2 11 11 0 0 0

3a 21,45 21,45 0 145 11

3b 31,45,169 31,45,169 0 145 0

3c TPMT 

allelic 

determination

491,46-54,57-

59,61,63,64,67,70,76,8

1,86,89,93,99,157,162,

165,170-191

341,46-54,57-

59,70,76,81,86,89,93,1

57,162,170-

180,190,191

1561,63,64,67,99,165

,181-189

1549,58,63,64,67,76,

162,165,178,183-

185,187-189

546-48,52,54,57

3c TPMT 

enzymatic 

activity

3646,49-

53,55,58,59,62,65,68,7

0,72,79,91,93,95,97,11

9,157,161,162,165,192

-203

2646,49-

53,55,58,59,70,72,79,9

1,93,95,119,157,161,1

62,192-197,203

1062,65,68,97,165,19

8-202

2046,49,55,58,59,62,

65,68,95,97,119,162,1

65,194,198-203

450-53

4 1140,83,204-212 1140,83,204-212 0 0 0

Overall across key questions

Companion 

records

(N=21)

60,69,71,73,75,77,78,80,82,84,85,87,88,90,92,94,96,98,100,101,103

Studies 

contributing 

to the 

evidence base 

(N=115)

1, 46-54, 56, 57, 59, 61, 70, 72, 74, 79, 81, 83, 86, 89, 91, 93, 99, 102, 104-138, 140, 142-148, 150-163, 167, 169-177, 179-182, 186, 

190-193, 195-197, 204-212

One companion record was considered unique paper for key question 1b74

Included 

records that 

did not report 

relevant data

N=33

45,55,58,62-65,67,68,76,95,97,139,141,149,164-166,168,178,183-185,187-189,194,198-203

Abbreviations: NA = not applicable; TPMT = thiopurine methyltransferase
35

Key Question 1a: In terms of the analytical performance 

characteristics of enzymatic measurement of TPMT activity and 

determination of TPMT allelic polymorphisms, what are the 

preanalytical requirements for enzymatic measurement of TPMT and 

determination of TPMT allelic polymorphisms? (e.g. specimen types 

and collection procedures, lab transportation, interference of 

coadministered drugs, patient preparation and identification etc.)

A total of 35 studies reported data relevant to answer this key question, all in relation to 

enzymatic analysis of TPMT activity (Table ). No evidence was available regarding 

preanalytical performance characteristics for determination of TPMT allelic polymorphisms. 

Genotyping is most commonly performed by using the restriction fragment length polymorphism 

(RFLP) technique, polymerase chain reaction (PCR), or direct sequencing. 

Stability. Thirteen studies assessing the stability of TPMT enzyme activity are summarized 

in Table 9.08,115,116,118,121,124,127-129,131,134,136,137 Two studies were conducted in the USA134,136, and 

eleven in Europe.108,115,116,118,121,124,127-129,131,137 TPMT stability was assessed at room 

temperature, 4°C; -20°C, -21°C, -23°C, -25°C, -30°C, -70°C, -80°C and -85°C. Time periods 

from a few hours to 16 months were studied. 

TPMT was found to be stable at room temperature for a maximum of seven days in control 

blood samples, while in a case of acute lymphocytic leukemia, patient blood TPMT was stable 

for three days.121 At -20°C, TPMT was stable for up to three months. Four studies of storage at 

-80°C showed that TPMT was stable from a minimum of a few days to 25 days,116,129,131 but 

TPMT activity decreased by 15 percent after 16 months of storage.116 Repeated freeze-thaw 

cycles of the red blood cell (RBC) lysate were reported to result in a 16.2 percent decrease in 

activity.115 However, the decrease was not statistically significant after three cycles (initial 

values: 9.2, 11.7, and 16.5 U /mL RBCs; final values: 8.1, 9.8, and 14.3 U/mL RBCs). TPMT 

activity was stable in six blood samples shipped via regular mail, and received two to seven days 

post sampling (coefficient of variance, 5.6 percent).121
36

Table 9. Stability of TPMT enzymatic activity 

Storage 

temperature Sample Storage time, anticoagulantstudy reference Stability

Room temperature Whole blood

24 h, heparin134

36 h, EDTA108

3 days, heparin121*

4 days, heparin127

5 days, heparin118

6 days, EDTA124

7 days, heparin121*

Stable

Whole blood 72 h, EDTA137 25% +/- 6% decrease 

in 24 hours

4°C Whole blood

24 h, heparin134

4 days, heparin127

6 days, EDTA124*

8 days, unspecified

anticoagulant136

Stable

-20°C to -30°C RBC lysate

7 days116

21 days115

3 month108

Several month124

Stable

RBC lysate 3 month128 7% decrease

-70°C to -80°C RBC lysate Few days129,131

25 days116 Stable

RBC lysate 16 month128 15% decrease

Notes: * Control blood was stable for 7 days. Leukemia patient blood was stable for 3 days, and the median activity showed a 

small but statistically significant decrease after 6 days.

Abbreviations: EDTA = ethylene diamine tetra-acetic acid; RBC = red blood cell; TPMT = thiopurine methyltransferase.

Gender. Eighteen studies evaluated gender-related differences in TPMT activity (Table 

10).106,107,110-113,116,117,122,124,125,127-129,131,133-135 Seventeen studies reported the TPMT activity in 

RBCs and one in renal tissue.133 In 16 studies, no significant differences associated with gender 

were found in TPMT activity.106,107,110-113,116,117,122,124,125,127,128,131,134,135 One study reported 

TPMT values to be higher for males in Caucasian and mixed-race groups (30 U/g Hb in females

versus 38 U/g Hb in males, and 33 U/g Hb in females versus 39 U/g Hb in males, respectively, 

for Caucasian and mixed race groups).129 TPMT values in renal tissue were 10 percent higher in 

males than in females.133 Fifteen studies reported TPMT values, whereas three studies stated 

only the outcomes of comparisons.107,122,127
37

Table 10. Gender related differences in TPMT enzymatic activity 

Study Male Female Significance

Hindorf

2004111

12.9 (range, 0.2–24.6) U/mL 

RBCs

(n=607)

12.7 (range, 0.4–25.4) U/mL 

RBCs

(n=544)

p = 0.08

Weinshilboum

1978134

10.0 ± 2.3 U/mL RBCs 

(n = 37)

10.4 ± 2.5 U/mL RBCs 

(n = 36)

Tinel

1991131

14.6 ± 6.7 U/mL RBCs 

(n=119)

16.8 ± 7.7 U/mL RBCs 

(n=184)

Jacqz-Aigrain

1994128

19.6 ± 4.9 U/mL RBC 

(n=134) 

18.9± 4.9 U/mL RBC 

(n=166) 

Kroplin

1998124

38.8 nmol 6-MTG/h/g Hb

(n=117)

36.9 nmol 6-MTG/h/g Hb 

(n=82)

KeizerGarritsen

2003116

15,8± 6,4 pmol/h/107 RBCs

(n=59)

15,1± 4,8 pmol/h/107 RBCs

(n=44)

GaniereMonteil

2004113

Adults: 19.55± 4.25 U/mL 

RBCs

(n= 229) 

Children: 18.34±4.21 U/mL 

RBCs 

(n=97)

Adults: 18.61±3.59 U/mL 

RBCs. 

(n=75) 

Children: 18.87±3.98 U/mL 

RBCs 

(n=50)

Adults: p=0.057. 

Children: p=0.310

Ford

2004112

Mean and median TPMT

activities: 34 and 34 nmol 6-

MTG/h/g Hb (n=469)

Mean and median TPMT 

activities: 33 and 32 nmol 6-

MTG/h/g Hb

(n=531) 

No significant 

difference

Zhang

2007106 Wild type Healthy: (n=155) 17.05±3.12 U/ml RBCs 

Heterozygous Healthy: 

(n=4)7.50±1.58 U/ml RBCs 

Wild type Healthy: (n=86)

15.90±2.87 U/ml RBCs. 

Heterozygous Healthy: (n=3)

8.17±1.30 U/ml RBCs 

p=0.01 

Zhang

2006135

12,36 U/mL RBCs 

(n=5)

13,16 U/mL RBCs 

(n=14)

Chocair

1993129 Caucasians: (n = 21) 38 U/g Hb

Black: (n = 24)

38 U/g Hb

Mixed-race: (n = 19) 

39 U/g Hb

Japanese: (n = 11) 

37 U/g Hb

Caucasians: (n = 12) 

30 U/g Hb

Black: (n = 15) 

36.5 U/g Hb

Mixed-race: (n = 11) 

33 U/g Hb

Japanese: (n = 21)

38 U/g Hb

Micheli

1997125

Adults (20-59 y): (n=7)

21 ± 5 U/g Hb 

Children (1-14y): (n = 6)

23 ± 5.8 U/g Hb 

Adults (20-59 y): (n=7)

15± 8 U/g Hb 

Children (1-14y): (n = 2)

18.21 U/g Hb 

Menor

2002117

19.7 - 6.2 U/mL RBCs 

(n=1671)

19.5 - 6.1 U/mL RBCs 

(n=1873)

Brouwer

2005110

ALL patients: Median TPMT: 

12.4 (range 1.7–30.7) pmol/hr 

/107 RBCs (n=116)

ALL patients: Median TPMT: 

12.8 (range 5.8–30.4) pmol/hr 

/107 RBCs (n=57)

No significant 

difference for 

ALL patients (p = 

0.841) 

Oselin

2006107

n=52 n=47 No significant 

difference

TPMT activity: 
38

Study Male Female Significance

21.5 to 129.6 

ng/h/mL RBCs 

Lennard

1994127

In children, boys versus girls TPMT activity for the entire group (n=100) not 

significantly different (p > 0.25, Mann-Whitney).

High TPMT activity group (n=100) boys versus girls not significantly different 

(p=0.8)

Alves

2001122

(n=76) (n=67) No statistically 

significant 

association (p = 

0.796, ANOVA).

Lee

1982133

Renal cell carcinoma 

patients

232 ± 9 U/g tissue or 

4.24 ± 0.15 U/mg protein 

(n=37 )

Renal cell carcinoma 

patients

210 ± 21 U/g of tissue or 

3.93 ± 0.4 U/mg of protein 

(n=14)

No significant 

differences.

Imbalance in 

sample numbers 

likely due to the 

2:1 male:female 

incidence of 

renal cell 

carcinoma. 

Abbreviations: 6-MTG = 6-methylthioguanine ALL = acute lymphoblastic leukemia; ANOVA = analysis of covariance; g 

= gram; mL RBCs = milliliter of red blood cells; p = probability; pmol = picomole; TPMT = thiopurine methyltransferase; U = 

Unit = nanomole of product per hour

Age. Ten studies investigated variation of TPMT activity with age (Table

11)

104,110,111,113,116,123,125,131,133,135 In eight studies TPMT activity was analyzed in RBCs, while one study 

examined each of renal tissue133 and lymphocytes.123 Only a single study reported a statistically 

significant difference in TPMT activities between adults and children (12.0 U/mL RBCs in 

children and teenagers, versus 12.9 U/mL RBCs in adults; p less than 0.001).111

Table 11. Effect of age on TPMT enzymatic activity

Study Children (<20 years) Adults (>20 years) Significance

Hindorf

2004111

12.0 (range 0.6–25.4) U/mL 

RBCs

(n = 192, <15 y)

12.9 (range 0.2 – 24.6) U/mL 

RBCs (n = 959, 

median age 33.4 y)

p < 0.001

Tinel

1991131

14.5 ± 6.7 U/mL RBCs 

(n = 175, 20-40 y; 

16.7 ± 7.6 U/mL RBCs (n = 

128) ages from 40 to 60 

years.

No significant difference

GaniereMonteil

2004113

18.49 ± 4.13 U/mL RBCs. 

range: 8.25–30.0 U/mL 

RBCs

(n = 165) 

19.34 ± 4.09 U/mL RBCs 

range: 0.43-30.38 U/mL 

RBCs 

(n = 304)

p = 0.310 ; no significant 

difference

Zhang

2006135

14.38 U/mL RBCs 

(22 - 38 y, n = 8)

11.90 U/mL RBCs for age 

40-59, n = 11. 

12.95 (SD, 3.07) U/mL RBCs 

entire sample

No significant difference
39

Micheli

1997125

22 ± 5 U/mL RBCs 

(1-14 y, n = 8)

18 ± 7 U/mL RBC 

(20-59 y, n=14)

No significant difference

Brouwer

2005110

2-31 U/mL RBCs

Patients with ALL, at 

diagnosis (5.9 ± 4.1 y, n = 

173) 

TPMT activity not correlated 

with age.

KeizerGarritsen

2003116

TPMT data

various ages.

Children, young adults 

(n=103)

No significant correlation 

between TPMT activity and 

age among children and 

young adults.

Gisbert

2007104

40 ± 16 y

(n = 14,545)

No significant correlation 

between TPMT activity and 

age

Lee

1982133 renal TPMT [average 225 ± 9U/g of tissue

(n = 51) 

No significant correlation of 

patient age with TPMT 

activity with patient age. 

Correlation coefficients = 

0.04 for all samples.

Coulthar

d

1998123

0.24 nU/mg lymphocytes

Range: 0.1 and 0.76 nU/mg 

protein 

11 months to 15.5 y

(n = 35)

0.16 nU/mg lymphocytes

Range: 0.04 - 0.86 nU/mg 

protein 

16 - 77 y

(n = 37)

No significant correlation 

between TPMT activity and 

age 

Abbreviations: ALL = acute lymphoblastic leukemia; g = gram; mg = milligram; mL RBCs = milliliter of red blood cells; nU = 

nanoUnit; p = probability; pmol = picomole; TPMT = thiopurine methyltransferase; U = Unit = nanomole per hour

 

Drugs. Ten studies evaluated the influence of drugs on TPMT activity in blood (Table

12).102,104,105,110,114,117,119,120,128,131 Four studies were conducted in vitro.102,110,114,128 The following 

drugs were studied: 5-aminosalycilate102,104, sulfasalazine102,114,122, mesalazine105,122, 

azathioprine105,117, mesalamine58, ac-5-aminosalicylate102, syringic acid128, prednisone128, 

prednisolone128, 6-methylprednisolone128, cyclophosphamide128, methotrexate110,128, 

trimethoprim-sulphamethoxazole128, SKF 525-A131, 3,4-dimethoxy-5-hydroxybenzoic acid131, 

trimethoprim110, vincristine110, dexamethasone110, and L-asparaginase.110 Two studies reported 

significant inhibition. 3,4-dimethoxy-5-hydroxybenzoic acid decreased TPMT activity by 97 

percent in vitro.131 One in vitro study showed concentration-dependent inhibition of TPMT from 

11 to 55 percent, in the presence of 80 to 640 mol/L sulfasalazine.114 One study reported 147 to 

148 percent stimulation of TPMT activity in vitro by methotrexate and trimethoprim.110

Interestingly, another study of methotrexate in vitro found no effect.116 The rest of the studies 

reported no significant inhibition by any of the drugs studied.

Table 12. Effects of drugs on TPMT enzymatic activity

Study Drug ID Effect on TPMT activity 

Tinel

1991131

SKF 525-A None

3,4-dimethoxy-5-

hydroxybenzoic acid

Decreased by 97%
40

Study Drug ID Effect on TPMT activity 

Dewit

2002120

Acetylated metabolite 

of 5-aminosalicylic 

acid, with either 

sulfasalazine or 

mesalazine.

No significant change after aminosalicylate withdrawal. 

TPMT activity before withdrawal of aminosalicylate: 

whole group: 12.29 U/mL RBCs (range 8.25±16.85); 

sulfasalazine subgroup: 12.14 U/mL RBCs; 

mesalazine subgroup 12.43 U/mL RBCs. 

TPMT activity after aminosalicylate withdrawal: 

whole group: 11.41 U/mL RBCs (7.3±14.5) (p=0.245, not 

significant); 

sulfasalazine subgroup: 11.43 U/mL RBCs; 

mesalazine subgroup: 11.39 U/mL RBCs.

Dilger

2007105

Azathioprine versus 

mesalazine

No significant differences between patients on 

azathioprine compared with those on mesalazine, at 

baseline or at any further visit.

Gisbert

2007104

5-aminosalycilates No differences between patients on azathioprine or 5-

aminosalicylates versus controls. 

Azathioprine: no treatment 20.7 U/mL RBCs versus 

treatment 21.2 U/mL RBCs 

5-aminosalycilates: no treatment 20.9 U/mL RBCs 

versus treatment 21.2 U/mL RBCs

Dubinsky

2002119

Mesalamine No differences between patients on mesalamine 

medications versus controls (median 32.4 (range 14.7-

49.2) ELISA units vs. 31.8 (15.3-49.1 ELISA units) 

Menor

2002117

azathioprine No significant difference between patients on 

azathioprine versus no treatment

19.9 ± 6.0 U/mL RBCs versus 19.7±5.8 U/mL RBCs

Xin

2005102

sulfasalazine, 

5-aminosalicylate, 

Ac-5-aminosalicylate 

No significant effects in vitro. 

IC50s: 

Sulfasalazine: 9.4 (±3.1) μM 

5-aminosalicylate: 236 (±55) μM

Ac-5-aminosalicylate: 73 (±20) μM

JacqzAigrain

1994128

syringic acid, 

prednisone, 

prednisolone, 6-

methylprednisolone, 

cyclophosphamide, 

methotrexate, 

trimethoprim 

sulphamethoxazole

No significant effect in vitro. 

Residual TPMT activity was always more than 70% of 

control activity.

Shipkova

2004114

sulfasalazine Significant, concentration-dependent inhibition 11% for 

80 μM to 45% for 640 μM sulfasalazine. 

Brouwer,

2005110

methotrexate, 

trimethoprim, 

vincristine, 

dexamethasone, 

L-asparaginase

TPMT activity significantly increased in vitro.

2.4 μM trimethoprim: 148% 

0.01 μM methotrexate: 147%. 

Vincristine, dexamethasone and L-asparaginase had 

nonsignificant inhibitory effects on TPMT activity.

Abbreviations: IC50 = concentration of inhibitor at which enzyme activity is 50 percent of uninhibited activity; mL RBCs = 

milliliter of red blood cells; p = probability; TPMT = thiopurine methyltransferase; U = Unit = nanomole per hour; μM = 

micromoles per liter.
41

Race. Two studies of TPMT activity differences among different races are summarized in 

Table 13.

109,129 No significant differences among studied races (Caucasians, blacks, Japanese, 

mixed races) were found.109,129

Table 13. Variation of TPMT activity with race 

Study Population

TPMT activity

(range)

(U/mL RBCs)

Heckmann

2005109

Black (n=50) 8.75 8.75 (6.5–10.1)

Mixed race (n=50) 10.15 (8.7–11.3)

Caucasian (n=15) 13.40 (10.9–15.1)

Chocair

1993129

Black (n=39) 37 (13-84)

Mixed race (n=30) 36.5 (14-65)

Japanese (n=32) 37.5 (29-48) ** 

Caucasian (n=33) 32 (15-54) **

Note: ** (p<0.04)

Abbreviations: n = number in group; RBCs = red blood cells; TPMT = thiopurine methyltransferase; U = Unit = nanomole per 

hour.

Hematocrit. The effect of hematocrit on TPMT activity was measured in three 

studies.117,126,104 One study proposed a decrease of TPMT activity by seven percent (range 1.2 

percent to 12.0 percent) when comparing the high and low hematocrit levels among 12 

participants.126 They studied TPMT activity between erythrocyte fractions of 0.1 and 0.5 and 

reported the slopes to be significantly different from zero (p ranging from 0.02 to 0.0001). A 

single study reported young RBCs to have TPMT activity 8.8 units higher than old RBCs 

(Wilcoxon median difference 8.8 units (95 percent CI 7.2 to 10.8, p=0.006).121 Among 10

participants, TPMT activities in the 60 percent, 63 percent, 66 percent and 69 percent gradients 

were analysed and the TPMT activities in each gradients differed (p less than 0.001). Another 

study described no hematocrit dependant difference in TPMT activity.104 

Morbidities. Two studies104,106 evaluated the effect of morbidities on TPMT activity (Table 

14). Inflammatory bowel disease (ulcerative colitis, Crohn disease, and indeterminate colitis), 

autoimmune hepatitis, multiple sclerosis, myasthenia gravis, pemphigus and chronic renal failure 

were studied. Statistically significant differences (p less than 0.001) in TPMT activity were 

observed among some disease groups, such as inflammatory bowel disease, autoimmune 

hepatitis, multiple sclerosis, myasthenia gravis, and pemphigus.104 Patients with chronic renal 

failure had almost double the TPMT activity compared with the healthy control before 

hemodialysis (33.88 ± 12.33 U/mL RBCs versus 16.03 ± 4.16 U/mL RBCs).106 Posthemodialysis 

TPMT activity levels were comparable to patients without renal failure.

Table 14. TPMT activity in various disease groups 

Study Population TPMT activity

(U/mL RBCs)

Gisbert

2007104

All study participants 20.1 ± 6 (Range 0-46.4)

Inflammatory bowel disease (n=7046) 20.4 ± 6 

Autoimmune hepatitis (n=359) 21.5 ± 6 

Multiple sclerosis (n=814) 18.4 ± 6 

Myasthenia gravis (n=344) 20.9 ± 6 

Pemphigus (n=133) 21.1 ± 6 
42

Zhang

2007106

Healthy control (n=241) 16.03 ± 4.16 

Chronic renal failure (n=30): before 

hemodialysis 

33.88 ± 12.33 

Chronic renal failure (n=30): after hemodialysis 17.89 ± 5.24 

Abbreviations: n = number in group; RBCs = red blood cells; TPMT = thiopurine methyltransferase; U = Unit = nanomole per 

hour.

Key points. TPMT is a stable enzyme that apparently can be stored for up to seven days at 

room temperature, and up to three months at -30°C. However, further research is required to 

confirm this.

Age and gender do not appear to affect TPMT activity. Furthermore, although the current 

data does not demonstrate racial differences between TPMT activities, further research is 

required to confirm this.

A number of drugs were studied for their effect on TPMT activity, including 5-

aminosalycilate, sulfasalazine, mesalazine, azathioprine, mesalamine, ac-5-aminosalicylate, 

syringic acid, prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, 

trimethoprim-sulphamethoxazole, SKF 525-A, 3,4-dimethoxy-5-hydroxybenzoic acid, 

trimethoprim, vincristine, dexamethasone, L-asparaginase. Studies showing potentially clinically 

significant effects were conducted in vitro, and therefore their in vivo influence on TPMT 

activity remains unknown. 

In patients with renal failure, TPMT activity is elevated prior to hemodialysis, and returns to 

normal levels following hemodialysis.

No studies were identified that addressed preanalytic variables for TPMT genotyping. 
43

Key Question 1b: In terms of the analytical performance 

characteristics of enzymatic measurement of TPMT activity and 

determination of TPMT allelic polymorphisms, what are the within and 

between laboratory precision and reproducibility of the available 

methods of enzymatic measurement of TPMT and determination of 

TPMT allelic polymorphisms (proficiency testing)?

Thirty-three studies reported information relevant to question 1b enzymatic 

measurement.74,106-108,111-116,118,121,124,128,131,135,136,138,140,142-148,150-156 Detailed information is 

available in Appendix C. Two studies were conducted in North-America,136,155 three studies in 

China,106,135,154 and the remaining 28 in Europe. 

TPMT enzymatic activity is usually determined by measuring the formation of 6-methyl 

mercaptopurine (6-MMP) from 6-mercaptopurine (6-MP), with S-adenosyl-L-methionine (SAM) 

as the methyl donor. This was originally described by Weinshilboum et al, who used 

radiolabelled SAM.134 More recently, 6-MMP has also been measured using high performance 

liquid chromatography (HPLC). TPMT is prevalent in many tissue types, but in clinical practice 

it is normally measured in red blood cells (RBCs).

Nine studies used radiolabelled SAM for TPMT assays.74,111,138,140,154,156 The reported interassay coefficient of variation (CV) ranged from 0.51 to 8.4 percent. The intra-assay CV ranged 

from 0.72 to 6.8 percent. One study reported regression values as y = 0.72x + 0.43 = 0.75, where 

x represents the value for first estimate of TPMT activity and y the value for the second 

estimate.131 This result indicates high reproducibility. One study reported a 95 percent 

confidence interval as 13.94 to 14.88 U/mL RBCs for a high TPMT enzyme sample and 8.07 to 

8.78 U/mL RBCs for an intermediate TPMT sample.148 One study144 used thin layer 

chromatography and quantitative scanning to measure TPMT activity, with 6-MP as a substrate, 

and radiolabelled SAM as cosubstrate. The day to day variance was 8.5±1.7 percent.

Seventeen studies used an HPLC assay to determine 6-MMP.106,108,113-

116,118,121,135,143,145,146,150-153,155 The inter-assay CV ranged from 0.2 to nine percent and the intraassay CV ranged from zero to 9.5 percent. One study reported the assay to be accurate, and 

highly reproducible when the samples were kept at -80°C for up to 25 days.116 Another study 

reported 4 percent precision and 96 to 103 percent accuracy.107 However, the authors did not 

clearly describe how the variability was measured. Five studies used an HPLC method to 

measure 6-methylthioguanine (6-MTG) formed using 6-thioguanine (6-TG) as a 

substrate,112,124,136,142,147 and reported CVs from two to five percent for intra-assay, and from four

to 10 percent for inter-assay performance.

Two studies measured the between laboratory precision and reproducibility.113,126 Two labs 

in France, Laboratoire de Pharmacologie Pediatrique et Pharmacogenetique (Hospital Robert 

Debre, France) and Laboratoire de Pharmacologie (Hotel-Dieu, France) studied HPLC precision 

with 6-MMP in erythrocyte lysate (25, 75, 125 ng/mL). The intra-day and inter-days CV values 

were 1.7 to 4.3 percent, and 0.8 to six percent respectively, for six samples. Inter-assay CV 

values were 5.8 percent for 20 samples, and 5.2 percent for five samples of quality control blood. 

Klemetsdal et al (Department of Pharmacology, Institute of Medical Biology, University of 

Tromso, Norway) compared RBC TPMT activity in five samples with those measured in Dr. 

Weinshilboum’s laboratory at the Mayo Clinic (Rochester, USA), finding CV values of 1.8

percent to 2.6 percent for 20 analyses.126
44

Overall, no obvious trend, depending on time or study characteristics, was observed between 

studies reporting lower versus those reporting higher accuracy and reproducibility. 

No study was found that specifically investigated the accuracy and precision of TPMT allelic 

polymorphism determination. However, three studies compared different genotyping methods to 

one other.105,139,141 One group developed a novel multiplex assay using matrix-assisted laser 

desorption/ionization, with a time-of-flight mass spectrometer (MALDI-TOF mass spectrometry) 

based on Sequenom iPLEX technology.139 All genotypes for 586 samples were 100 percent

concordant with the results from a previous denaturing HPLC genotyping assay. The new 

method was accurate, with 100 percent agreement for duplicate analyses. The other study used 

TaqMan® 5' nuclease assay for genotyping.105 Fifty samples genotyped by denaturing HPLC 

direct sequencing were in 100 percent concordance with TaqMan®.213 In the third study, a 

microchip based method that includes PCR, RFLP and capillary electrophoresis in a single 

platform was developed and tested. 100 percent concordance was reported for 80 patients, 

comparing the microchip method with both the conventional RFLP assay and the commercial 

TaqMan® assay.141

Key points. The various methods used to determine TPMT enzymatic activity are based on a 

method developed by Weinshilboum et al.134 While the enzymatic reaction has remained 

relatively unchanged, with a few minor adjustments, the method of product detection has 

evolved from radiolabel detection to HPLC. 

Both detection techniques produce reliable results, with intra- and inter-assay CVs of less 

than 10 percent.

Choice of TPMT substrate, 6-MP or 6-TG, does not appear to affect the precision of the 

assay significantly; however, 6-MP appears to be more widely used. 

Three studies have compared MALDI-TOF, TaqMan® and microchip assays with denaturing 

HPLC and RFLP, and demonstrated 100 percent concordance in genotypes. 
45

Key Question 1c: What is the diagnostic sensitivity and specificity of 

TPMT allelic polymorphism measurement compared to the 

measurement of TPMT enzymatic activity in correctly identifying 

chronic autoimmune disease patients eligible for thiopurine therapy 

with low or absent TPMT enzymatic activity? How do effect modifiers 

(e.g. underlying disease prevalence and severity, different activity 

thresholds, Hardy-Weinberg equilibrium, number and types of alleles 

tested) explain any observed heterogeneity in sensitivity and 

specificity?

Nineteen studies reported relevant data on testing of variable numbers and types of variant 

thiopurine methyl transferase alleles (Table 8). For certain combinations of alleles tested, only 

single studies provided data, and for those combinations, evidence was considered insufficient 

for any meaningful syntheses.159,160,163 One early study undertaken in 1993 did not report the 

specific variant alleles that were tested.93 In a sensitivity analysis we assumed the common 

TPMT *3A, *3B, *3C alleles were tested in this study, in keeping with the early date of 

publication when fewer alleles were being identified and tested. 

Characteristics of included studies and risk of bias. Study characteristics are summarized 

in Table 15. 

Over 50 percent of included studies were cross-sectional, and 21 percent were of prospective 

observational design. Studies did not specifically examine diagnostic accuracy of genetic testing 

with the TPMT enzymatic activity test as the reference standard, so we designated the activity 

test to be the reference standard and genotyping to be the index test. Over 75 percent of studies 

were conducted in patients with inflammatory bowel disease (IBD). Most studies used standard 

TPMT activity cutoffs. 

Risk of bias is summarized in Figure 4 with studies grouped according to items of the 

QUADAS risk of bias scale.35 Only one study clearly reported that genotyping and enzymatic 

activity measurements were not influenced by prior knowledge of the other test result.162Sixtythree percent of studies were considered to be of fair quality and the rest were rated poor. 

Diagnostic sensitivity and specificity of TPMT genotyping with respect to the enzymatic 

activity assay as the reference standard are presented below. Diagnostic groups are organized 

according to the specific set of TPMT variant alleles tested. Insufficient evidence was available 

for the following sets of single nucleotide polymorphisms (SNPs):

TPMT *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, *8160

TPMT *2, *3A, *3B, *3C, *3D163

TPMT *3A, *3B, *3C, *3D159

Table 15. Characteristics of studies of diagnostic accuracy of TPMT genetic testing compared with TPMT 

enzymatic activity

Characteristic Number of 

studies

References

Study Design

Cross-Sectional 11 1,50,53,56,59,70,93,160-162,167

NonRandomized 

Intervention Study

1 46

Prospective Observational 4 49,51,158,159
Table 15. Characteristics of studies of diagnostic accuracy of TPMT genetic testing compared with TPMT 

enzymatic activity (continued)

46

Characteristic Number of 

studies

References

Randomized Controlled 

Trial

1 52

Retrospective Review of 

Records

2 157,163

Chronic Autoimmune Disease

AntiNeutrophil Cytoplasmic 

Antibody Associated 

Vasculitis

1 157

Autoimmune Dermatologic 

Conditions

1 93

Autoimmune Hepatitis 2 162,167

Inflammatory Bowel 

Disease

14 1,46,49-52,56,59,70,158-161,163

Systemic Lupus 

Erythematosus 

1 53

Allelic Variants Tested

TPMT*2, *3A, *3B, * 3C 7 1,51-53,59,70,157

TPMT*2, *3A, *3B, *3C, *3D 1 163

TPMT*2, *3A, *3C 3 56,158,161

TPMT*3A, *3B, *3C 2 46,162

TPMT*3A, *3B, *3C, *3D 1 159

TPMT*2, *3A, *3B, *3C, *3D, 

*4, *5, *6, *7, *8

1 160

TPMT*2, *3A, *3B, *3C, *3D, 

*4, *5, *6, *7, *8, 10, *14, *15

3 49,167,214

Not Reported 1 93

TPMT Assay Type

High Performance Liquid 

Chromatography

7 1,51,53,59,157,163,167

Mass Spectrometry 2 46,70

Radioassay 10 49,50,52,56,93,158-162

Genotyping 

Method

Denatured High 

Performance Liquid 

Chromatography

1 163

Polymerase Chain Reaction 12 1,51-53,56,59,70,157-159,161,162

Pyrosequencing 4 49,50,160,167

Not Reported 2 46,93

Age Group

Adults 11 1,46,49,50,52,93,158,159,161,163,167

Mixed 4 51,59,160,162

Not Reported 4 53,56,70,157

Setting

Outpatient Specialty Clinics 9 1,46,49,51,56,59,161-163

Inpatients, and Outpatient 

Specialty Clinic

1 158

Not Reported 8 50,52,70,93,157,159,160,167,214

Region

Asia 1 53

Europe 14 1,46,49-52,56,59,70,159,160,162,163,167

North America 1 93
Table 15. Characteristics of studies of diagnostic accuracy of TPMT genetic testing compared with TPMT 

enzymatic activity (continued)

47

Characteristic Number of 

studies

References

Oceania 1 158

Not Reported 2 157,161

Risk of Bias

Fair 12 1,46,49,50,52,56,70,157,159,161,163,167

Poor 7 51,53,59,93,158,160,162

Abbreviations: TPMT = thiopurine methyltransferase

Figure 4. Risk of bias of studies of diagnostic accuracy of TPMT genetic testing compared with TPMT 

enzymatic activity

5

58

5

21

5

5

100

89

100

79

68

100

53

5

95

42

69

0

0

0

0

11

0

0

5

0

26

16

0

0

26

79

95

95

0

0

0

21

26

0

21

79

0% 20% 40% 60% 80% 100%

Deviation from Hardy Weinberg

equilibrium tested?

Study withdrawals explained? 

Reporting of uninterpretable or

equivocal test results?

Pre-testing clinical data availability

typical of practice?

Genotype determination was blinded

to patient TPMT activity?

Activity test interpretation was

blinded to patient genotype? 

Activity assay clearly described for

replication?

Genotyping method clearly described

for replication?

Independence of TPMT activity and

genotyping tests?

Activity tested regardless of

genotype?

All patients or random sample tested

for TPMT activity?

TPMT test accurate for enzymatic

activity?

Clearly described I/E criteria?

Representative of patient

population?

Yes (%) No (%) Unclear (%)

As described above in the “Methods” section, when possible, two meta-analyses were 

considered for each set of variant alleles. In meta-analysis 1, genotypes were dichotomized into 

noncarriers (or wild types) and carriers (or homozygotes and/or heterozygotes) and phenotypes 

were dichotomized into normal (or high) and subnormal (or intermediate and/or low and/or 

absent) enzymatic activities. In meta-analysis 2, we dichotomized genotypes into noncarriers 

and/or heterozygotes and homozygotes, and phenotype into normal and/or high and/or 

intermediate and low/absent activities (Table 16).
48

Table 16. Meta-analyses 1 and 2, for sensitivity and specificity of variant allelic determination to diagnose 

TPMT enzymatic activity

Meta-analysis 1

Normal/high activity Intermediate/low/ 

absent activity

Sensitivity 1 Specificity 1

Homozygotes 

and/or 

heterozygotes

A1 B1

B1/(B1+D1) C1/(C1+A1)

Noncarrier (or 

Wild types) C1 D1

Meta-analysis 2

Normal/high/intermediate 

activity

Low/absent 

activity

Sensitivity 2 Specificity 2

Homozygotes A2 B2

Noncarrier (or B2/(B2+D2) C2/(C2+A2) Wild types) and/or 

heterozygotes

C2 D2

TPMT *2, *3A, *3B, and *3C. These variant alleles were genotyped in seven studies 

involving 752 participants.1,51-53,59,70,157 In meta-analysis 1, the pooled sensitivity of the carrier 

genotypes (i.e. homozygosity and heterozygosity) to correctly identify all those patients with 

subnormal enzymatic activity as determined by activity assays was 70.70 percent (95 percent CI 

37.90 percent to 90.50 percent). Risk of bias was not found to be significantly associated with 

variation in effect estimates. No explanation was identified for this heterogeneity. The pooled 

specificity of the noncarrier or wild type genotype to correctly identify all of those with 

normal/high enzymatic activity was 99.90 percent (95 percent CI 97.40 percent to 99.60 percent) 

(Figure 5).

In meta-analysis 2, since only one patient was homozygous for a TPMT allele, only the 

pooled specificity of noncarrier and heterozygous carrier genotype to correctly identify all those 

who do not have low or absent enzymatic activities could be calculated. This approached 100 

percent (Figure 6).

In further sensitivity analyses, we assumed that studies that specifically tested the alleles 

TPMT *2, *3A, *3C (see below) also implicitly tested for *3B, because of shared *3B SNP 

460G→A, which is one of the two TPMT *3A alleles. We therefore widened meta-analysis 1 

and 2 to included the three studies that reported specifically testing for TPMT *2, *3A, *3C as 

additional sensitivity analyses.56,158,161 Still, only one of a total of 945 patients in nine studies 

was homozygous for a TPMT variant allele. In the sensitivity meta-analysis 1, the pooled 

sensitivity of the carrier genotype (i.e. homozygosity and heterozygosity) to correctly identify all 

those patients with subnormal enzymatic activity as determined by activity assays remained 

unchanged at 74.00 percent (95 percent CI 48.90 percent to 89.40 percent). Risk of bias was 

again not found to be significantly associated with variation in effect estimates. The pooled 

specificity of the noncarrier or wild type genotype to correctly identify all of those with 

normal/high enzymatic activity was 98.60 percent (95 percent CI 97.10 percent to 99.30 percent) 

(Figure 7).

In the sensitivity meta-analysis 2, since only one patient was homozygous for a TPMT allele, 

only the pooled specificity of noncarrier and heterozygous carrier genotype to correctly identify 

all those who do not have low or absent enzymatic activities could be calculated. This 

approached 100 percent (Figure 8)
49

Figure 5. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, *3B and *3C, to diagnose 

TPMT activity

† (Homozygotes or heterozygotes with intermediate or low to absent enzymatic activity)

 (all with intermediate or low enzymatic activity)

‡ (Noncarriers or wild type genotype with normal/high enzymatic activity)

 (all with normal or high enzymatic activity)
50

Figure 6. Meta-analysis 2 of specificity of genotyping TPMT *2, *3A, *3B and *3C, to diagnose TPMT activity

‡ (Noncarriers (wild type) and/or heterozygotes with normal/intermediate/high enzymatic activity)

 (all with normal, intermediate or high enzymatic activity)
51

Figure 7. Additional meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, *3B and *3C, or 

TPMT *2, *3A and *3C, to diagnose TPMT activity

† (Homozygotes or heterozygotes with intermediate or low to absent enzymatic activity)

 (all with intermediate or low enzymatic activity)

‡ (Noncarriers or wild type genotype with normal/high enzymatic activity)

 (all with normal or high enzymatic activity)
52

Figure 8. Additional meta-analysis 2 of specificity of genotyping TPMT *2, *3A, *3B and *3C, or TPMT *2, *3A 

and *3C, to diagnose TPMT activity

‡ (Noncarriers (wild type) and/or heterozygotes with normal/intermediate/high enzymatic activity)

 (all with normal, intermediate or high enzymatic activity)

TPMT *2, *3A, and *3C. These variant alleles were genotyped in three studies including 

386 patients.56,158,161 In meta-analysis 1, the pooled sensitivity of the carrier genotype (i.e. 

homozygosity and heterozygosity) to correctly identify all those patients with subnormal 

enzymatic activity was 82.10 percent (95 percent CI 40.40 percent to 96.90 percent). The pooled 

specificity of noncarrier or wild type genotypes to correctly identify all those with normal/high 

enzymatic activity was 97.80 percent (95 percent CI 93.30 percent to 99.30 percent).(Figure 9).

This group of studies did not include any homozygous participant; hence only the specificities of 

noncarrier and heterozygous carrier genotypes to correctly identify all those who do not have 

low/absent enzymatic activity could be pooled for meta-analysis 2. The pooled estimate 

approached 100 percent (Figure 10).
53

Figure 9. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, and *3C, to diagnose 

TPMT activity

† (Homozygotes or heterozygotes with intermediate or low to absent enzymatic activity)

 (all with intermediate or low enzymatic activity)

‡ (Noncarriers or wild type genotype with normal/high enzymatic activity)

 (all with normal or high enzymatic activity)

Figure 10. Meta-analysis 2 of specificity of genotyping TPMT *2, *3A, and *3C, to diagnose TPMT activity

‡ (Noncarriers (wild type) and/or heterozygotes with normal/intermediate/high enzymatic activity)

 (all with normal, intermediate or high enzymatic activity)

 

Sensitivity†

Specificity‡ Study Sensitivity & Specificity (95% CI)

Gardiner, 2008

Ansari, 2002

Marinaki, 2003

0% 100%

55.6 (25.1, 82.3)

95.5 (55.2, 99.7)

91.7 (37.8, 99.5)

82.1 (40.4, 96.9)

96.6 (87.4, 99.2)

98.4 (78.9, 99.9)

99.4 (90.0, 100)

97.8 (93.3, 99.3)

5 / 9

10 / 10

5 / 5

57 / 59

30 / 30

80 / 80

Pooled

 

 

 

Specificity‡ Study Specificity (95% CI)

Gardiner, 2008

Ansari, 2002

Marinaki, 2003

0% 100%

99.3 (89.5, 100)

98.8 (83.3, 99.9)

99.4 (91.4, 100)

99.2 (96.1, 99.8)

68 / 68

40 / 40

85 / 85

Pooled

 

 
54

TPMT *3A, *3B, and *3C. These variant alleles were genotyped in two studies that 

included a total of 245 participants.46,162 In meta-analysis 1, the pooled sensitivity of the carrier 

genotype (i.e. homozygosity and heterozygosity) to correctly identify all those patients with 

subnormal enzymatic activity was 71.80 percent (95 percent CI 54.00 percent to 84.60 percent). 

The pooled specificity of noncarrier or wild type genotypes to correctly identify all those with 

normal/high enzymatic activity was 97.30 percent (95 percent CI 69.60 percent to 99.80 percent) 

(Figure 11). In a sensitivity analysis, when we included an early study by Snow et al.,93 a study 

that did not report variant alleles it tested, under the assumption that it tested TPMT *3A, *3B, 

*3C, the results remained essentially unchanged for pooled estimates of both sensitivity and 

specificity (N = 26). Because no homozygous carriers were included, meta-analysis 2 was only 

possible for specificity of noncarrier and heterozygous carrier genotypes to correctly identify all 

those who do not have low/absent enzymatic activity. The pooled specificity approached 100 

percent. 

Figure 11. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *3A, *3B and *3C, to diagnose 

TPMT activity

† (Homozygotes or heterozygotes with intermediate or low to absent enzymatic activity)

 (all with intermediate or low enzymatic activity)

‡ (Noncarriers or wild type genotype with normal/high enzymatic activity)

 (all with normal or high enzymatic activity)

 

Sensitivity†

Specificity‡ Study Sensitivity & Specificity (95% CI)

Ansari, 2008

Langley, 2002

Pooled

0% 100%

73.9 (52.8, 87.8)

66.7 (33.3, 88.9)

71.8 (54.0, 84.6)

99.4 (95.9, 99.9)

90.9 (78.2, 96.5)

97.3 (69.6, 99.8)

17 / 23

6 / 9

168 / 169

40 / 44

 

 
55

TPMT *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, *8, *10, *14, and *15. These variant alleles 

were genotyped in three studies that included a total of 341 patients of whom seven were 

homozygous for an SNP.49,50,167 In meta-analysis 1, the pooled sensitivity of the carrier genotype 

(i.e. homozygosity and heterozygosity) to correctly identify all those patients with subnormal 

enzymatic activity was 81.30 percent (95 percent CI 59.90 percent to 92.70 percent). The pooled 

specificity of noncarrier or wild type genotypes to correctly identify all those with normal/high 

enzymatic activity was 8.50 percent (95 percent CI 96.10 percent to 99.40 percent) (Figure 12). 

In meta-analysis 2, the sensitivity of the homozygous genotype to correctly identify all patients 

with low/absent enzymatic activity was 87.10 percent (95 percent CI 44.30 percent to 98.30 

percent), and the specificity of noncarrier and heterozygous carrier genotype to correctly identify 

all those who do not have low/absent enzymatic activity approached 100 percent (Figure 13).

No other combinations of variant TPMT alleles were tested.

Figure 12. Meta-analysis 1 of sensitivity and specificity of genotyping TPMT *2, *3A, *3B, *3C, *3D, *4, *5, *6, 

*7, *8, *10, *14, and *15, to diagnose TPMT activity

† (Homozygotes or heterozygotes with intermediate or low to absent enzymatic activity)

 (all with intermediate or low enzymatic activity)

‡ (Noncarriers or wild type genotype with normal/high enzymatic activity)

 (all with normal or high enzymatic activity)
56

Figure 13. Meta-analysis 2 of specificity of genotyping TPMT *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, *8, *10, *14, 

and *15, to diagnose TPMT activity

† (Homozygous with low/absent enzymatic activity)

 (all with low/absent enzymatic activity)

‡ (Noncarriers (wild type) and/or heterozygotes with normal/intermediate/high enzymatic activity)

 (all with normal, intermediate or high enzymatic activity)

Key points. A total of 16 studies, mostly of cross-sectional and prospective observational 

design, contributed to quantitative syntheses.1,46,49-53,56,59,70,93,157,158,161,162,167 Studies did not 

specifically examine diagnostic accuracy of genetic testing with the TPMT enzymatic activity 

test as the reference standard, so we designated the activity test to be the reference standard and 

genotyping to be the index test. Overall, homozygosity for variant allele(s) was quite low in the 

study samples. Different combinations of TPMT variant alleles were analyzed in various studies. 

Thirty-seven percent of the studies were rated as of poor quality.

Across all the combinations of alleles tested, an imprecise pooled sensitivity of the carrier 

genotype (i.e. homozygous plus heterozygous patients) to correctly identify all those patients 

with subnormal (intermediate, or low to absent) enzymatic activity, as determined by TPMT 

assays, was in the range of 70.70 to 82.10 percent (95 percent CI, lower bound range 37.90 to 

54.00 percent; upper bound range 84.60 to 96.90 percent). 

The pooled sensitivity of a homozygous TPMT genotype to correctly identify patients with 

low to absent enzymatic activity was based on two studies with two percent of 341 patients 

identified as homozygous for variant allele. The pooled sensitivity was 87.10 percent (95 percent 

CI 44.30 to 98.30 percent). 

Meta-regression analysis did not identify any significant effect modifiers. 

Compared with the reference standard of TPMT enzymatic activity, the specificity of TPMT 

genotyping to correctly identify patients with normal/high enzymatic activities, or normal/high 

and intermediate enzymatic activities, is very high (greater than 90 percent) across all 

combinations of alleles tested.
57

There is insufficient data to determine the optimum combination of TPMT alleles for testing. 

Most studies tested at least the TPMT *3A, *3B, and *3C variant alleles, irrespective of testing

additional polymorphisms. 
58

Key Question 2: Does the measurement of TPMT enzymatic activity or 

determination of TPMT allelic polymorphisms change the 

management of patients with chronic autoimmune disease when 

compared with no determination of TPMT status?

The evidence to answer this question came from one randomized controlled trial of fair 

quality in 333 patients with inflammatory conditions, mostly inflammatory bowel disease (Figure 

14 and Appendix C, Tables C7-9).

1 Patients were randomized into either a group that had prior 

TPMT genotyping or one without pretesting. Where applicable, therapy was advised to be 

guided by genotyping results, however, all treatment decisions were at the discretion of treating 

physicians. Over a 4 month period, no significant differences between starting doses 

administered with or without prior knowledge of TPMT genotype were observed in both the 

noncarriers and heterozygous carrier patients. However, in the genotyped treatment arm, 

heterozygotes received significantly lower doses of TPMT when compared with noncarriers. 

Most patients in both groups were given starting doses lower than 2mg/kg/day including those 

with predisclosed noncarrier genotype. About seven percent of those in whom noncarrier 

genotype was predisclosed received AZA doses ≥ 2mg/kg/day as compared to 8.4 percent of 

those in whom noncarrier genotype was found out after the fact. Furthermore, there was no 

significant difference between the two groups in terms of mean AZA prescribed dose at the end 

of the study period. There is limited applicability of this evidence because there was just one 

homozygous carrier in the whole sample of mostly IBD patients. 
59

Figure 14: Risk of bias of the randomized controlled trial answering KQ 2 

100

0

0

100

100

100

100

0

0

100

0

100

100

0

0

0

0

100

0

0

0

0

0

0

0

0

0

0

100

0

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Thiopurine adherence assessed?

High loss to follow-up or important differential loss to follow-up

between groups? 

Potential for conflict of interest?

Intention to treat analysis?

Outcomes described? 

Groups similar at baseline? 

Blinded assessment of outcomes?

Blinding of investigators and participants?

Adequacy of allocation concealment?

True randomization?

Yes (%) No (%) Unclear (%)
60

Key Question 3a: In chronic autoimmune disease patients prescribed 

thiopurine-based drugs (AZA or 6-MP), does the assessment of TPMT 

status to guide therapy, when compared with no pretreatment 

assessment, lead to reduction in rates of mortality, infection, 

hospitalization, withdrawal due to adverse events (WDAE), serious 

adverse events (SAE) and improvement in health-related quality of 

life?

The evidence to answer this question came from one randomized controlled trial of fair 

quality in 333 patients with inflammatory conditions, mostly inflammatory bowel disease (Figure 

15 and Appendix C, Tables C 10-12).1 Patients were randomized into either a group that had 

prior TPMT genotyping or another without pretesting. Where applicable, therapy was advised to 

be guided by genotyping results; however, all treatment decisions were at the discretion of 

treating physicians. Over a 4 month period, no significant differences were seen in the outcomes 

of mortality [1/167 versus3/166; odds ratio 0.33 (95 percent CI, 0.03 to 3.18 percent)]; SAE 

[4/167 versus 8/166; odd ratio 0.48 (95 percent CI 0.14 to 1.64 percent)]; and WDAE (0/167 

versus 0/166). Other outcomes were not reported including the important outcome of healthrelated quality of life.

Figure 15: Risk of bias of the randomized controlled trial answering KQ 3a

100

0

0

100

100

100

100

0

0

100

0

100

100

0

0

0

0

100

0

0

0

0

0

0

0

0

0

0

100

0

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Thiopurine adherence assessed?

High loss to follow-up or important differential loss to follow-up

between groups? 

Potential for conflict of interest?

Intention to treat analysis?

Outcomes described? 

Groups similar at baseline? 

Blinded assessment of outcomes?

Blinding of investigators and participants?

Adequacy of allocation concealment?

True randomization?

Yes (%) No (%) Unclear (%)
61

Strength of evidence answering key question 3a. The evidence was rated following 

published guidance39 as follows:

Table 17. Rating the strength of evidence-key question 3a

Outcome N of 

studies

N of 

Subjects Domains pertaining to strength of evidence

OR 

(95% 

CI)

Strength of 

evidence

Risk of 

Bias

Consistency Directness Precision 

Mortality 11 333 Medium Unknown Direct Imprecise 0.33 

(0.03, 

3.18)

Insufficient 

Serious 

adverse 

events 

11 333 Medium Unknown Direct Imprecise 0.48 

(0.14, 

1.64)

Insufficient

Healthrelated 

quality 

of life

0 0 - - - - - Insufficient 

There is limited applicability of this evidence as there was just one homozygous carrier of 

TPMT variant allele in the entire sample of mostly IBD patients observed for just 4 months. 

Also, patients likelier to experience adverse events were excluded. 
62

Key Question 3b: In chronic autoimmune disease patients prescribed 

thiopurine-based drugs (AZA or 6-MP), does the assessment of TPMT 

status to guide therapy, when compared with no pretreatment 

assessment, lead to reduction in rates of myelotoxicity, liver toxicity, 

and pancreatitis? 

The evidence to answer this key question was available from one randomized controlled trial 

and one retrospective cohort study (Appendix C, Tables C13-15).1,169 No significantly reduced 

event rates were noted for intermediate outcomes in the pretested versus the nonpretested groups.

One trial of fair quality in 333 patients with inflammatory conditions, mostly inflammatory 

bowel disease, in which patients were randomized into either a group that had prior TPMT 

genotyping or another without pretesting (Figure 16).

1 Where applicable, therapy was advised to 

be guided by genotyping results, however, all treatment decisions were at the discretion of 

treating physicians. Over a 4 month period, No significant differences were seen in the outcomes 

of neutropenia [2/167 versus 1/166; odd ratio 2.00 (95 percent CI 0.18 to 22.27 percent)]; and 

pancreatitis [1/167 versus 4/166; odd ratio 0.24 (95 percent CI 0.03 to 2.21 percent)]; while 

significantly higher odds were observed for hepatitis in the group that underwent prior TPMT 

genotyping [19/167 versus 8/166; odds ratio 2.54 (95 percent CI 1.08 to 5.97 percent)]. Other 

intermediate outcomes were not reported. There is limited applicability of this evidence as there 

was just one homozygous carrier in the whole sample of patients observed for just 4 months. 

Also, patients likelier to experience adverse events were excluded.

Banerjee et al.’s was a retrospective cohort study in a pediatric population with IBD, treated 

with at least 4 months of stable dose treatment with azathioprine (AZA) or 6-mercaptopurine (6-

MP).169 Ninety percent of patients received AZA. The study group was comprised of 64 patients 

who received initial AZA dosing based on TPMT enzymatic activity as measured by the high 

performance liquid chromatography (HPLC) method, with subsequent dose titrations based on 6-

thioguanine nucleotide and 6-methylmercaptopurine levels. A historical control of 37 patients 

had been started on AZA 1.5 mg/kg/day and subsequent doses were adjusted based on clinical 

response and drug toxicity. Fourteen percent of patients had intermediate TPMT enzymatic 

activity and none had low/zero activity. On average, the study group received 1.7 mg/kg/day of 

AZA treatment and the control 1.2 mg/kg/day. A statistically significant difference was not 

observed for the outcome of leukopenia (study versus control group; 4.7 percent versus 0 

percent) and hepatotoxicity (study versus control group; 9.4 percent versus 16.2 percent). The 

overall risk of bias assessment rated this evidence as poor, based on several limitation (Figure 

17).
63

Figure 16: Risk of bias of the single randomized controlled trial answering KQ 3b 

Figure 17: Risk of bias of the single retrospective cohort study answering KQ 3b 

100

0

0

0

0

0

100

100

100

0

0

0

0

0

0

100

100

0

0

0

0

0

0

100

100

100

0

0

0

0

0

100

100

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Appropriateness of the TPMT test?

Methods to control for confounding?

Selection bias minimized? 

Adequate sample size?

High loss to follow-up or differential loss to follow-up between

groups? 

Potential for conflict of interest?

Intention to treat analysis?

Outcomes adequately described?

Groups comparable at baseline? 

Blinded assessment of outcomes?

Investigators and participants blinded to testing?

Yes (%) No (%) Unclear (%)

100

0

0

100

100

100

100

0

0

100

0

100

100

0

0

0

0

100

0

0

0

0

0

0

0

0

0

0

100

0

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Thiopurine adherence assessed?

High loss to follow-up or important differential loss to follow-up

between groups? 

Potential for conflict of interest?

Intention to treat analysis?

Outcomes described? 

Groups similar at baseline? 

Blinded assessment of outcomes?

Blinding of investigators and participants?

Adequacy of allocation concealment?

True randomization?

Yes (%) No (%) Unclear (%)
64

Strength of evidence answering key question 3b. The evidence was rated following 

published guidance39 as follows: 

Table 18. Rating the strength of evidence-key question 3b

Outcome

N of 

studie

s

N of 

Subject

s

Domains pertaining to strength of 

evidence

OR 

(95

% 

CI)

Strength 

of 

evidence

Ris

k of 

Bia

s

Consistenc

y 

Directnes

s 

Precisio

n 

Myelotoxicit

y

0 0 - - - - - Insufficien

t 
65

Key Question 3c: In the absence or inconclusiveness of evidence 

answering key question 3a and/or 3b above, is there an association 

between TPMT status (as determined by TPMT enzymatic activity 

and/or TPMT allelic determination) and/or the following amongst 

chronic autoimmune disease patients treated with thiopurines:

i) the clinical outcomes of mortality, infections, hospitalization, 

withdrawal due to adverse events (WDAE), serious adverse 

events (SAE) and health-related quality of life?

ii) surrogate outcomes of myelotoxicity, liver toxicity, and 

pancreatitis? 

TPMT Enzymatic Activity Determination

Sixteen of the eligible studies reported relevant data (Table 19). No evidence was available 

for the outcomes of mortality, hospitalization, SAE, and HQOL. The sparse data available for the 

outcomes of infection, neutropenia and thrombocytopenia, did not permit a meaningful analysis. 

Characteristics of included studies and quality are summarized in Table 19. Approximately 

50 percent of studies were in populations with IBD. In 12 studies reporting gender distribution, 

female representation was on average 50 percent.50-52,70,72,79,91,93,161,193,196,197 Racial distribution 

was very poorly reported. In 80 percent of included studies, thiopurine treatment was restricted 

to AZA, and when reported dosages were generally in the range 1.5-

2.5mg/kg/day.50,52,53,70,72,91,93,157,161,192,193,195,197 The adverse event observation period on 

thiopurine treatment ranged between six and 48 months (average 20 months) while four studies 

did not report this information.53,93,193,195 Over sixty percent of studies were cross-sectional in 

design, and most studies were rated as fair quality. Most importantly, biased enzymatic activity 

determination based on prior knowledge of outcomes or outcomes assessment based on prior 

knowledge of enzymatic activity results could not be clearly ruled out in any one of the studies 

addressing this question. No study was rated as good quality, 25 percent were rated as poor and 

the rest were judged as fair. 

Figure 18 depicts the distribution of studies by individual quality scoring items. Detailed 

results of all 16 studies are available in Appendix C.

Table 19. Summary characteristics of TPMT enzymatic activity and thiopurine toxicity association studies

Characteristic Number of 

studies References

Study Design

Case Control 1 197

Chart Review 1 157

Cohort 1 195

Cross-Sectional 10 50,51,53,70,79,93,161,192,193,196

NonRandomized Intervention 

Study

1 91

Prospective Observational 1 72

Randomized Controlled Trial 1 52
66

Characteristic Number of 

studies References

Chronic 

Autoimmune 

Disease

AntiNeutrophil Cytoplasmic 

Antibody Associated 

Vasculitis

1 157

Autoimmune Dermatologic 

Conditions

1 93

Autoimmune Disorders 2 195,197

Autoimmune Hepatitis 1 79

Inflammatory Bowel Disease 8 50-52,70,72,161,192,196

Pemphigus Vulgaris 1 193

Rheumatoid Arthritis 1 91

Systemic Lupus 

Erythematosus 

1 215

Not Reported

Thiopurine 

Treatment

6-MP, AZA 1 196

AZA 14 50,50,52,53,70,72,91,93,157,161,192,193,195,197

Mixed Thiopurines 1 51

Assay Type High Performance Liquid 

Chromatography

4 51,53,157,193

Mass Spectrometry 1 70

Radioassay 9 50,52,72,79,91,93,161,195,197

Not Reported 2 192,196

Outcomes 

Assessed

Anemia 2 193,197

Any Infection 1 91

Hepatitis 9 50-52,70,72,91,93,193,196

Leukopenia 8 53,70,93,157,193,195-197

Myelotoxicity 7 50-52,72,91,195,197

Neutropenia 1 161

Pancreatitis 6 50-52,70,72,193

Thrombocytopenia 1 197

Withdrawal due to Adverse 

Events

4 52,79,192,193

Age Group Adults 8 50,52,72,79,91,93,195,216

Mixed 3 51,53,196

Not Reported 5 70,157,161,192,193

Setting Outpatient Specialty Clinics 5 51,70,91,161,193

Not Reported 11 50,52,53,72,79,93,157,192,195-197

Region Asia 1 53

Europe 9 50-52,70,72,79,91,192,195

Europe and North America 1 217

Middle East and North Africa 1 193

North America 2 93,196

Not Reported 2 157,161

Risk of Bias Fair 12 50-53,70,72,79,91,157,161,193,195

Poor 4 93,192,196,197

Abbreviations: 6-MP = 6-mercaptopurine; AZA = azathioprine; TPMT = thiopurine methyltransferase
67

Figure 18. Risk of bias of TPMT enzymatic activity, and thiopurine toxicity association studies

81

6

6

0

19

6

50

69

0

0

0

88

25

0

69

94

31

25

6

0

19

6

69

100

13

0

19

6

94

100

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Appropriateness TPMT activity testing method?

Methods to control for confounding?

Unbiased selection of participants?

Adequacy of sample size?

High loss to follow-up or important differential loss to follow-up

between groups? 

Potential for financial conflict of interest?

Intention to treat analysis?

Outcomes adequately described?

Comparability of groups at baseline? 

Clearly there is no biased assessment of outcomes and/or

phenotyping results based on a prior knowledge of each other

Yes (%) No (%) Unclear (%)

Withdrawal due to adverse events. Insufficient evidence addressed withdrawal due to 

adverse events in patients with low/absent TPMT activity. Compared with normal TPMT 

enzymatic activity, intermediate activity was not significantly associated with withdrawals (OR 

1.75, 95 percent CI 0.75 to 4.11) (Figure 19). 52,79,192,193
68

Figure 19. Odds ratio of withdrawal due to adverse events during thiopurine treatment of chronic 

autoimmune disease; intermediate versus normal enzymatic activities

Myelotoxicity. Myelotoxicity was defined variably, in some studies including at least two 

myeloid cell lines (e.g. leukopenia and thrombocytopenia)50-52,91,195 or was not clearly 

defined.72,197 A nonsignificant odds ratio was noted for the outcome of myelotoxicity when 

intermediate enzymatic activity was compared with normal TPMT activity,50,51,72,91,195 but 

significant odds favoring higher activities were found when low enzymatic activity was 

compared with normal (OR 13.6, 95 percent CI 3.52 to 52.80) and intermediate (OR 10.2, 95 

percent CI 2.23 to 46.60) activities (Figure 20).50,50,197 The overall event rate of myelotoxicity 

was 5 percent among 996 patients. 

Study name Odds ratio and 95% CI

0.01 0.1 1 10 100

 

Events / Total n

Intermediate Normal

Favors 

Intermediate

Favors 

Normal

 

Firooz, 2008 1 / 11 13 / 127 0.88 (0.10, 7.41)

Shah, 2008 1 / 12 18 / 119 0.51 (0.06, 4.20)

Czaja, 2006 2 / 12 9 / 73 1.42 (0.27, 7.56)

von Ahsen, 2005 14 / 21 18 / 50 3.56 (1.21, 10.4)

1.75 (0.75, 4.11)
69

Figure 20. Odds ratios of myelotoxicity during thiopurine treatment of chronic autoimmune disease; 

intermediate/normal, low/intermediate and low/normal comparisons

Study name Odds ratio and 95% CI

Events / Total n

Intermediate Normal

Favors 

Intermediate

Favors 

Normal

Intermediate vs. Normal

Study name Odds ratio and 95% CI

Events / Total n

Low Intermediate

Favors 

Low

Favors 

Intermediate

Low vs. Intermediate

Study name Odds ratio and 95% CI

Events / Total n

Low Normal

Favors 

Low

Favors 

Normal

Low vs. Normal

Hindorf, 2006 3 / 45 22 / 313 0.95 (0.27, 3.29)

Schedel, 2006 1 / 28 1 / 65 2.37 (0.14, 39.3)

Stolk, 1998 0 / 8 1 / 24 0.92 (0.03, 24.9)

Gisbert, 2006 4 / 28 13 / 366 4.53 (1.37, 14.9)

Stocco, 2005 0 / 3 3 / 25 0.92 (0.04, 21.9)

1.96 (0.90, 4.25)

Hindorf, 2006 3 / 6 3 / 45 14.0 (1.93, 101.7)

Schedel, 2006 0 / 3 1 / 28 2.62 (0.09, 77.6)

Lennard, 1989 3 / 5 0 / 5 15.4 (0.56, 425.5)

10.2 (2.23, 46.6)

0.01 0.1 1 10 100

0.01 0.1 1 10 100

0.01 0.1 1 10 100

Hindorf, 2006 3 / 6 22 / 313 13.2 (2.52, 69.4)

Schedel, 2006 0 / 3 1 / 65 6.14 (0.21, 179.8)

Lennard, 1989 3 / 5 0 / 11 32.2 (1.23, 841.8)

13.6 (3.52, 52.8)
70

Leukopenia. Leukopenia was experienced by 12 percent of participants, and a significant 

dose response relationship was observed. When low and intermediate activities were compared 

with normal activities and with each other, a significantly higher number of patients with lower 

enzymatic activities experienced leukopenia. In a total of 538 patients in seven studies, 

significant odds ratios of intermediate versus normal were 2.96 (95 percent CI 1.18 to 

7.42);53,70,93,157,193,195,196 low versus intermediate of 29.2 (95 percent CI 4.55 to 188) in three

studies with 247 participants;70,195,197 and a low versus normal activity odds ratio of 80.00 (95 

percent CI 11.5 to 559) in three studies with 247 participants (Figure 21).70,195,197

 

Figure 21. Odds ratios of leukopenia during thiopurine treatment of chronic autoimmune disease; 

intermediate/normal, low/intermediate and low/normal comparisons

Study name Odds ratio and 95% CI

Events / Total n

Intermediate Normal

Favors 

Intermediate

Favors 

Normal

Intermediate vs. Normal

Study name Odds ratio and 95% CI

Events / Total n

Low Intermediate

Favors 

Low

Favors 

Intermediate

Low vs. Intermediate

Study name Odds ratio and 95% CI

Events / Total n

Low Normal

Favors 

Low

Favors 

Normal

Low vs. Normal

0.1 1 10 100

Winter, 2007 1 / 16 8 / 113 0.88 (0.1, 7.5)

Firooz, 2008 0 / 11 1 / 127 3.7 (0.14, 95)

Kader, 2000 1 / 2 1 / 20 19 (0.62, 583)

Schedel, 2006 2 / 28 0 / 65 12.4 (0.57, 266)

Okada, 2005 1 / 3 2 / 15 3.25 (0.19, 54.8)

Snow, 1995 2 / 5 1 / 21 13.3 (0.91, 196.4)

2.96 (1.18, 7.42)

Winter, 2007 1/ 1 1 / 16 31 (0.84, 1149)

Schedel, 2006 2 / 3 2 / 28 26 (1.58, 427)

Lennard, 1989 4 / 5 0 / 5 33 (1.06, 1023)

29.2 (4.55, 188)

Winter, 2007 1 / 1 8 / 113 37 (1.4, 986)

Schedel, 2006 2 / 3 0 / 65 218 (7.0, 6815)

Lennard, 1989 4 / 5 0 / 11 69 (2.4, 2029)

80 (11.5, 559)

1000

0.1 1 10 100 1000

0.1 1 10 100 1000

Stassen, 2009 3 / 7 34 / 101 1.48 (0.31, 6.98)
71

Anemia. Two studies in 246 patients found no significant difference between patients with 

intermediate enzymatic activity and normal activity, in development of anemia during thiopurine 

treatment (Figure 22).157,193 Insufficient evidence was available to compare low enzymatic 

activity with higher activity. 

Figure 22. Odds ratio of anemia during thiopurine treatment of chronic autoimmune disease; intermediate 

versus normal TPMT enzymatic activities

Hepatitis or elevated hepatic transaminases. No significant association between enzymatic 

activities and odds of developing hepatoxicity was evident in seven studies. (Figure 23). 

Study name Odds ratio and 95% CI

0.01 0.1 1 10 100

Events / Total n

Intermediate Normal

Favors 

Intermediate

Favors 

Normal

 

Firooz, 2008 0 / 11 1 / 127 3.67 (0.14, 95.2)

Stassen, 2009 1.78 (0.37, 8.42) 3 / 7 30 / 101

2.03 (0.50, 8.27)
72

Figure 23. Odds ratios of hepatitis or elevated hepatic transaminases during thiopurine treatment of chronic 

autoimmune disease: intermediate/normal, low/intermediate and low/normal comparisons

Study name Odds ratio and 95% CI

 

Events / Total n

Intermediate Normal

Favors 

Intermediate

Favors 

Normal

Intermediate vs. Normal

Study name Odds ratio and 95% CI

Events / Total n

Low Intermediate

Favors 

Low

Favors 

Intermediate

Low vs. Intermediate

Study name Odds ratio and 95% CI

Events / Total n

Low Normal

Favors 

Low

Favors 

Normal

Low vs. Normal

Hindorf, 2006 4 / 45 15 / 313 1.94 (0.61, 6.12)

Winter, 2007 1 / 16 8 / 113 0.88 (0.10, 7.50)

Firooz, 2008 1 / 11 10 / 127 1.17 (0.14, 10.1)

Kader, 2000 1 / 2 2 / 20 9.00 (0.39, 206.5)

Snow, 1995 0 / 5 1 / 21 1.24 (0.04, 34.9)

Stolk, 1998 1 / 8 1 / 24 3.29 (0.18, 59.6)

von Ahsen, 2005 1 / 20 2 / 50 1.20 (0.10, 14.0)

1.74 (0.80, 3.77)

Hindorf, 2006 0 / 6 4 / 45 0.71 (0.03, 14.8)

Winter, 2007 0 / 1 1 / 16 3.44 (0.09, 127.7)

1.36 (0.13, 13.9)

Hindorf, 2006 0 / 6 15 / 313 1.48 (0.08, 27.5)

Winter, 2007 0 / 1 8 / 113 4.14 (0.16, 109.5)

2.34 (0.26, 20.7)

0.01 0.1 1 10 100

0.01 0.1 1 10 100

0.01 0.1 1 10 100
73

Pancreatitis. Insufficient evidence was available to compare low enzymatic activity with 

normal activity. Odds of pancreatitis were nonsignificant when intermediate activity was 

compared with normal activity (OR 1.79, 95 percent CI 0.65 to 4.99) and low with intermediate 

activity (OR 2.01, 95 percent CI 0.19 to 21.60) (Figure 24). 

Figure 24. Odds ratios of pancreatitis during thiopurine treatment of chronic autoimmune disease: 

intermediate/normal, and low/intermediate comparisons

Study name Odds ratio and 95% CI

Events / Total n

Intermediate Normal

Favors 

Intermediate

Favors 

Normal

Intermediate vs. Normal

Study name Odds ratio and 95% CI

Events / Total n

Low Intermediate

Favors 

Low

Favors 

Intermediate

Low vs. Intermediate

Hindorf, 2006 2 / 45 13 / 313 1.07 (0.23, 4.92)

Winter, 2007 1 / 16 0 / 113 22.0 (0.86, 563.3)

Firooz, 2008 0 / 11 1 / 127 3.67 (0.14, 95.2)

Von Ahsen, 2005 1 / 20 1 / 50 2.45 (0.15, 41.1)

Gisbert, 2006 0 / 28 11 / 366 0.54 (0.03, 9.44)

Stocco, 2005 0 / 3 1 / 25 2.33 (0.08, 69.2)

1.79 (0.65, 4.99)

Hindorf, 2006 1.34 (0.06, 31.1) 0 / 6 2 / 45

Winter, 2007 3.44 (0.09, 127.7) 0 / 1 1 / 16

2.01 (0.19, 21.6)

0.01 0.1 1 10 100

0.01 0.1 1 10 100
74

Key points. Fifteen studies contributed to quantitative syntheses regarding association of 

thiopurine treatment toxicity with TPMT enzymatic activity.50-53,70,72,79,91,93,157,192,193,195-197

A dose response relationship was demonstrated for the association of subnormal TPMT 

enzymatic activities and the outcome of leukopenia. The greatest odds of the outcome were 

noted when the enzymatic activity was low followed by when it was intermediate in comparison 

with normal enzymatic activity. 

Greater odds for the outcome of myelotoxicity were also noted with low activity when 

compared with intermediate and normal TPMT enzymatic activities. 

No evidence was available for the outcomes of mortality, hospitalization, SAE, and HQOL. 

Sparse data were available for the outcome of infection, neutropenia and thrombocytopenia, not 

permitting a meaningful analysis. 

TPMT Allelic Determination

Genotyping was reported in thirty-four of the eligible studies (Table ). Data reported for 

individual allelic variants were too sparse to permit a meaningful allele specific evidence 

synthesis. Insufficient evidence of association was available for the outcomes of mortality, 

hospitalization, serious adverse events (SAE), health related quality of life (HQOL) and 

neutropenia. Five studies reported outcomes data associated with compound heterozygosity, so 

these participants were evaluated as homozygous carriers.57,159,160,172,176

Characteristics of included studies and risk of bias are summarized in Table 20. The majority 

(68 percent) of studies were in IBD patients. Overall, females were approximately equally

represented, although this data could not be ascertained in nine studies53,57,61,81,157,170,176,179,182 and 

in five studies females were overrepresented.89,99,171,174,190 Racial distribution was very poorly 

reported. 

In 17 studies, thiopurine treatment was restricted to AZA mostly in the doses varying from 1-

2 mg/kg/day of AZA1,46,48,52,59,70,86,89,93,170-175,179,190 while eight studies did not report AZA 

dose.50,53,61,99,157,180,182,191 One study employed 6-MP in a dose of 50mg/kg/day,54 while the rest 

used mixed thiopurines. Adverse event observation periods on thiopurine treatment varied but 

when reported were mostly greater than 2 months in duration. Nine studies did not report the 

period of on-thiopurine-treatment observation for adverse events.53,57,61,81,93,99,171,177,182 Most 

studies were cross-sectional in design, in which patients’ past thiopurine treatment and related 

adverse events were correlated with study genotyping results. These were generally rated as fair 

risk of bias. Most importantly, biased genotyping based on prior knowledge of outcomes or 

outcomes assessment based on prior knowledge of genotyping results could be ruled out only in 

five studies.1,46,48,89,177

Figure 25 depicts the distribution of studies by individual quality scoring items. Except for 

Ansari et al.’s study for which data on the homozygous carrier state was not reported, we tested 

studies for Hardy-Weinberg equilibrium (HWE) but did not consider it an item for sensitivity 

analyses.46 Six studies were not found in equilibrium,47,50,86,172,173,190. Detailed results of all 34 

studies are available in Appendix C. Below we first present the pooled results from studies that 

tested for the most common of TPMT variant alleles (TPMT*2, *3A, *3B, *3C), whether or not 

testing additional polymorphisms. Subsequently we report results from subgroups of studies 

testing specific sets of variant alleles. 
75

Table 20. Summary characteristics of TPMT allelic determination and thiopurine toxicity association studies

Characteristic

Number 

of 

studies

Studies 

Study Design

Case Control 4 81,86,181,182

Chart Review 1 157

Cross-Sectional 18 48,50,51,53,57,59,70,93,99,170-176,190,191

NonRandomized 

Intervention Study

2 46,54

Prospective 

Observational

7 1,47,61,89,177,180,186

Randomized 

Controlled Trial

1 52

Unclear whether 

prospective or 

retrospective 

design

1 179

Chronic 

Autoimmune 

Disease

AntiNeutrophil 

Cytoplasmic 

Antibody 

Associated 

Vasculitis

1 157

Autoimmune 

Dermatologic 

Conditions

2 93,170

Autoimmune 

Hepatitis

1 171

Inflammatory 

Bowel Disease

23 1,46-48,50-52,54,59,61,70,81,86,172,173,175-177,180-182,186,191

Rheumatoid 

Arthritis

2 57,179

Rheumatic 

Diseases

2 89,190

Systemic Lupus 

Erythematosus 

4 53,99,174,179

Thiopurine 

Treatment

6-MP 1 54

6-MP, AZA 5 47,176,177,181,186

AZA 25 1,46,48,50,52,53,59,61,70,86,89,93,99,157,170-175,179,180,182,190,191

Mixed Thiopurines 2 51,81

Not Reported 1 57

Allelic 

Variants 

Tested

TPMT*1, *2, *3A, 

*3B, *3C

4 173,179-181

TPMT*1, *2, *3A, 

*3C

1 81

TPMT*2, *3A 1 89
Table 20. Summary characteristics of TPMT allelic determination and thiopurine toxicity association studies 

(continued)

76

Characteristic

Number 

of 

studies

Studies 

TPMT*2, *3A, *3B, 

*3C

17 1,48,51-54,59,70,99,157,170-172,176,186,190,191

TPMT*2, *3A, *3B, 

*3C, *3D

1 182

TPMT*2, *3A, *3B, 

*3C, *3D, *4, *5, *6, 

*7, *8, *10, *14, *15

1 50

TPMT*2, *3A, *3B, 

*3C, *3D, *6

1 174

TPMT*2, *3A, *3B, 

*3C, *7, *8

1 57

TPMT*2, *3A, *3C 1 86

TPMT*3A, *3B, *3C 4 46,47,175,177

Not Reported 2 61,93

Genotyping 

Method

High Performance 

Liquid 

Chromatography

1 181

Polymerase Chain 

Reaction

28 1,47,48,51-54,57,59,61,70,81,86,89,157,170-177,179,180,186,190,191

Pyrosequencing 1 50

Not Reported 4 46,93,99,182

Outcomes 

Reported

Mortality 1 1

SAE 1 1

Anemia 2 157,170

Any Infection 3 59,170,190

Hepatitis 20 1,46-48,50-52,54,59,61,70,81,86,89,93,99,172-175

Leukopenia 25 46-48,53,54,61,70,81,89,93,99,157,170,172-177,179-181,186,190,191

Myelotoxicity 6 50-52,59,171,180

Neutropenia 3 Insufficient evidence with sparse events1,86,170

Pancreatitis 14 1,46-48,50-52,54,59,70,81,86,172,182

Thrombocytopenia 4 48,61,157,190

Withdrawal due to 

Adverse Events

5 1,46,57,170,190

Age Group

Adults 15 1,46,47,50,52,54,81,93,171,173,175,180,186,190,191

Children 3 99,172,177

Mixed 5 48,51,53,86,181

Not Reported 11 57,59,61,70,89,157,170,174,176,179,182

Setting

Inpatient 1 170

Outpatient 

Specialty Clinics

15 1,48,51,54,57,59,70,81,86,89,173,175-177,181

Not Reported 18 46,47,50,52,53,61,93,99,157,171,172,174,179,180,182,186,190,191

Region

Asia 6 53,171,174,176,179,181

Europe 20 1,46-48,50-52,54,57,59,70,86,89,170,172,173,175,180,190,191

North America 2 93,177
Table 20. Summary characteristics of TPMT allelic determination and thiopurine toxicity association studies 

(continued)

77

Characteristic

Number 

of 

studies

Studies 

Oceania 1 81

Not Reported 5 61,99,157,182,186

Risk of Bias

Good 1 1

Fair 29 46-48,50-54,57,59,61,70,81,86,89,157,170-177,179-181,190,191

Poor 4 93,99,182,186

Abbreviations: 6-MP = 6-mercaptopurine; AZA = azathioprine; TPMT = thiopurine methyltransferase

Figure 25. Risk of bias of TPMT allelic determination and thiopurine toxicity association studies

6

0

44

0

6

3

6

6

18

62

15

79

3

59

53

50

76

79

85

91

79

21

32

0

0

0

35

47

6

24

15

12

3

15

62

6

85

21

97

0% 20% 40% 60% 80% 100%

Potential for financial conflict of Interest? 

Possibility of survival bias?

Lost-to-followup reporting?

Different sources of DNA -- potential measurement bias?

Thiopurine adherence assessed? 

Gene-gene interaction assessed? 

Deviation from Hardy-Weinberg equilibrium tested?

Ambiguous results of genotyping reported? 

Reliability of genotyping method?

Genotyping method described adequately? 

Outcomes and genotyping blinded to each other? 

Participants enrolled without prior knowledge of genotype?

Genotype groups comparable?

Yes (%) No (%) Unclear (%)

Infection. Three studies of cross-sectional study design reported infection in association with 

thiopurine treatment, including 155 patients with chronic autoimmune diseases.59,170,190 No 

significant association was noted between heterozygous and noncarrier carrier states when most 

common variant alleles (TPMT*2, *3A, *3B, *3C) were tested (OR 1.37, 95 percent CI 0.22 to 

8.64). Insufficient data were available to evaluate homozygous carrier state (Figure 26). 
78

Figure 26. Meta-analysis of odds ratios of infections during thiopurine treatment of chronic autoimmune 

disease, heterozygotes versus noncarriers

Study name Odds ratio and 95% CI

Bezier, 2008 0.47 (0.02, 10.8)

Stocco, 2004 1.19 (0.05, 27.0)

Tani, 2009 5.37 (0.19, 151.4)

1.37 (0.22, 8.64)

Events / Total n

Heterozygotes Non-carriers

9 / 31

3 / 40

4 / 76

0 / 2

0 / 4

0 / 1

0.01 0.1 1 10 100

Favors 

Non-carriers

Favors 

Heterozygotes

Withdrawal due to adverse events. Four studies with an analyzable sample size of 317 

reported this outcome. There were no withdrawals due to adverse events in 166 patients on 

azathioprine for 4 months administered in doses not guided by prior TPMT status determination.1

The remaining studies of cross-sectional design, that tested for the most common variant alleles 

(TPMT*2, *3A, *3B, *3C) in 151 participants demonstrated no significant difference between 

heterozygous carriers and noncarriers in withdrawal due to adverse events on thiopurine 

treatment (OR 3.46, 95 percent CI 0.50 to 23.80) (Figure 27).57,170,190 Insufficient data were 

available to investigate the homozygous carrier state.

Figure 27. Odds ratio of withdrawal due to adverse events during thiopurine treatment of chronic 

autoimmune disease, heterozygotes versus noncarriers

Study name Odds ratio and 95% CI

16.0 (1.87, 136.7)

1.07 (0.06, 18.6)

1.05 (0.04, 27.0)

3.46 (0.50, 23.8)

 

Events / Total n

Heterozygotes

3 / 35

15 / 31

18 / 76

3 / 5

1 / 2

0 / 1

Favors 

Non-carriers

Favors 

Heterozygotes

Corominas, 2003

Bezier, 2008

Tani, 2009

0.01 0.1 1 10 100

Non-carriers

Myelotoxicity. Myelotoxicity was reflected in at least two cell lines (e.g. leukopenia and 

thrombocytopenia). The pooled estimate from four studies in 193 participants testing for the 

most common allelic variants (TPMT*2, *3A, *3B, *3C) demonstrated no significant difference 

between heterozygous carriers and noncarriers (OR 0.77, 95 percent CI 0.22 to 2.65).50,51,180,218
79

When homozygous carriers were compared with noncarriers, the nonsignificant odds ratio was 

2.20 (95 percent 0.15 to 32.80).50,171 (Figure 28 and Figure 29). Data were insufficient to derive a 

pooled estimate for the difference in rates of myelotoxicity between the two carrier states.50 

Figure 28. Odds ratio of myelotoxicity during thiopurine treatment of chronic autoimmune disease, 

heterozygotes versus noncarriers

Figure 29. Odds ratio of myelotoxicity during thiopurine treatment of chronic autoimmune disease, 

homozygotes versus noncarriers 

Study name Odds ratio and 95% CI

Events / Total n

Favors 

Non-carriers

Favors 

Heterozygotes

Hindorf, 2006 0.82 (0.15, 4.56)

Joji, 2003 3.27 (0.10, 103.4)

Stocco, 2005 0.71 (0.04, 14.1)

Stocco, 2004 0.23 (0.01, 4.52)

Heterozygotes Non-carriers

3 / 6 22 / 40

0 / 2 1 / 25

0 / 5 7 / 65

0 / 4 13 / 40

0.77 (0.22, 2.65)

0.01 0.1 1 10 100

Study name Odds ratio and 95% CI

Events / Total n

Hindorf, 2006 0.82 (0.15, 4.56)

Tamori, 2007 15 (0.39, 576.7)

Homozygotes Non-carriers

3 / 6 22 / 40

1 / 1 1 / 8

2.20 (0.15, 32.8)

Favors 

Non-carriers

Favors 

Homozygotes

0.01 0.1 1 10 100
80

Leukopenia. Sixteen studies including a total of 1483 analyzable patients of whom 281 (19 

percent) experienced leukopenia tested the four common variant alleles with or without 

additional variants.48,53,54,70,157,170,172-174,176,179-181,186,190,191Odds of leukopenia were significantly 

higher when genotypes were heterozygous for TPMT mutations compared with noncarriers or 

wild type, and individual study results were quite consistent across studies (OR 4.62, 95 percent 

CI 2.34 to 9.16) (Figure 30). Meta-regression indicated significant differences in odds ratios with 

the method of genotyping (PCR versus HPLC). The study by Jae Hak et al., 2008 was the only 

study that used HPLC method for genotyping. A dose response relationship was indicated when 

homozygous carriers were compared with noncarriers (OR 18.60, 95 percent CI 4.12 to 83.60) 

(Figure 31).54,172,173,176,190 However, no significant differences in likelihood of leukopenia 

between the two carrier states were observed from pooling four small studies (Figure 

32).54,172,173,176

Figure 30. Odds ratio of leukopenia during thiopurine treatment of chronic autoimmune disease, 

heterozygotes versus noncarriers 
81

Figure 31. Odds ratio of leukopenia during thiopurine treatment of chronic autoimmune disease, 

homozygotes versus noncarriers 

Figure 32. Odds ratio of leukopenia during thiopurine treatment of chronic autoimmune disease, 

homozygotes versus heterozygotes 

Anemia. Two studies in 140 patients did not demonstrate a significant difference in 

incidence of anemia when patients heterozygous for the common TPMT allelic variants were 

compared with noncarrier patients (OR 1.44, 95 percent CI 0.36 to 5.82) (Figure 33). There were 

no data pertaining to TPMT homozygosity for this outcome.

Study name Odds ratio and 95% CI

Events / Total n

De Ridder, 2006 6.33 (0.22, 185.3)

Derijks, 2004 39.0 (1.06, 1436)

Zelinkova, 2006 81.4 (3.08, 2147)

Tani, 2009 3.09 (0.12, 82.8)

Homozygotes Non-carriers

0 / 3 1 / 67

1 / 1 1 / 20

1 / 1 8 / 238

0 / 1 7 / 76

18.6 (4.12, 83.6)

Favors 

Non-carriers

Favors 

Homozygotes

0.01 0.1 1 10 100

Hibi, 2003 1 / 1 5 / 74 37.9 (1.38, 1045)

Study name Odds ratio and 95% CI

Events / Total n

De Ridder, 2006 0.14 (0.003, 5.95)

Derijks, 2004 3.00 (0.08, 115.3)

Zelinkova, 2006 17.6 (0.59, 524.1)

Homozygotes Heterozygotes

0 / 3 1 / 2

1 / 1 2 / 4

1 / 1 3 / 23

1.66 (0.22, 12.7)

Favors 

Heterozygotes

Favors 

Homozygotes

0.01 0.1 1 10 100

Hibi, 2003 1 / 1 6 / 7 0.69 (0.02, 26.9)
82

Figure 33. Odds ratio of anemia during thiopurine treatment of chronic autoimmune disease, heterozygotes 

versus noncarriers

Study name Odds ratio and 95% CI

Events / Total n

Bezier, 2008 0.65 (0.03, 15.2)

1.44 (0.36, 5.82)

Stassen, 2009 1.75 (0.37, 8.30)

Heterozygotes

0 / 2 7 / 31

3 / 7 30 / 70

Favors 

Non-carriers

Favors 

Heterozygotes

0.01 0.1 1 10 100

Non-carriers

Thrombocytopenia. Based on a total of 256 patients in three studies, the pooled odds ratio 

for thrombocytopenia was 3.43 (95 percent CI, 0.52 to 22.60) when heterozygous genotypes 

were compared with noncarriers or wild type (Figure 34). Insufficient data were available to 

investigate the homozygous carrier state.

Figure 34. Odds ratio of thrombocytopenia during thiopurine treatment of chronic autoimmune disease, 

heterozygotes versus noncarriers

Study name Odds ratio and 95% CI

Events / Total n

Stocco, 2007 6.33 (0.22, 185.3)

Tani, 2009 16.8 (0.47, 605.2)

3.43 (0.52, 22.6)

Stassen, 2009 0.73 (0.04, 14.0)

Heterozygotes

0 / 3 1 / 67

0 / 1 1 / 76

0 / 7 8 / 101

Favors 

Non-carriers

Favors 

Heterozygotes

0.01 0.1 1 10 100

Non-carriers

Hepatitis or elevated hepatic transaminases. Based on a total of 984 patients in ten studies, 

comparing heterozygous carriers with noncarriers, the pooled odds ratio for hepatitis or elevated 

hepatic transaminases was 1.35 (95 percent CI 0.59 to 3.11) (Figure 35).1,48,50-52,54,59,70,173,174

Meta-regression suggested a significant relationship between the genotyping method and the 

outcome (p<0.05). The study by Hindorf et al., which used a pyrosequencing method as opposed 

to the PCR used in the other studies, showed a nonsignificant point estimate in favor of 

heterozygosity because of zero events in the heterozygous group.50 When homozygous 

participants were compared with noncarriers, similar nonsignificant odds were noted (

Figure 36). Data were insufficient to compare the two carrier states.
83

Figure 35. Odds ratio of hepatotoxicity during thiopurine treatment of chronic autoimmune disease, 

heterozygotes versus noncarriers

Figure 36. Odds ratio of hepatotoxicity during thiopurine treatment of chronic autoimmune disease, 

homozygotes versus noncarriers

Pancreatitis. When heterozygous carriers were compared with noncarriers, no 

significant difference in incidence of patients with pancreatitis was noted in ten studies of a 

total of 807 participants (OR 1.20, 95 percent CI 0.49 to 2.97) (Figure 37).

Figure 371,48,50-52,54,59,70,172,182 However, meta-regression indicated a significant relationship 

with the overall risk of bias score with poor quality studies tended to report lower rates of 
84

pancreatitis in heterozygous participants compared with noncarriers.182With fewer studies, no 

significant difference was noted in pancreatitis events when homozygous carriers were compared 

with noncarriers (Figure 38). Data were insufficient to compare the two carrier states of 

homozygosity and heterozygosity.

Figure 37. Odds ratio of pancreatitis during thiopurine treatment of chronic autoimmune disease, 

heterozygotes versus noncarriers
85

Figure 38. Odds ratio of pancreatitis during thiopurine treatment of chronic autoimmune disease, 

homozygotes versus noncarriers

Subgroup meta-analyses by specific sets of alleles tested. We also analyzed subgroups of 

studies testing specific sets of variant alleles (Table 21). Concordant with the main meta-analysis 

above in which studies that tested for TPMT*2, *3A, *3B, *3C with or without additional 

variants were pooled, studies that exclusively tested for TPMT *2, *3A, *3B, * 3C also 

demonstrated a pooled odds of leukopenia significantly greater with either carrier states in 

comparison with noncarriers. For other outcomes, comparatively fewer studies contributed 

subgroup specific evidence, with odds ratios failing to reach statistical significance. 

Table 21. Pooled odds ratios of outcomes on thiopurine treatment between TPMT variant allele carriers and 

noncarriers, as well as between the two carrier states (allele specific subgroups)

TPMT 

alleles 

tested

Outcome

Homozygous 

carriers vs. 

noncarriers

Heterozygous carriers vs. 

noncarriers 

Homozygous 

carriers vs. 

heterozygous 

N of 

studies 

metaanalyzed

Odds 

Ratio 

(95% 

CI) 

N of studies 

meta-analyzed

Odds 

Ratio 

(95% CI) 

N of 

studies 

metaanalyzed

Odds 

Ratio 

(95% 

CI) 

TPMT 

*2, *3A, 

*3B, * 

3C 

Infection X 359,170,190 1.37 (0.22 

to 8.64)

X

WDAE X 31,170,190 1.06 (0.12 

to 9.06)

X

Myelotoxicity 

(11% of 

patients with 

events)

X 251,59 0.40 (0.05 

to 3.33) 

X
86

TPMT 

alleles 

tested

Outcome

Homozygous 

carriers vs. 

noncarriers

Heterozygous carriers vs. 

noncarriers 

Homozygous 

carriers vs. 

heterozygous 

Leukopenia

(13% of 

patients with 

events)

454,172,176,

190

12.50 

(2.29 

to 

68.00)

Favors 

noncar

rier

1248,53,54,70,99,157,170,

172,176,186,190,191

5. 60 

(2.35 to 

13.30)

Favors 

noncarrie

rs

354,172,176 0.68 

(0.08 

to 

5.70)

Anemia X 2157,170 1.44 (0.36 

to 5.82)

X

Thrombocyto

penia

X 348,157,190 3.43 (0.52 

to 22.60)

X

Hepatitis or 

raised hepatic 

transaminase

s

21,54 4.98 

(0.44 

to 

56.30)

81,48,51,52,54,59,70,99 1.11 (0.39 

to 3.10)

X

Pancreatitis 31,54,172 3.27 

(0.49 

to 

21.60)

848,51,52,54,59,70,172 2.22(0.78 

to 6.32)

X

TPMT 

*3A, 

*3B, *3C

Leukopenia X 246,47,177 5.13 (0.82 

to 32.00)

I

2 > 50% 

and pvalue for 

test of 

heteroge

neity 

<0.10

X

Hepatitis or 

raised hepatic 

transaminase

s

246,47 0.98 (0.18 

to 5.33)

X

Pancreatitis X 246,47 0.48 

(0.06, 

3.60)

X

TPMT*1

, *2, 

*3A, 

*3B, * 

3C

Leukopenia X 4173,179-181 5.23 (1.99 

to 13.70)

X

Notes: underlined odds ratios reached statistical significance. 

Abbreviations: TPMT = thiopurine methytransferase; vs. = versus; X =absent or insufficient evidence 

Key points. Thirty studies contributed to quantitative syntheses.1,46-48,50-54,57,59,70,99,157,170-

177,179-182,186,190,191,219
87

A dose response relationship was suggested between TPMT genotypic status and leukopenia. 

In studies testing TPMT *2, *3A, *3B, * 3C plus/minus additional variants, homozygosity for 

the variant alleles yielded the highest odds ratio for leukopenia when compared with noncarrier 

status. Lower, but still significantly increased odds were seen for heterozygous patients versus 

noncarriers. However, direct comparison between the two carrier states did not yield statistically 

significant results. 

For all other outcomes of mortality, hospitalization, serious adverse events (SAE), health 

related quality of life (HQOL), neutropenia, infection, withdrawal due to adverse events, 

myelotoxicity, anemia, thrombocytopenia, hepatitis or elevated hepatic transaminases, 

and pancreatitis, evidence was either absent, insufficient or lacked power to demonstrate 

significant differences between heterozygous and homozygous carriers in comparisons with 

noncarriers, and between themselves.
88

Key Question 4: What are the costs of determining TPMT enzyme 

activity and/or genotyping for patients with chronic autoimmune 

disease being considered for thiopurine-based therapy (e.g., costs of 

testing, costs of care, and costs of treating drug-associated 

complications)?

Eleven studies relevant to question 4 were included (Table 22). The studies were conducted 

in Canada,204,207,210 Korea,208 Europe,83,204,211 New Zealand,205 and the U.S.A.40,206,212 Five of the 

included studies were costing studies,40,204,207,210,211 while the rest were cost-effectiveness 

analyses. All of the studies examined the costs associated with azathioprine (AZA). 

Table 22. Characteristics of TPMT testing, care and drug-associated complications costs studies

Study Country of

conduct Type of study Population characteristics Perspective

Prakshar 

199540

USA Costing study Theoretical population 

of RA patients receiving 

AZA

NR

Tavadia 

2000210

Canada Costing study Based on one BP 

patient with AZA 

toxicity

NR

Marra

2002209

Canada CEA Theoretical population 

of RA and SLE patients 

receiving AZA

Third-party payer

Oh

2004208

Korea CEA Theoretical cohort of 

adults with moderate to 

severe RA or SLE 

receiving AZA

Societal

Winter

200483

Scotland CEA Theoretical population 

of 1000 patients 

receiving AZA for IBD

NR

Dubinsky

2005206

USA CEA Theoretical population 

of adults with moderate 

to severe CD patients 

treated with steroids 

and AZA

Third-party payer

Sayani 

2005207

Canada Costing study IBD patients 

participating in a 

randomized controlled 

trial

NR

Priest

2006205

New 

Zealand

CEA Theoretical population 

of 1000 Caucasian 

patients with moderate 

to severe IBD receiving 

AZA

Payer’s perspective 

(New Zealand 

government and IBD 

patients)

Compagni 

2008204

Italy SR of costing 

studies; 

costing study

Theoretical population 

of RA or IBD patients 

receiving AZA

NR 

Gurwitz 

2010211

UK and 

Spain

Costing study RA or IBD patient 

cohorts from Spain and 

the UK

NR
Table 22.. Characteristics of TPMT testing, care and drug-associated complications costs studies (continued)

89

Study Country of

conduct Type of study Population characteristics Perspective

Hagaman 

2010212

USA CEA Theoretical population 

of IPF patients

NR

Abbreviations: AZA = azathioprine; BP = bullous pemphigoid; CD = Crohn’s disease; CEA = cost-effectiveness analysis; IBD = 

inflammatory bowel disease; IPF=idiopathic pulmonary fibrosis; NR = not reported; RA = rheumatoid arthritis; SLE = systemic 

lupus erythematosus; SR = systematic review; UK=United Kingdom, USA = United States of America.

One study based its information on a patient with bullous pemphigoid,210 while the other 

studies based their information on rheumatoid arthritis,40 inflammatory bowel disease,83,205,207

Crohn’s disease,206 rheumatoid arthritis or inflammatory bowel disease,204,211 idiopathic 

pulmonary fibrosis,212 and rheumatoid arthritis or systemic lupus erythematosus208,209 patients. 

For the costing analysis, one of the studies used the societal perspective,208 one used the payer’s 

perspective,205 two used the third-party payer perspective,206,209 and the rest did not report their 

perspective.

Costs of adverse events and care. One included study provided costs associated with 

treatment failure.209 This study obtained information from the Canadian provincial guide to 

medical fees. Treatment failure with AZA was estimated to cost $578.04 USD (2009) per 

rheumatoid arthritis or systemic lupus erythematosus patient. Another study provided a cost 

estimate for idiopathic pulmonary fibrosis disease progression, which was $15,805.94.212.

Costs of testing. Eight studies reported nine cost estimates for TPMT genotyping (Appendix 

C, Table C-26)

83,204-212 Most of the genotyping cost information was obtained from public 

laboratories, while one was based on data from a hospital208 and another was from a literature 

search of studies reporting costs from private laboratories.206 The cost of obtaining a genotype 

test per patient ranged from $28.03 USD to $617.03 USD (2009). The highest cost was to obtain 

the TPMT genotype test from private laboratories.206 Excluding the costing item from the private 

laboratory, the average cost for the genotype test per patient was $89.94 USD (Figure 39).

One study also provided other costing estimates related to TPMT genotyping.204 The 

pharmacogenetic kits for detecting TPMT*1, *2, *3A, *3B, *3C cost $46.25 USD/patient, 

pharmacogenetic kits for detecting TPMT*2, *3A, *3B, *3C cost $29.43 USD/patient.204
90

Figure 39. Costs of genotype test per patient from public laboratory data in USD (2008), in chronological 

order 

Four studies provided five estimates for the cost of TPMT phenotyping,205,207,211,212 which 

were obtained from laboratories or the government. The cost of obtaining the TPMT phenotype 

test per patient ranged from $46.36 to $320.98. It was not reported where the highest cost was 

obtained. Excluding the highest cost, the average cost for TPMT phenotyping per patient was 

$53.13 (Figure 40).

Figure 40. Costs of phenotype test per patient from public laboratory data in USD (2009), in chronological 

order 

Costs of treating azathioprine-related complications. Seven of the included studies 

reported eight cost estimates related to the costs associated with treating AZA-related 

complications (Appendix C, Table C-27).40,83,205,208-210,212 All costs were obtained from hospitals 

or from government agencies. In order to compare across studies, the one-time cost of adverse 

$100.88 $100.88

$60.02

$131.51

$94.48

$-

$20.00 

$40.00 

$60.00 

$80.00 

$100.00 

$120.00 

$140.00 

Cost (USD 2009)

Tavadia 2000 Marra 2002 Winter 2004 Oh 2004 Priest 2006

$-

$10.00 

$20.00 

$30.00 

$40.00 

$50.00 

$60.00 

$70.00 

Sayani 2005 Priest 2006 Gurwitz 2009

USD (2009)
91

events associated with AZA were computed.204 This cost was substantial and ranged from 

$1,366.82 to $7,110.02 USD (2009) (Figure 41). The average one-time cost of AZA-induced 

adverse events was $4,019.29. 

Figure 41. One-time cost estimates of adverse events associated with AZA in USD (2009), in chronological 

order

One study reported the costs of leucopenia leading to death, which was $15,691.46.212 In 

addition, another study reported two cost estimates for the average cost per identified TPMTdeficient individual, which was $11,982.10 in a hospital in the United Kingdom and $11,714.92 

in UCB Pharma in Spain.211

Key points. The costs related to TPMT genotyping range from $28.03 to $617.80 USD 

(2009). This heterogeneity was likely due to different methodological choices.

TPMT phenotyping usually costs less than TPMT genotyping and ranges from $46.36 to 

$320.98. Similar to TPMT genotype testing costs, the heterogeneity observed across studies 

likely reflected methodological choices. 

The one-time cost of adverse events associated with AZA was substantial, ranging from 

$1,366.82 to $7,110.02 across studies.

The costs related to identifying a TPMT-deficient individual is approximately $11,848.51.
92

Survey of Laboratories Conducting Analyses of TPMT Enzymatic 

Activity and Genotyping

Responses were received from six out of the seven laboratories that were sent a link to the 

online questionnaire: three in Canada; and three in the United Kingdom. Results are summarized 

in Table .

Among the responding laboratories, yearly volumes of TPMT measurements range from 50 

to 1500 for allelic determinations, and from 600 to 19,000 for enzymatic activity analyses. 

Analytical methodologies. One laboratory conducts only genotyping and two laboratory 

conduct only phenotyping. The two labs also refer samples to other laboratories for genotyping. 

One lab refers out low enzymatic activity (i.e. below 10 U/g Hb) specimens for confirmatory 

genotyping. The other three laboratories conduct both types of analyses. 

Between one and four specific genotypes are determined: TPMT*2 (four laboratories); 

TPMT*3A (six laboratories); TPMT*3B (four laboratories); and TPMT*3C (five laboratories). 

Genotype determination is performed by polymerase chain reaction/restriction fragment length 

polymorphism (PCR/RFLP), genetic sequencing and site-directed PCR. The methods are equally 

popular, each being used by two laboratories.

TPMT enzymatic activity is determined on red blood cell (RBC) lysates using enzymatic 

assay followed by high performance liquid chromatography (HPLC) (three laboratories), or mass 

spectrometry (one laboratory). Three laboratories report results as nmol/g Hb/h, and one 

laboratory reports results as pmol/h/mg Hb. Both are numerically equivalent to 1 U/g Hb, the 

unit we are using in the present report. 

Preanalytical requirements. All laboratories collect whole blood, with EDTA 

anticoagulant. Specimens are stored at 4oC or room temperature for between one and eight days.

One of the laboratories requires a list of patients’ current medications prior to conducting 

analyses.

Quality control. All six participating laboratories report a procedure for internal quality 

control, which involves the inclusion of positive and negative controls within each run, in some 

cases in duplicate (two laboratories report conducting multiple runs per day). Results of internal 

quality control procedures are similar across participating laboratories, with enzymatic analysis 

repeatability ranging from three to ten percent within runs, and from five to ten percent between 

runs. Two of the laboratories conducting both genotyping and phenotyping analyses reported 

between 95 and 100 percent concordance between genotyping and phenotyping overall. One 

laboratory clarified that concordance was lower (60 percent) for intermediate carriers.

Positive and negative internal control samples are obtained from known staff, patients or 

pooled known samples.

Two of the six laboratories participate in external quality assurance, consisting of either 

specimen exchange with a comparable laboratory or participation in a formal external quality 

assurance program. The United Kingdom National External Quality Assessment Service has 

received funding for global availability for TMPT activity analyses in 2010.

Reporting of results. All of the laboratories report abnormal results to the responsible 

physician; however, one laboratory only reports homozygous (severe deficiency) results. 

Furthermore, of the five laboratories that perform enzymatic activity analyses, four also highlight 

suspected heterozygous patients. 

Enzymatic activity reference intervals differ between laboratories. Three laboratories employ 

three reference intervals with two of the laboratories defining the intervals as: (low, intermediate 
93

and normal) and one laboratory defining them as: (normal, heterozygous, deficient); One 

laboratory uses four reference intervals (deficient, low, normal and high). Two laboratories 

define low activity as below 10 U/g Hb, while the third defines low activity as between 6 and 34 

U/g Hb. Two laboratories define normal activity as above 35 U/g Hb, while three other 

laboratories define normal activity as between 26 and 50 U/g Hb; between 25 and 120 U/g Hb; 

and between 38.5 and 62.5 U/g Hb respectively. The two laboratories that include a deficient 

interval define deficient TPMT activity as below 5 U/g Hb and below 19.2 U/g Hb. The 

laboratory that includes a heterozygous interval defines heterozygous as between 19.2 – 33.7 U/g 

Hb. Finally the laboratory that includes a high reference interval defines high TPMT activity as 

above 80 U/g Hb. In each case, reference intervals have been determined by the individual 

laboratories from large population-based studies. 

Analysis turnaround times range from one day to less than three weeks, with four labs 

reporting a turnaround time of a week.

Costs associated with testing. Two laboratories reported on costs of testing. One reported a 

cost of £29 for both TPMT enzymatic activity plus genotyping if required for low activity values. 

The other charges £33 for TPMT enzymatic activity and £96 for genotyping.

Key points. A questionnaire, developed with expert input, was completed by six laboratories 

that provide TPMT analytic services, in Canada and the United Kingdom. 

The preferred specimen type across laboratories is whole blood, with EDTA anticoagulant. 

Specimens are stored at 4o

C or room temperature for between one and eight days.

Each laboratory follows a procedure for internal quality control. Two also participate in 

external quality control programs.

Enzymatic analysis repeatability ranges from three to ten percent within runs, and from five 

to 20 percent between runs. 

Genotyping concordance with phenotyping up to 100 percent has been reported, although 

lower concordance was reported for intermediate carriers.

Analysis turnaround times range from one day to less than three weeks.

All laboratories report abnormal results to the responsible physician on an urgent basis.
94

Table 23. Laboratory questionnaire responses

Questionnaire item Answer N affirmative 

responses

Thiopurine 

methyltransferase (TPMT) 

analyses 

Phenotyping, with low enzymatic activity 

specimens referred to external laboratory 

for genotyping

2

Genotyping only 1

Both genotyping and phenotyping 3

TPMT analytical method Enzymatic activity 

assay

Mass spectrometry 1

HPLC 3

Genetic analysis PCR/RFLP 2

Sequencing 2

Site-directed PCR 2

Alleles targeted in 

mutation specific genetic 

analysis

TPMT*2, *3A, *3B, *3C 3

TPMT*2, *3A, *3C 1

TPMT*3A, *3B, *3C 1 (external 

lab)

TPMT*3A 1

Yearly TPMT testing volume Enzymatic activity 

assay

600 1

3000 1

4000 1

15,000 1

19,000 1

Genetic analysis 50 1

550 1

1500 1

Unknown referrals 

for low enzymatic 

activity samples

1

Preferred specimen for 

analysis

EDTA whole blood 6

Preanalytical requirements 

a) Specimen stability prior 

to analysis

24-48 Hr at (4°C) 1

Stable 4 days (temperature not specified) 1

Stable 5 days at room temperature 1

Stable 1 week at room temperature 1

Stable 8 days refrigerated (4°C) 1

N/A 1

Preanalytical requirements 

b) List of patients’ current 

medications

No 5

Yes 1

Internal quality control Positive and negative controls with each 

run 

4

(2 specify 

duplicates)

Proficiency of TPMT 

analyses

Enzymatic activity 

analysis repeatability

Within runs:

3-10% 4
Table 23. Laboratory questionnaire responses (continued)

95

Questionnaire item Answer N affirmative 

responses

Between runs: 

5-20% 5

Genotyping 

concordance: 

100% 

(comparator not 

reported)

1

97% -100% 

(compared with 

phenotyping, 

overall)

2

60% for

intermediate 

carrier samples

1

External quality control Yes (externally supplied / exchanged 

unknowns)

2

No 4

Abnormal results called to 

the physician

Yes 6

(1 calls only 

for 

homozygotes)

No 0

Reference intervals for 

TPMT enzymatic activity

Deficient <5 U/g Hb 1

Deficient <19.2 U/g Hb 1

Low 6-34 U/g Hb 1

Low <10 U/g Hb 2

Carrier 11-25 U/g Hb 1

Intermediate 10-35 U/g Hb 1

Heterozygous 19.2-33.7 U/g Hb 1

Normal 26-50 U/g Hb 1

Normal 38.5-62.5 U/g Hb 1

Normal 35-79 U/g Hb 1

Normal >35 U/g Hb 1

High >80 U/g Hb 1

Highlight suspected 

heterozygous patients from 

enzymatic assay 

Yes 4

No 1

N/A 1

Turn around time for 

testing

1-2 days 1

1 week 4

<3 weeks 1

Charges associated with 

laboratory testing

Enzymatic activity - £33 / Genotyping - £96 1

Enzymatic activity - £29 (the price includes 

genotyping if required)

1

Abbreviations: EDTA = ethylenediaminetetraacetic acid; HPLC = high performance liquid chromatography; N/A = not 

applicable; PCR/RFLP = polymerase chain reaction/restriction fragment length polymorphism TPMT = thiopurine 

methyltransferase; U/g Hb = Units per gram of hemoglobin = nanomoles of product per hour per gram hemoglobin.

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Chapter 4. Discussion

This review was nominated by the American Association for Clinical Chemistry (AACC), 

and commissioned by the Agency for Healthcare Research and Quality (AHRQ), to examine 

testing for thiopurine methyltransferase (TPMT) enzymatic activity (phenotype), and allelic 

polymorphism determination (genotype) in chronic autoimmune disease, with an overall view 

potentially to optimize use of thiopurine medications. The objectives were to examine the 

analytical aspects of TPMT status determination (genotype or phenotype); and resultant change 

in patient management and clinical outcomes of thiopurine toxicity in light of pretreatment 

knowledge of TPMT status; and costs associated with TPMT testing, along with costs of adverse 

events arising from thiopurine toxicities. Inclusion of evidence to meet these objectives was 

restricted to chronic autoimmune disease populations. 

TPMT Status Determination

TPMT status may be determined through either genetic or TPMT enzymatic activity analysis. 

Genetic analysis involves detection of variant alleles coding for TPMT enzymes with reduced 

enzymatic activity, while enzymatic assays are able to determine directly the activity of the 

TPMT enzyme. Both genotyping and phenotyping can be determined from routine blood 

specimens, as white blood cells providing the required genetic material and red blood cells as the 

source of the TPMT enzyme. From a clinical laboratory perspective, determination of TPMT 

status encompasses preanalytical, analytical and postanalytical phases of testing.

Preanalytical testing requirements are often overlooked while focusing on analytical 

performance. However, the majority of errors in laboratory medicine occur during the 

preanalytical phase.220 The preanalytical phase of testing covers from the moment a specimen is 

collected to the point that it is analyzed, and includes sample collection method, anticoagulant 

used, transportation conditions, time between specimen collection and analysis, storage, 

specimen preparation, and preanalysis storage time and conditions. If appropriate preanalytical 

conditions are not met, then significant error can be introduced. These sources of error are often 

not recognized, as they are not identified by routine quality control monitoring of the assay. We 

therefore included examination of relevant preanalytical requirements, and potential 

confounders.

The analytic phase of testing involves the actual analysis of enzymatic activity or detection of 

variant TPMT alleles. Each method has individual analytical performance characteristics 

including precision, reproducibility, and diagnostic sensitivity and specificity, which are 

reviewed here.

Postanalytical requirements generally involve procedures to report results.

Preanalytic Requirements and Sample Stability 

Thirteen studies examined relationships between storage conditions and TPMT enzymatic 

activity, with mixed results.108,115,116,118,121,124,127-129,131,134,136,137 Six studies reported TPMT to be 

stable at room temperature in anticoagulated whole blood for periods up to seven 

days,108,118,121,124,127,134 whereas in one study, reported only as an abstract, TPMT activity 

decreased by 25 percent over 24 hours.137 Similarly, in red blood cell (RBC) lysate stored at -
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20°C for 3 months, two studies reported TPMT activity to be stable,108,124 while one study 

reported a seven percent decrease.128 Four studies reported that TPMT activity was stable in 

heparinised whole blood,127,134, EDTA whole blood124, or an unspecified anticoagulant136 at 4°C. 

When RBC lysate was stored at -80°C, TPMT enzymatic activity was reported to be stable for up 

to 25 days, whereas a 15 percent decrease in activity was measured after 16 months.128,129,131

One explanation for these disparate results is that only one study was actually designed to 

evaluate the effect of storage on TPMT activity.137 The available data suggests that TPMT 

activity is stable in EDTA and heparin anticoagulated whole blood for up to 7 days at room 

temperature or 4°C. RBC lysate is stable for 3 months at -20 °C. Longer storage should be at 

-80°C, although in the range of 15 percent of TPMT enzymatic activity may be lost after 16 

months. Repeated freeze-thaw cycles were reported to decrease the results by up to 16 percent, 

although the drop was not statistically significant.115

No studies were identified that addressed any preanalytical requirements for TPMT allelic 

polymorphism determination. However, since preanalytical requirements are common for 

genetic testing, previously published guidelines can be used. The Clinical and Laboratory 

Standards Institute (CLSI) has published excellent guidelines covering all preanalytical 

requirement for collection, transportation, preparation and storage of specimens for genetic 

testing.221

Six of the seven laboratories asked to participate in the survey returned responses, three from 

Canada and three from the United Kingdom. Among the responding, yearly volumes ranged 

from 50 to 1500 allelic determinations and 600 to 19,000 enzymatic TPMT determinations.

TPMT Variation Amongst Patient Populations

Gender, age, and race. All studies reported no gender difference for TPMT. One study of a 

small sample size reported a difference between TPMT enzyme activity of whites and mixed 

race that was not statistically significant.129 Of ten studies, a single report showed a significant 

difference (p less than 0.001) in TPMT enzymatic activity between 192 children (12.0 U/mL 

RBCs (range 0.6 to 25.4 U/mL RBC)) and 959 adults (12.9 U/mL RBCs (range 0.2 to 24.6 U/mL 

RBCs)).111 However this difference was small and not clinically relevant. Two studies observed 

no significant differences in TPMT activity across races, including blacks, whites, mixed races, 

and Japanese.109,129 However, more races with appropriate sample sizes should be included to 

confirm the lack of racial differences. One large study published in July 2004 in the journal 

Pharmacogenetics was excluded from the review on population. It analyzed 1200 healthy 

German individuals and demonstrated a statistically significant difference in TPMT activity 

between males and females. They also showed a statistial difference between smokers vs nonsmokers; male and female smokers. However, clinically the differences are likely unimportant. 

Coadministered drugs. Fifteen drugs (5-aminosalycilate, sulfasalazine, mesalazine, 

azathioprine, mesalamine, ac-5-aminosalicylate, syringic acid, prednisone, prednisolone, 6-

methylprednisolone, cyclophosphamide, methotrexate, trimethoprimsulphamethoxazole, SKF 

525-A, 3,4-dimethoxy-5-hydroxybenzoic acid, trimethoprim, vincristine, dexamethasone, Lasparaginase) have been evaluated in ten studies.102,104,105,110,114,117,119,120,128,131 Only six of the 

studies were conducted in vivo,104,117,119,120,131 in which no clinically relevant interactions were 

demonstrated.

Hematocrit. Three studies investigated the effect of hematocrit on TPMT enzymatic 

activity.104,121,126 Two reported a positive correlation of hematocrit with TPMT enzyme 
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activity121,126 and one104 observed no difference when comparing high and low hematocrit levels. 

Although two studies did demonstrate a correlation of hematocrit with TPMT activity, the effect 

was small (less than 7 percent in the normal hematocrit range) and likely not clinically 

relevant.126 Standardizing TPMT measurement to grams of hemoglobin or milliliters of packed 

RBCs should correct for any significant effect of hematocrit on TPMT measurement.

Morbidities. Two studies assessed the effect of concomitant diseases on TPMT 

activity.104,106 Inflammatory bowel disease (ulcerative colitis, Crohn disease, or indeterminate 

colitis), autoimmune hepatitis, multiple sclerosis, myasthenia gravis, pemphigus and chronic 

renal failure were shown to influence TPMT activity. Although the differences between disease 

groups showed statistical significance, the differences were minor and not clinically relevant, 

with the exception of patients requiring dialysis. Patients with renal failure showed elevated 

TPMT enzymatic levels prior to hemodialysis, which dropped by approximately 50 percent 

following hemodialysis to levels comparable to normal individuals’. The mechanism responsible 

for the elevated TPMT activity prehemodialysis is unclear, but may involve unidentified TPMT 

activating uremic compounds.106 Although there are no comparative studies of harms in dialysis 

population directly evaluating TPMT testing pre- and postdialysis, the available evidence 

suggests that dialysis patients should be measured postdialysis, as the levels most closely match 

those that would otherwise be seen in them as healthier individuals. Measurement of TPMT 

activity prior to dialysis may result in falsely identifying a low/absent or intermediate 

metabolizer as a normal metabolizer, potentially placing them at increased risk of drug toxicity. 

The remaining disease states studied to date are organ transplant and acute lymphoblastic 

leukemia, which were not included in this review. 

Analytic Performance

The enzymatic measurement of TPMT was originally developed by Weinshilboum et al134

and has since undergone only minor modifications. In brief, RBCs are concentrated by 

centrifugation, washed, resuspended and lysed to release the TPMT enzyme. The lysate is added 

to a buffered solution of radioactively labeled S-adenosyl-L-[

14C]methionine and substrate 6-

mercaptopurine (6-MP). TPMT methylates 6-MP to form radioactively labeled 6-methylMP 

which can then be measured. Modifications include use of 6-thioguanine monophosphate (6-TG) 

as substrate, or nonradioactive detection by high performance liquid chromatography (HPLC). 

The rate of product formation is dependent upon TPMT enzymatic activity, and is independent 

of the detection method - radiochemical or HPLC. Enzymatic assays using either 6-MP or 6-TG, 

regardless of the detection method (radiochemical or HPLC) were reasonably precise, with interassay coefficients of variance (CVs) of less than 10 percent in all cases. With an analytical 

coefficient of variance less than 50 percent of the biological variability, the amount of variation 

added to the true test variability is 11.8 percent.222 In comparison with other enzymatic assays, 

the currently achievable intra-laboratory CV for TPMT enzymatic analysis is better than the 

minimal acceptable performance for routine enzymatic analysis specified by the U.S. 

Department of Health and Human Services: Clinical Laboratory Improvement Amendments of 

1988 (e.g. total creatinine kinase (CK) below 30 percent, or aspartate aminotransferase (AST) 

below 20 percent).223

Three studies that partially addressed the reproducibility and accuracy of variant TPMT 

allelic polymorphism detection reported 100 percent concordance between denaturing HPLC and 

restriction fragment length polymorphism (RFLP) genotyping tests. The dichotomous nature of 
100

genetic results, reported as either present or absent, does not allow for traditional precision and 

accuracy determination as is done for enzymatic determination. However, a number of guidelines 

are available that address the complex issues. Recently, the Centers for Disease Control released 

a Morbidity and Mortality Weekly Report detailing good laboratory practices for molecular 

genetic testing. It reviewed the need for adequate quality control of genetic testing and put 

forward recommendations for laboratories performing molecular genetic testing.224 The Clinical 

and Laboratory Standards Institute (CLSI) has also published guidelines for Molecular 

Diagnostic Methods for Genetic Disease225 and Validation and Verification of Multiplex Nucleic 

Acid Assays.226 It is recommended that any laboratories performing allelic polymorphism 

detection of TPMT review the above guidelines to ensure the accuracy of their results.

Diagnostic Sensitivity and Specificity

Enzymatic analysis was selected as the reference standard, as this method should identify all 

patients with reduced or absent TPMT activity, regardless of the mechanism. To date at least 30

mutant alleles have been identified within the coding region14,27-29,213,227-231 . Others have been 

identified in the 3’ untranslated and promoter regions.29 These mutations likely do not directly 

affect the activity of the TPMT enzyme molecule, but may influence the quantity of enzyme 

present and thereby indirectly decrease the overall in vivo TPMT enzymatic activity. 

Reporting units and ranges of enzymatic activity are not standardized, so in our analyses the 

activity cutoff values stated in each article were used to assign patients to one of three groups: 

low/absent; intermediate; or normal/high. None of the studies were specifically designed to 

determine the diagnostic accuracy of genotyping in comparison to enzymatic activity. Thus, not 

surprisingly, using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool35 a 

substantial (37 percent) of the studies were rated poor quality. 

Diagnostic groups were organized in our analyses according to TPMT allelic variant(s) tested 

rather than by specific point mutation, to correspond to reporting in clinical practice. The relative 

abundance of specific allelic variants in a population has a direct impact on the diagnostic 

sensitivity and specificity of genetic testing, and therefore should be considered when developing 

genetic testing strategies. Sahasranaman et al. reviewed the relative frequency of the four most 

common alleles, *2A, *3A, *3B, and *3C.227 Pooled data suggests that the most common allele 

in Caucasians is *3A, with a mean frequency of 3.89 percent in a general population of 5076 

(range 2.1 to 8.6 percent), while the most common in Africans is *3C, with a mean frequency of 

4.7 percent in a population of 884 (range 2.4 - 7.6 percent). Pooled frequencies in a general 

population of 356 Asians and South Asians were lower than those seen in Caucasians and 

Africans, with a mean frequency of 1.0 percent (range zero to 2.3 percent) for *3C, and 0.17 

percent (range zero to one) for *3A. 

A total of 16 studies were included in the quantitative syntheses, assessing diagnostic

sensitivity and specificity.1,46,49-53,56,59,70,93,157,158,161,162,167 From seven studies, the pooled 

sensitivity of genotyping for homozygosity or heterozygosity of the common TPMT *2, *3A, 

*3B, and *3C alleles, to correctly identify patients with absent to intermediate TPMT enzymatic 

activity, was 70.7 percent (95 percent confidence interval (CI) 37.9 to 90.5 percent) (metaanalysis 1). The pooled specificity of noncarrier genotype to correctly identify those with normal 

or high enzymatic activity approached 100 percent. With other combinations of alleles, pooled 

specificities of genotyping remained close to 100 percent, but sensitivity did not improve 

convincingly, as inadequate relevant evidence led to substantial imprecision in estimates 
101

[sensitivity ranged from 70.70 to 82.10 percent (95 percent CI, lower bound range 37.90 to 54.00 

percent; upper bound range 84.60 to 96.90 percent]. Studies by Okada et al,53 and von Ahsen et 

al52 reported markedly lower sensitivities compared with the other studies. Okada et al analyzed 

a Japanese cohort, previously shown to have a low frequency of the common alleles, which 

suggest that other relatively common unidentified alleles may be present in the Japanese 

population. von Ahsen et al examined a German Caucasian cohort, and remarked upon the lower 

sensitivity observed, relative to previous reports. No plausible explanation could be identified for 

the heterogeneity in effect estimates. 

Few individuals exhibited homozygous TPMT variant alleles. The pooled sensitivity of a 

homozygous TPMT genotype to correctly identify patients with low to absent enzymatic activity 

was based on two small studies with two percent of 341 patients identified as homozygous for 

variant allele. The pooled sensitivity was 87.10 percent (95 percent CI 44.30 to 98.30 percent). 

The pooled specificities of the noncarrier and heterozygous carrier states to correctly identify 

those without low or absent TPMT enzymatic activity were determined for the different 

combinations of tested allelic variants (meta-analysis 2). The specificities were high, 

approaching 100 percent.

For a screening test, genotyping appears to have moderate sensitivity to detect those with 

subnormal (i.e. intermediate plus low plus absent) enzymatic activities while possibly high 

sensitivity of 87 percent to identify only those with low to absent activities. However, the 

available evidence is imprecise and of uncertain validity given that 37 percent of studies were 

rated as poor during risk of bias categorization. These limitations in diagnostic sensitivity of 

genotyping are not unexpected as genetic analysis of TPMT most often targets only the common 

polymorphisms and will fail to identify new or rare mutations. Furthermore, the commonly 

employed genotypic tests while able to identify a specific SNP, are unable to determine the 

allelic location of it. Therefore, a patient typed as a heterozygote for TPMT*3A (i.e. wild 

type/*3A) may have been misdiagnosed as such while actually being a compound heterozygote 

TPMT*3B/*3C for the observed TPMT activity.232,233

As discussed later, with a dearth of relevant primary literature, it remains unclear how 

incidence rates of thiopurine related adverse events may be affected by pretreatment genotyping. 

Therefore, this evidence should not be interpreted to conclude that prior genotyping is not 

effective in reducing thiopurine related drug toxicity in the treatment of chronic autoimmune 

diseases, especially when data associated with homozygosity were scant. 

Currently, there is insufficient data to determine the optimum combination of TPMT alleles 

that must be tested in order to identify patients with reduced or absent enzymatic activity. There 

is also a lack of well powered, good quality studies comparing the diagnostic accuracy and 

relative effectiveness of the two methods of genotyping and phenotyping to determine TPMT 

status. 

Postanalytic Requirements

This review did not identify any relevant studies that addressed postanalytic requirements in 

terms of reporting units, common reference intervals, or result reporting for either enzymatic 

testing or allelic polymorphism measurement. In general, results should be communicated to 

ordering practitioners as soon as possible after testing is completed. However, as this test is 

normally used prior to administration of thiopurine drugs, there is no critical requirement to 

contact practitioners directly to communicate abnormal results. Ideally, reports should include 
102

both lower and upper reference limits, information on how the reference interval was 

determined, and an indication of overlap that is seen between normal and intermediate 

metabolizers. At a minimum, information on reference interval determination and overlap 

between intermediate and normal activities should be available on request.

Enzymatic assays are currently reported using one of two commonly used units, nmol/h/g Hb 

and Unit/mL RBC. One Unit is defined as generation of 1 nmol of product per hour (6-

methylMP or 6-methylTG). Thus, the key difference is the standardization of the product 

generation, per gram of hemoglobin in RBC lysate, or per milliliter of packed RBCs. Hence, 

conversion between the two is neither easy, nor exact. It is recommended that a single preferred 

unit of measure be identified and used in the future to simplify reporting and interpretation, and 

to allow comparisons between laboratories. 

Interpretation of enzymatic testing results is highly dependent on the stated reference interval 

provided by the performing laboratory. Individuals with very low or absent TPMT enzymatic

activity (homozygous abnormal) are relatively easily identified, as they are clearly separated 

from patients with normal activity. Those patients with intermediate activity, however, are more 

difficult to identify as theirs’ often overlap with the enzymatic activity of normal metabolizers. 

Therefore, determination of the lower reference limit for normal metabolizers is most important. 

Currently, there is no universally agreed upon lower limit of normal for TPMT activity, however 

many studies used similar lower limits. Standardization of analytical methods and reporting units 

will aid in identifying a universal lower limit of normal, and should be a future goal.

Clinical Laboratory Survey 

A survey of laboratories was conducted to gather information regarding current clinically 

available TPMT analysis. Following identification of potential laboratories, two organizations 

and seven laboratories were contacted to determine their willingness either to complete a 

questionnaire or to disseminate the questionnaire to other relevant laboratories. Six of seven 

laboratories invited to participate returned a completed questionnaire. Preanalytical 

requirements, acceptable specimen type, storage times and conditions, were in keeping with the 

results of this review. Stated analytical precision of enzymatic analysis by the surveyed 

laboratories, as expected, was similar to that reported within published articles and ranged from 3 

to 10 percent within runs, and from 5 to 20 percent between runs. Among the surveyed 

laboratories, reported concordance between enzymatic analysis genotyping ranged from 60 

percent to 100 percent. Although the range is in keeping with the published reports, no data was 

provided to directly support these conclusions.

Knowledge of TPMT Status to Guide Therapy

A single fair quality randomized trial in 333 patients demonstrated that over a four month 

observation period, pretreatment genotyping did not significantly alter prescribing practice 

compared with no pretesting. There was no significant difference between the two groups in 

terms of starting doses and mean AZA prescribed dose at the end of the study period. Despite 

prior knowledge of noncarrier TPMT status in the tested group of patients, most patients were 

administered starting doses lower than 2mg/kg/day, similar to the nontested group. This was 

because physicians were free to practice as per routine, and which they did just as cautiously as 

the nontested group despite prior knowledge obtained from genotyping. Knowledge of 
103

heterozygous status, however, did result in prescription of lower starting doses compared with 

noncarriers in the group tested before therapy. There is limited applicability of this evidence 

because there was just one homozygous carrier in the whole sample of mostly IBD patients. The 

finding conforms with an earlier national survey in the United Kingdom in which the uptake of 

prior TPMT testing differed substantially by clinical specialty – there was a higher uptake of 

TPMT enzyme-level testing by dermatologists, compared with gastroenterologists and 

rheumatologists, and this might explain why the group that underwent prior genotyping still 

ended up receiving doses of azathioprine similar to the nongenotyped control group.234 Since 

most patients had inflammatory bowel disease, it appears that gastroenterologists, specifically, 

tend to exercise a cautious prescribing approach over one primarily guided by prior knowledge 

of the TPMT status. 

When compared with no pretesting for TPMT status, testing did not demonstrate a significant 

difference in the odds of mortality and serious adverse events in 333 randomized patients. The 

evidence was rated as insufficient given a medium risk of bias and strong possibility of type II 

error. The applicability of the evidence was deemed limited as there was just one homozygous 

carrier of TPMT variant allele in the entire sample of mostly IBD patients, the followup period 

was just 4 months and eligibility criteria of the trial excluded patients who would most likely 

have experienced adverse events. Evidence was also rated insufficient for the outcomes of 

health-related quality of life and myelotoxicity as no data were available. 

Evidence from one RCT with low event rate showed no significant advantage of prior 

genotyping with respect to the intermediate outcomes of neutropenia and pancreatitis. The

applicability of this evidence is quite limited because there was just one homozygous carrier in 

the whole sample of 333 patients, the followup period was just 4 months and eligibility criteria 

of the trial excluded patients who would most likely have experienced adverse events. Also, type 

II error cannot be ruled out. As TPMT status determination may not identify all individuals at 

increased risk of drug toxicity,235 direct and conclusive evidence of the utility of pretesting in 

terms of drug related harms reduction is wanting for evidence-based guidelines on thiopurine 

therapy. For the outcome of liver toxicity, significantly higher odds were observed in the group 

that underwent prior TPMT genotyping, odds ratio 2.54 (1.08, 5.97)]. There was no significant 

difference in starting or mean doses received between the tested and nontested group. Although 

this finding merits further investigation, but it appears to be a type I error and unrelated to the 

intervention of pretreatment genotyping. 

Various recent guidelines, as well as the product monograph for azathioprine, have advocated 

determination of TPMT status prior to treatment with thiopurine drugs.30,243 The proposition that 

knowledge of TPMT status prior to therapy would lead to decreased rates of dose-dependent 

toxicity is rational and based on evidence of strong genotypic and phenotypic associations in 

observational studies of limited validity. Compared with non-carriers and heterozygous carriers, 

homozygous are considered to be most at risk of developing neutropenia. However, from an 

evidence-based perspective, guideline recommendations of pretreatment TPMT testing are 

premature for several reasons. First and foremost, the direct evidence base for these

recommendations is lacking – especially the crucial evidence that TPMT pretesting before 

thiopurine therapy decreases myelotoxicity specific mortality. Also, given just one homozygous 

carrier in the only available direct evidence investigating usefulness of pretreatment, evidence is 

equally lacking for this particular subgroup of patients. Second, patients on thiopurine drugs are 

required to undergo complete blood count monitoring on a regular basis in an attempt to prevent 

severe myelotoxicity by early detection. Third, azathioprine and 6-MP had been used 
104

successfully for a number of years prior to the availability of TPMT testing and management (i.e. 

testing or not before therapy) varies across clinical specialties. Fourth, thiopurine related 

toxicities are also partially explained by mutations in other enzymes, drug interactions, 

intercurrent infections, and immune mediated drug reactions. Fifth, direct evidence of 

effectiveness of pretesting in the specific subpopulation of patients homozygous or compound 

heterozygous for the TPMT variant alleles is lacking the most, albeit not surprisingly, because of 

the low prevalence of homozygosity. Furthermore, the use of TPMT status to guide treatment has 

the potential to reduce the efficacy of thiopurine drugs if physicians are overzealous in reduction 

of thiopurine dosage. Indeed, the 2004 guidelines from the British Society of Gastroenterology 

recognized this and stated, “It cannot yet be recommended as a prerequisite to therapy, because 

decades of experience has shown clinical [azothioprine] to be safe in [ulcerative colitis] or 

[Crohn’s disease]”.244 As far as the utility of pretesting for TPMT status before thiopurine 

treatment is concerned, our review is indeterminate because of insufficient evidence and calls for 

urgent further research. This is at odds with previously published economic evaluations 

recommending testing. However, those evaluations have been criticized for incorporating clinical 

data from retrospective studies and expert opinion instead of prospective empiric evidence – the 

latter, as our review shows, are lacking236 

Association of TPMT Status With Thiopurine Toxicity 

In the presence of insufficient direct evidence of prior knowledge of TPMT status to guide 

thiopurine therapy, possible associations between TPMT status and the clinical outcomes of 

mortality, infections, hospitalization, withdrawals due to adverse events, serious adverse events 

and health-related quality of life, as well as the surrogate outcomes of myelotoxicity, liver 

toxicity, and pancreatitis were examined. 

Toxicity of thiopurine drugs is thought to be mediated primarily through their 

pharmacologically active metabolites, 6-tGNs, which can be considered a dose-dependent 

toxicity. Incorporation of 6-tGN into DNA triggers cell-cycle arrest and apoptosis through the 

mismatch repair pathway.8 Recent evidence has also shown that thiopurine drugs can induce 

apoptosis in T-cells through modulation of Rac1 activation upon CD28 costimulation. Therefore 

accumulation of 6-tGNs can clearly induce various degrees of myelosuppression. Doseindependent toxicity, until recently was not understood, and appears to be either immune 

mediated or due to metabolites previously thought to be inactive. Both hepatotoxicity and 

pancreatitis are thought to be caused through dose-independent toxicity. Immune mediated 

reactions with AZA include hepatitis, pancreatitis, rash etc. and usually occur with 4 weeks of 

initiation of therapy. In some patients, this reaction can be overcome by switching to 6-MP, 

implying a role for the imidazole moiety in toxicity.237 The mechanisms of hepatotoxicity have 

been studies in most depth. A link between hepatotoxicity and increased levels of 6-methlyMP 

ribonucleotide (6-MMPR) has been suggested. Seidman et al. identified a link between TPMT 

activity and levels of 6-MMPR, suggesting that TPMT may play a role in hepatotoxicity.238 

However, in this case higher TPMT activity may be more relevant to induction of toxicity than 

lower activity (see Figure 1). In a subset of patients with subtherapeutic levels of 6-tGNs, dose 

escalation of 6-MP did not increase 6-tGN levels, however levels of 6-MMPR did increase. In 

one study, 24 percent of patients with elevated 6-MMPR levels showed higher rates of 

hepatotoxicity.119 Mardini et al also found that elevated levels of 6-MMPR correlated with 
105

hepatotoxicity.239 While others have found no relation between levels of 6-MMP and 

hepatotoxicity.240,241

Thirty-four studies were identified that provided relevant data on allelic variants and 16 

studies were identified that provided data on TPMT enzymatic activity in relation to clinical 

outcomes. There is insufficient evidence examining association of TPMT status, as determined 

by either allelic determination or enzymatic activity, with the outcomes of mortality, 

hospitalization rates, serious adverse events, health related quality of life and neutropenia 

precluding meaningful conclusions. Furthermore, insufficient data were available for infection 

and thrombocytopenia by enzymatic analysis; however limited data showing no effect were 

available for allelic determination. The majority of the studies were of cross-sectional design and 

fair quality. 

The available evidence confirms our previous understanding that there is strong association 

between the outcome of leukopenia and presence of variant TPMT alleles or subnormal 

enzymatic activity, and dose response relationships with both allelic variants and TPMT 

enzymatic activity.227 The strongest association was for homozygous carriers or low to absent 

enzymatic activity, compared with noncarrier or normal enzymatic activity patient groups. There 

is also some indication that lower levels of TPMT enzymatic activity may be associated with the 

composite outcome of myelotoxicity, defined as decreased levels of at least two hematopoietic 

cell lines. For most other outcomes, there was no significant association with either a presence of 

a TPMT allelic variant or subnormal enzymatic activity. Given the small number of studies 

involving few patients with events, type II error cannot be ruled out for most outcomes other 

than hepatotoxicity and pancreatitis. For these two outcomes, our findings of no association of 

these outcomes with either low enzymatic activity or presence of TPMT carrier states are 

consistent with extant literature.72,82,163
106

Costs of Determining TPMT Status Versus Costs of Treating DrugAssociated Complications

Global interest in costs of TPMT phenotyping and genotyping is reflected in studies from 

around the world, published between 1995 and 2010. 

Most cost estimates were based on theoretical populations, although one study was based on 

a bullous pemphigoid patient with AZA toxicity.210 Across all studies, there was some consensus 

on the cost of genotype and phenotype testing, although the cost perspective was often not 

reported. The one study reporting costs from a societal perspective showed higher costs than the 

others.208 Heterogeneous estimates of the total cost likely arose from differing methodological 

choices. For example, the cost of treating AZA-associated complications was estimated to be 

between $1,325 and $5,877 in USD. This four-fold difference has the potential to result in 

disparate total cost estimates. The average cost of TPMT phenotyping was approximately half of 

the average cost of TPMT genotyping, but these costs may not be generalizable to all TPMT 

tests. These costs will have to be taken into consideration, along with the relative sensitivity and 

specificity of TPMT genotyping and TPMT phenotyping, when deciding upon which test to use. 

Strengths and Limitations

This is the first comprehensive systematic review answering the question whether testing 

TPMT status prior to thiopurine therapy changes management and thiopurine toxicity outcomes. 

It is also the first review of the analytical performance characteristics of enzymatic measurement 

of TPMT activity and determination of TPMT allelic polymorphisms. These key questions were 

developed from a conceptual framework of the topic with input from the Technical Expert Panel. 

This panel included clinical, genetics, biochemistry and systematic review methodology experts. 

We contacted authors and obtained additional data that we incorporated in meta-analyses. A 

survey of laboratories, although not part of the original work plan, further added to our 

understanding of laboratory practices related to TPMT testing. 

Despite our rigorous methodological approach, this review has several limitations. From a 

clinical perspective, the most important equipoise about the utility of prior TPMT testing remains 

insufficiently answered due to a dearth of comparative effectiveness literature on TPMT testing 

and its limited applicability. Evidence relating to other key questions originated in observational 

studies of poor to fair quality, so is of limited strength. The genetic associations established in 

this review, while confirming previous literature, are still of limited reliability.242 Lastly, we 

pooled diverse studies, with the assumption that most thiopurine toxicity is determined 

genetically and biochemically. There was insufficient primary evidence to identify important 

effect modifiers or to carry out separate subgroup meta-analyses on studies with lower risk of 

bias.

Recommendations and Future Research 

There is insufficient evidence examining the effectiveness of TPMT pretreatment enzymatic 

or genetic testing, to minimize thiopurine related toxicity in patients with chronic autoimmune 

diseases. As a priority, well powered, good quality, randomized controlled studies need to be 

conducted, in diverse and representative patient populations, to compare the effectiveness of 

TPMT genotyping and phenotyping with one another, and with no TPMT testing. These studies 
107

should be large enough to include a sizable number of patients homozygous for the variant 

alleles and should be pragmatic in conduct, mimicking routine clinical practice. Outcomes would 

include both treatment efficacy and harms associated with thiopurine therapy. Another objective 

would be to establish the optimum initial dose adjustment for a given TPMT status. These 

studies should ensure that outcomes are truly assessed without prior knowledge of results of 

TPMT testing and administered drug dose, by employing appropriate blinding procedures. The 

recently concluded pragmatic TARGET study by Newman and associates was under-powered to 

detect differences in clinically important outcomes, largely because it faced recruitment 

problems. In future such recruitment problems may be mitigated by educating the public and 

clinicians that the evidence base for pretreatment TPMT testing is lacking and that it is unclear 

whether pretreatment testing does more good (i.e. reduction in thiopurine related toxicity) than 

harm (i.e. reduction in thiopurine efficacy because of overzealous dose reductions based on prior 

testing). 

Until such experimental high quality evidence becomes available, alternative evidence may 

be sought in prospectively designed observational studies that estimate health related quality of 

life, drug prescription patterns, and myelotoxicity related mortality as important outcomes

associated with and with no pretreatment TPMT testing. With availability of empiric evidence 

from such studies, decision-analytic modeling that comprehensively consider alternative 

strategies such as regular blood cell count and liver enzyme testing, metabolite monitoring, and 

dose adjustments for concomitant medications that impact the TPMT enzymatic pathway can 

help guide practice until evidence becomes available from well powered pragmatic trials. 

Subsequent models might also need to consider new information as technologies develop and 

knowledge evolves. 

TPMT genotyping should test for the most common TPMT polymorphisms in the population 

of interest. There is little direct evidence identifying the optimum set of alleles to be tested, and 

this may need to be established for specific populations if TPMT genotyping turns out to be 

effective in future studies. 

TPMT activity analyses are reported on one of two bases: per milliliter of packed red blood 

cells; or per gram of hemoglobin. These are not readily or exactly comparable. Common 

reporting units are needed, as well as cutoffs for low/absent, intermediate, normal TPMT 

enzymatic activity, and high enzymatic activities. 

Future studies should clearly report numbers of uninterpretable or equivocal test results.

Conclusions 

This is the first comprehensive systematic review answering the question whether testing 

TPMT status prior to thiopurine therapy changes management and thiopurine toxicity outcomes, 

including leukopenia and myelotoxicity. There is currently insufficient evidence regarding 

effectiveness of determining TPMT status prior to thiopurine treatment in terms of improvement 

in clinical outcomes and incident myelotoxicity in comparison with routine monitoring of full 

blood counts and adverse events. It is also unclear whether pretesting guides appropriate 

prescribing. Indirect evidence confirmed previously known strong associations between lower 

levels of TPMT enzymatic activity and the presence of TPMT variant alleles with thiopurine 

related leukopenia. 

Sufficient preanalytical data are available to recommend preferred specimen collection, 

stability and storage conditions for determination of TPMT status. There was no clinically 
108

significant effect of age, gender, various coadministered drugs, or most comorbid conditions 

(with the exception of renal failure and dialysis). The currently available methods for 

determination of TPMT enzymatic activity show good precision, with coefficients of variation

generally below 10 percent. Based upon limited evidence, the reproducibility of TPMT allelic 

polymorphism determination is acceptable. However, the sensitivity of genetic testing to identify 

patients with low and/or intermediate TPMT enzymatic activity cannot be precisely estimated. 

Thus, if knowledge of TPMT status is desired, and if recent RBC transfusion is excluded, the 

available evidence suggests that enzymatic assay should be preferred over the determination of 

allelic polymorphism.

Despite widespread interest, precise costs associated with TPMT phenotyping are unknown. 

More research has been conducted examining TPMT genotyping but the cost estimates are 

heterogeneous, likely due to different methodological choices.
109

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Apr;26(2):231-5. 

219. Kurzawski M, Dziewanowski K, Lener A, et al. 

TPMT but not ITPA gene polymorphism 

influences the risk of azathioprine intolerance in 

renal transplant recipients. Eur J Clin Pharmacol 

2009 May;65(5):533-40. 

220. Plebani M. Errors in clinical laboratories or errors 

in laboratory medicine? Clin Chem Lab Med 

2006;44(6):750-9. 

221. U.S.Food and Drug Administration. Collection, 

Transport, Preparation, and Storage of Specimens 

for Molecular Methods; Approved Guideline. 

MM13-A Vol. 25. (31). 2005. 

222. Fraser CG. Biological variation: from principles 

to practice. Washington D.C.: Amer Assn for 

Clinical Chemistry; 2001.

223. The Federal Registrar. U.S. Department of 

Health and Human Services: Clinical Laboratory 

Improvement Amendments of 1988; Final Rules 

and Notice. Vol.57. 42 CFR Part 493. 1992. 

224. Chen B, Gagnon M, Shahangian S, et al. Good 

laboratory practices for molecular genetic testing 

for heritable diseases and conditions. MMWR 

Recomm Rep 2009 Jun 12;58(RR-6):1-37. 

225. Grody W and Richards C. Molecular Diagnostic 

Methods for Genetic Disease; Approved 

Guideline. MM01-2A. 2006. 

226. Wilson J, Zoccoli M, Jacobson J et al. 

Verification and Validation of Multiplex Nucleic 

Acid Assays; Approved Guideline. MM17-A. 

Vol 28 (9). 2008. 

227. Sahasranaman S, Howard D, Roy S. Clinical 

pharmacology and pharmacogenetics of 

thiopurines. [Review] [153 refs]. Eur J Clin 

Pharmacol 2008 Aug;64(8):753-67. 

228. Hamdan-Khalil R, Gala JL, Allorge D, et al. 

Identification and functional analysis of two rare 

allelic variants of the thiopurine Smethyltransferase gene, TPMT*16 and 

TPMT*19. Biochem Pharmacol 2005 Feb 

1;69(3):525-9. 

229. Garat A, Cauffiez C, Renault N, et al. 

Characterisation of novel defective thiopurine Smethyltransferase allelic variants. Biochem 

Pharmacol 2008 Aug 1;76(3):404-15. 

230. Lindqvist M, Skoglund K, Karlgren A, et al. 

Explaining TPMT genotype/phenotype 

discrepancy by haplotyping of TPMT*3A and 

identification of a novel sequence variant, 

TPMT*23. Pharmacogenet Genomics 2007 

Oct;17(10):891-5. 

231. Kham SK, Soh CK, Liu TC, et al. Thiopurine Smethyltransferase activity in three major Asian 

populations: a population-based study in 

Singapore. Eur J Clin Pharmacol 2008 

Apr;64(4):373-9. 

232. Sanderson J, Ansari A, Marinaki T, et al. 

Thiopurine methyltransferase: should it be 

measured before commencing thiopurine drug 

therapy?[see comment]. [Review] [80 refs]. Ann 

Clin Biochem 2004 Jul;41(Pt 4):294-302. 
119

233. Ford LT, Berg JD. Thiopurine Smethyltransferase (TPMT) assessment prior to 

starting thiopurine drug treatment; a 

pharmacogenomic test whose time has come. J 

Clin Pathol 2010;63(4):288-95. 

234. Fargher EA, Tricker K, Newman W, et al. 

Current use of pharmacogenetic testing: a 

national survey of thiopurine methyltransferase 

testing prior to azathioprine prescription. J Clin 

Pharm Ther 2007 Apr;32(2):187-95. 

235. Colombel JF, Ferrari N, Debuysere H, et al. 

Genotypic analysis of thiopurine Smethyltransferase in patients with Crohn's disease 

and severe myelosuppression during azathioprine 

therapy. Gastroenterol 2000 Jun;118(6):1025-30. 

236. Payne K, Newman WG, Gurwitz D, et al. TPMT 

testing in azathioprine: A 'cost-effective use of 

healthcare resources'? Personaliz Med 

2009;6(1):103-13. 

237. McGovern DP, Travis SP, Duley J, et al. 

Azathioprine intolerance in patients with IBD 

may be imidazole-related and is independent of 

TPMT activity. Gastroenterol 2002 

Mar;122(3):838-9. 

238. Seidman EG. Clinical use and practical 

application of TPMT enzyme and 6-

mercaptopurine metabolite monitoring in IBD. 

Rev Gastroenterol Disord 2003;3 Suppl 1:S30-

8:S30-S38.

239. Mardini HE, Arnold GL. Utility of measuring 6-

methylmercaptopurine and 6-thioguanine 

nucleotide levels in managing inflammatory 

bowel disease patients treated with 6-

mercaptopurine in a clinical practice setting. J 

Clin Gastroenterol 2003 May;36(5):390-5. 

240. Reinshagen M, Schutz E, Armstrong VW, et al. 

6-thioguanine nucleotide-adapted azathioprine 

therapy does not lead to higher remission rates 

than standard therapy in chronic active crohn 

disease: results from a randomized, controlled, 

open trial.[see comment]. Clin Chem 2007 

Jul;53(7):1306-14. 

241. Goldenberg BA, Rawsthorne P, Bernstein CN. 

The utility of 6-thioguanine metabolite levels in 

managing patients with inflammatory bowel 

disease. Am J Gastroenterol 2004 

Sep;99(9):1744-8. 

242. Ioannidis JP, Ntzani EE, Trikalinos TA, et al. 

Replication validity of genetic association 

studies. Nat Genet 2001 Nov;29(3):306-9. 

243. Chakravarty K, McDonald H, Pullar T, et al. 

BSR/BHPR guideline for disease-modifying antirheumatic drug (DMARD) therapy in 

consultation with the British Association of 

Dermatologists. Rheumatology (Oxford) 2008 

Jun;47(6):924-5. 

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the management of inflammatory bowel disease 

in adults. Gut 2004 Sep;53 Suppl 5:V1-16. 

121

List of Acronyms/Abbreviations

6-MMP 6-methylmercaptopurine

6-MMPR 6-methlyMP ribonucleotide 

6-MP 6-mercaptopurine

6-MTG 6-methylthioguanine

6-TG 6-thioguanine

6-tGN deoxy-6-thioguanosine 5’ triphosphate 

6-tIMP 6-thiomercaptopurine 

AACC American Association for Clinical Chemistry 

AHRQ Agency for Health Research Quality

ALL acute lymphoblastic leukemia

AO aldehyde oxidase 

AST aspartate aminotransferase

AZA azathioprine 

CI confidence interval

CK creatinine kinase

CLSI Clinical and Laboratory Standards Institute 

CV coefficient of variance

CLIB Cochrane Library

FN false negative

FP false positive

g gram

GD guanine deaminase

h hour

Hb haemoglobin

HGPRT hypoxanthine-guanine phosphoribosyl transferase 

HPLC high performance liquid chromatography

HQOL health related quality of life

HWE Hardy-Weinberg equilibrium 

IBD inflammatory bowel disease 

IC50 concentration of inhibitor at which enzyme activity is 50 percent of uninhibited 

activity

ICU intensive care unit

IMPDH inosine monophosphate dehydrogenase KQ key question

MALDI-TOF Matrix-assisted laser desorption/ionization, with time-of-flight mass spectrometry 

mg milligram

n number in group with particular characteristic

N total number in study or group

nmol nanomole

OR odds ratio

p probability

PCR polymerase chain reaction

pmol picomol

QUADAS Quality Assessment of Diagnostic Accuracy Studies 

RBCs red blood cells
122

RFLP restriction fragment length polymorphism 

SAE serious adverse events

SAM S-adenosyl-L-methionine 

SNPs single nucleotide polymorphisms 

TARGET TPMT: Azathioprine Response to Genotyping and Enzyme Testing study

TN true negative

TP true positive

TPMT thiopurine methyltransferase

U unit (nmol/h production in an enzymatic reaction)

WDAE withdrawal due to adverse events

XO xanthine oxidase
A - 1

Appendix A

Exact Search Strings

Ovid MEDLINE(R) 1950 to May Week 3 2010

1 (TPMT* or thiopurine methyltransferase* or thiopurine s-methyltransferase* or 

thiopurine methyl-transferase* or thiopurine s-methyl-transferase* or 

Thiopurinemethyltransferase*).mp. [mp=title, original title, abstract, name of substance 

word, subject heading word] 

2 animal/

3 human/

4 2 not (2 and 3) 

5 1 not 4

EMBASE 1980 to 2010 Week 21

1 exp Thiopurine Methyltransferase/

2 (TPMT* or thiopurine methyltransferase* or thiopurine s-methyltransferase* or 

thiopurine methyl-transferase* or thiopurine s-methyl-transferase* or 

Thiopurinemethyltransferase*).mp. [mp=title, abstract, subject headings, heading word, 

drug trade name, original title, device manufacturer, drug manufacturer name]

3 1 or 2

4 human.sh.

5 nonhuman.sh.

6 animal.sh. (18273)

7 animal experiment.sh.

8 or/5-7 

9 8 not (4 and 8)

10 3 not 9 

Central (CLIB 2009 2)

(Thiopurine Methyltransferase*):ti,ab,kw or (TPMT* OR 

Thiopurinemethyltransferase*):ti,ab,kw or (thiopurine s-methyltransferase*):ti,ab,kw or 

(thiopurine methyl-transferase*):ti,ab,kw or (thiopurine s-methyl-transferase*):ti,ab,kw

Ovid Healthstar 1966 to April 2010

1 (TPMT* or thiopurine methyltransferase* or thiopurine s-methyltransferase* or 

thiopurine methyl-transferase* or thiopurine s-methyl-transferase* or 

Thiopurinemethyltransferase*).mp. 

2 animal/ 

3 human/ 

4 2 not (2 and 3) 

5 1 not 4 
A - 2

Genetic Abstracts. May 7 2009.

Query: KW=(TPMT* OR thiopurine methyltransferase* OR thiopurine smethyltransferase* OR thiopurine methyl-transferase* OR thiopurine s-methyltransferase* OR Thiopurinemethyltransferase*) OR TI=(TPMT* OR thiopurine 

methyltransferase* OR thiopurine s-methyltransferase* OR thiopurine methyltransferase* OR thiopurine s-methyl-transferase* OR Thiopurinemethyltransferase*) OR 

AB=(TPMT* OR thiopurine methyltransferase* OR thiopurine s-methyltransferase* OR 

thiopurine methyl-transferase* OR thiopurine s-methyl-transferase* OR

Thiopurinemethyltransferase*)OR DE=(TPMT* OR thiopurine methyltransferase* OR 

thiopurine s-methyltransferase* OR thiopurine methyl-transferase* OR thiopurine smethyl-transferase* OR Thiopurinemethyltransferase*)

BioSYS May 5 2009

# 1 Topic=(TPMT* OR thiopurine methyltransferase* OR thiopurine smethyltransferase* OR thiopurine methyl-transferase* OR thiopurine s-methyltransferase* OR Thiopurinemethyltransferase*) OR Title=(TPMT* OR thiopurine 

methyltransferase* OR thiopurine s-methyltransferase* OR thiopurine methyltransferase* OR thiopurine s-methyl-transferase* OR Thiopurinemethyltransferase*)

Databases=PREVIEWS Timespan=All Years

# 2 DE=(TPMT* OR thiopurine methyltransferase* OR thiopurine smethyltransferase* OR thiopurine methyl-transferase* OR thiopurine s-methyltransferase* OR Thiopurinemethyltransferase*) Databases=PREVIEWS 

Timespan=All Years

# 3 #2 OR #1 Databases=PREVIEWS Timespan=All Years

# 4 TS=(econom* or cost or costs or costly or costing or costed or cost-benefit* or 

price or prices or pricing or priced or discount or discounts or discounted or discounting 

or expenditure or expenditures or budget* or afford* or pharmacoeconomic* or 

pharmaco-economic*) Databases=PREVIEWS Timespan=All Years

# 5 TI=(econom* or cost or costs or costly or costing or costed or cost-benefit* or 

price or prices or pricing or priced or discount or discounts or discounted or discounting 

or expenditure or expenditures or budget* or afford* or pharmacoeconomic* or 

pharmaco-economic*) Databases=PREVIEWS Timespan=All Years

# 6 DE=(econom* or cost or costs or costly or costing or costed or cost-benefit* or 

price or prices or pricing or priced or discount or discounts or discounted or discounting 

or expenditure or expenditures or budget* or afford* or pharmacoeconomic* or 

pharmaco-economic*) Databases=PREVIEWS Timespan=All Years

# 7 TI=(markov or markow or monte carlo) Databases=PREVIEWS 

Timespan=All Years
A - 3

# 8 TI=(markov or markow or monte carlo) Databases=PREVIEWS 

Timespan=All Years

# 9 DE=(markov or markow or monte carlo) Databases=PREVIEWS 

Timespan=All Years

# 10 TS=sensitivity analys* Databases=PREVIEWS Timespan=All Years

# 11 TI=sensitivity analys* Databases=PREVIEWS Timespan=All Years

# 12 DE=sensitivity analys* Databases=PREVIEWS Timespan=All Years

# 13 TS=quality adjusted life Databases=PREVIEWS Timespan=All Years

# 14 TI=quality adjusted life Databases=PREVIEWS Timespan=All Years

# 15 DE=quality adjusted life Databases=PREVIEWS Timespan=All Years

# 16 TS=CE analys?s Databases=PREVIEWS Timespan=All Years

# 17 TI=CE analys?s Databases=PREVIEWS Timespan=All Years

# 18 Databases=PREVIEWS Timespan=All Years

# 19 TS=(decision tree* or decision analy* or decision model*)

Databases=PREVIEWS Timespan=All Years

# 20 TI=(decision tree* or decision analy* or decision model*)

Databases=PREVIEWS Timespan=All Years

# 21 DE=(decision tree* or decision analy* or decision model*)

Databases=PREVIEWS Timespan=All Years

# 22 TS="quality of life" Databases=PREVIEWS Timespan=All Years

# 23 TI="quality of life" Databases=PREVIEWS Timespan=All Years

# 24 DE="quality of life" Databases=PREVIEWS Timespan=All Years

# 25 TS="willingness to pay" Databases=PREVIEWS Timespan=All Years

# 26 TI="willingness to pay" Databases=PREVIEWS Timespan=All Years

# 27 DE="willingness to pay" Databases=PREVIEWS Timespan=All Years
A - 4

# 28 Major Concepts=(Economics) Databases=PREVIEWS Timespan=All 

Years

# 29 #28 OR #27 OR #26 OR #25 OR #24 OR #23 OR #22 OR #21 OR #20 OR #19 

OR #18 OR #17 OR #16 OR #15 OR #14 OR #13 OR #12 OR #11 OR #10 OR #9 OR 

#8 OR #7 OR #6 OR #5 OR #4 Databases=PREVIEWS Timespan=All Years

# 30 TS=(markov or markow or monte carlo) Databases=PREVIEWS 

Timespan=All Years

# 31 #30 OR #29 Databases=PREVIEWS Timespan=All Years

# 32 #31 AND #3 Databases=PREVIEWS Timespan=All Years
B - 1

Appendix B

Data extraction and related forms

Contents

Key Questions 1a and 1b ................................................................................................................ 1

Key Question 1c.............................................................................................................................. 2

Laboratory Survey Introductory Letter........................................................................................... 6

Survey for “Thiopurine Methyltransferase” Laboratories.............................................................. 7

Key Question (2), 3a, 3b............................................................................................................... 10

Key Question 3c - phenotyping .................................................................................................... 12

Key Question 3c - genotyping ...................................................................................................... 15

Key Question 4 ............................................................................................................................. 19

Key Questions 1a and 1b

Pre-analytical requirements for enzymatic measurement of TPMT and determination of 

TPMT allelic polymorphisms; and 

Within and between laboratory precision and reproducibility of enzymatic measurement of 

TPMT and determination of TPMT allelic polymorphisms

Data element Comments, coding for data extraction

Ref id 

First author, year

Companion ref id If applicable

Contains information about (select 

all that apply)

1= pre-analytic requirement; 2 = post-analytic 

requirement, 3 = within laboratory precision and 

reproducibility, 4 = between laboratory precision 

and reproducibility

Full or partial industry funding 0 = no, 1 = yes, 2 = not reported 

Study region 1= North America; 2 = Europe, 3 = Central and 

South America, 4 = sub Saharan Africa, 5 = 

Middle East and North Africa, 6 = South Asia, 7 

= Asia, 9 = Oceania, 10 = 1+ 2, 11 = mixed 

/others, 12 = not reported 

Summary description of TPMT 

activity assay

Summary description of TPMT 

genotyping assay

Summary of pertinent paper (capture 

as much as possible in as few 

words)

Study limitations 

If not eligible for extraction, provide 

reasons 

Abbreviations: Ref id = unique reference identification number; TPMT = thiopurine methyltransferase
B - 2

Key question 1c

Diagnostic sensitivity and specificity of TPMT allelic polymorphism measurement 

compared to the measurement of TPMT enzymatic activity

1. General information for key question 1c

Data element Comments, coding

Ref id 

First author, year

Companion ref id 

Author contacted 1 = yes, 0 = no

Full or partial industry funding 0 = no, 1= yes, 2 = not reported 

Region of participants residence

1= North America; 2 = Europe, 3 = central and 

South America, 4 = sub Saharan Africa, 5 = Middle 

East and north Africa, 6 = South Asia, 7 = Asia, 9 = 

Oceania, 10 = 1+ 2, 11 = mixed others, 12 = not 

reported 

Diagnostic study design

1 = cross-sectional, 2 = case-control, 3 = 

prospective observational (including cohort), 4 = 

RCT, 5 = can't tell, 6 = other (specify)

Is enzymatic activity assay the 

reference/gold standard? 1 = yes, 0 = No

Enzymatic activity assay: testing 

method?

1 = Radioassay, 2 = HPLC, 3 = other (name the 

method), 4 = NR

Enzymatic activity assay: 

manufacturer?

Enzymatic activity: categories?

1 = high, normal, intermediate, low, absent; 2 = 

high, normal, intermediate, low/absent; 3 = 

high/normal, intermediate, low, absent; 4 = 

high/normal, intermediate, low/absent; 5 = other 

(specify) 

Enzymatic activity: cut off values? specify different cut-offs or state NR

Genotyping: method 

specify (e.g. RFLP, RT-PCR (probe hybridization or 

fluorescence), INVADER-PCR etc.) 1= PCR, 2 = 

pyrosequencing, 3 = other

Source of DNA for genotyping? specify

Number of SNPs genotyped 

Specify genotyped SNPs
1. General information for key question 1c (continued)

B - 3

Data element Comments, coding

Setting 1= inpatient, 2 = outpatient primary care, 3 = 

outpatient specialty clinic, 4 = NR or can't tell

Participants (a few words to describe the study population)

Participants, summary 

inclusion/exclusion criteria 

Underlying disease (s) including severity 

Thiopurine treatment 0= no; 1 = AZA, 2 = 6-MP, 3 = mixed thipurines, 4 = 

other, 5 = NR

Concomitant treatment potentially 

affecting TPMT activity

1 = no, 2 = allopurinol, 3 = 5-ASA 

(mesalazine/mesalamine/sulphasalazine, 

olsalazine), 4 = furosemide/frusemide, 5 = NSAID, 6 

= other (specify), 7 = NR 

Age group 1 = adults (>=16) only, 2 = children only (<16), 3 = 

adolescence only, 4 = mixed, 5 = not sure/ NR

Age in years mean or median or range

Females %

Caucasians %

African descent %

Hispanic %

Asians % 

Other % specify race and % of participants 

This report also has relevant 

subgroup data of genotyping and 

phenotyping results

specify subgroups

Applicability based on population 

characteristics and testing 

methodology 

1 = applicable, 2 = not applicable (explain why), 3= 

questionable applicability (explain why)

Abbreviations: 6-MP = 6-mercaptopurine; ASA = acetylsalicylic acid or aspirinTM; AZA = azathioprine; NR = not 

reported; NSAID = nonsteroidal anti-inflammatory drug; Ref id = unique reference identification number; TPMT = 

thiopurine methyltransferase
B - 4

2. Quantitative evidence for key question 1c

Data element Comments, coding for data extraction

Ref id

First author, year

Total number included in the study

Dropouts and withdrawals or untested

Activity assay: number of patients with 

non-interpretable test results

Genotyping: number of patients with noninterpretable test results

SNPs tested Specify SNPs

First comparison

Genotypes Heterozygous or homozygous for variant(s) 

/ wild type

Normal or hyperactivity (or controls) Numbers of participants with particular 

enzyme activity, with genotype

Intermediate or low/absent activity (or 

cases)

Numbers of participants with particular 

enzyme activity, with genotype

Second comparison

Genotype Homozygous for variant(s) / Wild type or 

heterozygous for variants

Normal/hyperactivity/intermediate activity 

(or controls)

Numbers of participants with particular 

enzyme activity, with genotype

Low/absent activity (or cases) Numbers of participants with particular 

enzyme activity, with genotype

Number of allele specific subgroups

Abbreviations: Ref id = unique reference identification number; TPMT = thiopurine methyltransferase
Key question 2, 3a, 3b (continued)

B - 5

3. Quality assessment for key question 1c

Data element Comments, coding for data 

extraction

Ref id 

First author, year

Is the spectrum of patients representative of those with 

one or more chronic autoimmune disease in terms of 

eligibility criteria, demographics, disease severity?

1 = yes, 0 = no, 2 = unclear

Clearly describes I/E criteria? 1 = yes, 0 = no, 2 = unclear

Was the reference test (activity assay) likely to correctly 

identify enzymatic activity? 1 = yes, 0 = no, 2 = unclear

Did the whole or a random sample of patients receive 

TPMT activity test? 1 = yes, 0 = no, 2 = unclear

Was the TPMT activity test administered regardless of 

genotyping results? 1 = yes, 0 = no, 2 = unclear

Were the TPMT activity and genotyping tests 

independent of each other? 1 = yes, 0 = no, 2 = unclear

Was the genotyping method clearly described for 

replication? 1 = yes, 0 = no, 2 = unclear

Was the activity assay clearly described for replication? 1 = yes, 0 = no, 2 = unclear

Activity test results were not interpreted with prior 

knowledge of genotyping results? 1 = yes, 0 = no, 2 = unclear

Genotyping results were not interpreted with prior 

knowledge of enzymatic activity? 1 = yes, 0 = no, 2 = unclear

Were the clinical data availability before testing was 

undertaken similar to that in usual practice setting in 

which the testing will be performed? 

1 = yes, 0 = no, 2 = unclear

Were non-interpretable or equivocal test results 

reported? 1 = yes, 0 = no, 2 = unclear

Were withdrawals from the study explained? 1 = yes, 0 = no, 2 = unclear

Was deviation from Hardy-Weinberg equilibrium 

reported? 1 = yes, 0 = no, 2 = unclear

Reviewer’s overall risk of bias assessment 1 = Good; 2 = Fair, 3 = Poor

Reason for overall risk of bias assessment (explain why 

good if good, explain why not good if fair or poor) specify

Abbreviations: I/E = inclusion / exclusion; Ref id = unique reference identification number.
Key question 2, 3a, 3b (continued)

B - 6

Laboratory Survey Introductory Letter

Sent by email attachment, on Ottawa Health Research Institute letterhead

DATE

Dear Dr. X,

The University of Ottawa Evidence-based Practice Center (EPC) is conducting a systematic 

review on the assessment of Thiopurine Methyltransferase (TPMT) in patients prescribed 

Azathiopurine or other Thiopurine-based Drugs. 

In addition to obtaining relevant data from the literature, we are conducting a survey to obtain 

specific data from laboratories that are involved in measuring TPMT. We are very interested in 

gathering data from your organization and invite you to participate in our survey. We would be 

happy to share with you the results of the survey as well as the final report. All data from the 

survey will be pooled ensuring anonymity. No organization will be identified in the report. The 

survey should take no more than 15-20 minutes to complete and we will give you three weeks to 

complete it. Please note all data will be housed for 15 years after termination of the study.

At this time we’d like to inquire into whether you are interested in participating in the survey. If 

so, we would greatly appreciate a response on this matter by DATE.

In your response, please include the proper name of your organization as well as the contact 

person/details.

Name of Organization: ________________________________________________

Contact name and details: ______________________________________________

If you have any questions about your rights as a research participant, you may contact the Board 

Chairman at 613-798-5555 ext 14902.

Sincerely,

Sophia Tsouros

Research Coordinator, University of Ottawa Evidence-Based Practice Centre

Clinical Epidemiology Program, Ottawa Hospital Research Institute

501 Smyth Road W0575

Box 208

Ottawa, Ontario, CANADA K1H 8L6 

Ph: 613.737.8899 ext. 73920

Fax: 613.737.8781

Email: stsouros@ohri.ca

On behalf of Dr. Ronald Booth, Clinical Chemist, Ottawa Hospital Research Institute, Clinical 

Lead, TPMT review and Dr. David Moher, Director of EPC
Key question 2, 3a, 3b (continued)

B - 7

Survey for “Thiopurine Methyltransferase” Laboratories

The University of Ottawa Evidence-based Practice Center is conducting a systematic review on 

assessment of Thiopurine Methyltransferase (TPMT) in patients prescribed Azathiopurine or 

other Thiopurine-based Drugs. 

In addition to obtaining relevant data from the literature, we are conducting a survey to obtain 

specific data from laboratories that are involved in measuring TPMT. We are very interested in 

gathering data from your organization and invite you to participate in our survey. We would be 

happy to share with you the results of the survey as well as the final report. All data from the 

survey will be pooled ensuring anonymity. No organization will be identified in the report. The 

survey should take no more than 15-20 minutes to complete.

Name of Organization (text box) 

Contact name (text box) 

1) Does your laboratory perform analysis of Thiopurine Methyltransferase (TPMT) inhouse?

 No 

 Genotyping only 

 Phenotyping (Enzymatic analysis) only

Both Genotyping and Phenotyping

a. If no, do you refer specimens out for analysis of TPMT?

Yes

No

b. If yes to 1a, where do you send your specimens for analysis of TPMT, and what 

type of analysis is done (genotyping or phenotyping)?

(Text box) 

2) What method is used for analysis of TPMT?

a. Enzymatic assay - please select detection method (select all that apply):

i. Radiometric, 

ii. HPLC, 

iii. Mass Spec., 

iv. Other (please specify) (Text box)

b. Genetic analysis - please select method (select all that apply):

i. Sequencing

ii. PCR/RFLP

iii. Real Time PCR

iv. Site directed PCR (please indicate mutations screened for)

v. Other (please specify) (Text box)
Key question 2, 3a, 3b (continued)

B - 8

c. If mutation specific genetic analysis is performed please indicate all alleles 

targeted (select all that apply):

i. TPMT*2

ii. TPMT*3A

iii. TPMT*3B

iv. TPMT*3C

v. Other (please list) (Text box)

3) What is your yearly TPMT testing volume (indicate separately for both genotyping and 

phenotyping if applicable)? (Text box)

4) What is the preferred specimen type for analysis 

a. Enzymatic analysis

i. Not applicable

ii. EDTA whole blood

iii. Heparinized whole blood

iv. Other (please indicate) (Text box)

b. Genotyping

i. Not applicable

ii. EDTA whole blood

iii. Heparinized whole blood

iv. Other (please indicate) (Text box)

5) What are the pre-analytical requirements for analysis

a. Specimen stability prior to analysis 

i. Enzymatic analysis

1. Not applicable

2. Specimen stability time (please indicate number of hours) 

(Text box) 

3. Storage temperature (room temperature, 4 C, -20 C, or -70 C) 

(Text box) 

ii. Genotyping

1. Not applicable

2. Specimen stability time (please indicate number of hours) 

(Text box) 

3. Storage temperature (room temperature, 4 C, -20 C, or -70 C) 

(Text box) 

b. Do you require a list of medications the patient is currently taking?

i. Yes

ii. No

6) What are the quality control results for:

a. Enzymatic analysis 

i. Coefficient of Variation

1. Within run (Text box)
Key question 2, 3a, 3b (continued)

B - 9

2. Between runs (Text box) 

b. Genotyping 

i. Percent concordance with another genotyping method and/or genotyping 

standards from an external source. Please indicate the percentage the

comparator genotyping methods and the basis of the comparison.

(Text box)

c. Enzymatic analysis in comparison with genotyping

i. Percent concordance between TPMT phenotyping and for each range of 

enzymatic activity reported by your laboratory.

(Text box)

7) Do you participate in an External Quality Assurance (EQA) program?

(Please indicate details of the program) (Text box)

8) What type of quality control is performed?

a. In-house (patient pool or other – please indicate) (Text box) 

b. Commercial (please indicate supplier) (Text box)

c. Please indicate number of quality control levels (1, 2, 3, more) and quality 

control frequency (per-run, every day, multiple per day) (Text box) 

9) Reporting of results

a. Are abnormal results called to the physician

i. No

ii. Yes (under what circumstances) (Textbox)

b. Please indicate your reference intervals for normal (homozygous wild type), 

heterozygote, and deficient as well as units of measure. (Text box)

c. How was your reference interval determined?

i. Correlation with known homozygous normal patients

ii. Other (please explain) (Text box)

d. Do you highlight suspected heterozygous patients when analyzed by enzymatic 

assay 

i. No

ii. Yes (under what circumstances) (Text box)

10) Please provide your expected turn around time of the laboratory testing you provide. 

(Text box)

11) Please provide details of the charges associated with laboratory testing in your native 

currency. (Please specify currency)

a. TPMT enzymatic activity test (Text box) 

b. TPMT genotyping(Text box)
B - 10

Key Question (2), 3a, 3b

(No studies were identified to answer Key Question 2)

TPMT status to guide therapy - Cohort studies

Data element Comments, coding for data extraction

Ref id 

First author, Year

Study site and setting 

Full or partial industry funding 0 = no, 1= yes, 2 = not reported 

Total number of participants included 

in the study

Describe population characteristics 

and setting

(a few words to describe the study population 

in terms of age, ethnicity, underlying diseases 

and their severity, and practice setting)

Followup period

Number of person weeks by groups 

Number lost to followup

Intervention group description 

(including type of TMPT testing and 

treatment modification). n

Intervention group is the group which had 

TPMT pre-testing followed by thiopurine 

treatment adjustment 

Control group description (no testing 

and no treatment modification OR

testing but no treatment modification 

based on its results). n

Control group had no treatment adjustment 

based on TPMT pre-testing

Treatment and dosage intervention 

group

0= no; 1 = AZA, 2 = 6-MP, 3 = mixed 

thiopurines, 4 = other, 5 = NR

Treatment and dosage in the control 0= no; 1 = AZA, 2 = 6-MP, 3 = mixed 

thipurines, 4 = other, 5 = NR

Outcome, and definition mortality, SAE, WDAE, all infections, sepsis, 

hospitalization, ICU admission, myelotoxicity, 

leukopenia, neutropenia

n experiencing the outcome

Confounding factors controlled for state factors 

Crude RR (intervention/control)

Adjusted RR (adjusted for?) 

(intervention/control)

Risk difference

Proportion of those with outcome of 

interest in the intervention group n/N

N = total number in the group

Proportion of those with the outcome 

of interest in the control group, n/N

N = total number in the group

Were investigators and participants 

blinded to testing?

0 = no, 1 = yes, 2 = unclear
Key question 2, 3a, 3b (continued)

B - 11

Data element Comments, coding for data extraction

Was there a blinded assessment of 

outcomes?

0 = no, 1 = yes, 2 = unclear

Were groups similar in characteristics 

at baseline 

0 = not similar, 1 = similar, 2 = unclear

Were outcomes adequately described? 1 = reasonably adequately, 0 = inadequately, 

2 = unclear

Was there an intention to treat 

analysis?

0= no, 1 = yes, 2 =unclear

Is there a conflict of interest? 1 = yes, 2 = likely, 3 = unlikely

Was there a high loss to follow-up or 

important differential loss to follow-up 

between groups? 

0 = no, 1 = yes, 2 = unclear

Is the sample size adequate? 0 = no, 1 = yes, 2 = unclear

Was the selection of participants 

unlikely to be biased? (e.g. inception 

cohort or method to avoid selection 

bias)

0 = no, 1 = yes, 2 = unclear

Were methods to control confounding 

(e.g. matching, restriction to 

subgroups, analytic methods of 

stratification, propensity scores etc.) 

employed? 

0 = no, 1 = yes, 2 = unclear

Were the method of TPMT testing (as 

applicable, e.g. genotyping, or 

phenotyping or both) appropriate?

0 = no, 1 = yes, 2 = unclear

Reviewer’s overall risk of bias 

assessment for the study

1 = Good; 2 = Fair, 3 = Poor

Reason for overall risk of bias 

assessment (explain why good if good, 

explain why not good if fair or poor)

specify

Applicability based on population 

characteristics and testing 

methodology 

1 = applicable, 0 = not applicable (explain 

why), 2 questionable applicability (explain 

why)

Abbreviations: 6-MP = 6-mercaptopurine; AZA = azathioprine; ICU = intensive care unit; n = number with 

characteristic of interest; N = total number in group or trial or study; NR = not reported; RR = relative risk; Ref id = 

unique reference identification number.
B - 12

Key question 3c - phenotyping

Association between TPMT status as determined by phenotyping, and adverse events

. 

Data element Comments, coding for data extraction

Ref id 

First author, year

Thiopurine treatment tailored 

according to TPMT genotype?

0 = no, 1 = yes (if yes do not data extract the 

study on this sheet. That is, the study does not 

qualify for Q3c)

Companion ref id If applicable

Author contacted 1 = yes, 0 = no

Full or partial industry funding 0 = no, 1= yes, 2 = not reported 

Region of participants 

residence

1= North America; 2 = Europe, 3 = central and 

South America, 4 = sub Saharan Africa, 5 = 

Middle East and north Africa, 6 = South Asia, 7 

= Asia, 9 = Oceania, 10 = 1+ 2, 11 = mixed 

others, 12 = not reported 

Setting 1= inpatient, 2 = outpatient primary care, 3 = 

outpatient specialty clinic, 4 = NR or can't tell

Participants (a few words to describe the study population)

Participants, summary 

inclusion/exclusion criteria 

summary 

Underlying disease(s) including severity 

Thiopurine treatment following 

TPMT testing?

0= no; 1 = AZA, 2 = 6-MP, 3 = mixed thipurines, 

4 = other, 5 = NR

Concomitant treatment 

affecting TPMT activity 

1 = no, 2 = allopurinol, 3 = 5-ASA 

(mesalazine/mesalamine/sulphasalazine, 

olsalazine), 4 = furosemide/frusemide, 5 = 

NSAID, 6 = other (specify), 7 = NR 

Drug dosage e.g. X mg/day

Age group 1 = adults (>=16) only, 2 = children only (<16), 3 

= adolescence only, 4 = mixed, 5 = not sure/ NR

Age in years mean or median or range

Females % 

Caucasians %

African descent %

Hispanic %

Asians % 

Other % specify race and % of participants 

This report also has relevant 

subgroup data for

specify subgroup

Study duration, or duration of 

followup for outcome 

assessment

weeks

Describe TPMT activity assay 1 = RA; 2 = Mass spectrometry; 3 = HPLC, 4 = 

Other (specify)

Cut of, or range, for TPMT high 

activity

state NR if not reported
Key question 3c – phenotyping (continued)

B - 13

Data element Comments, coding for data extraction

Cut of, or range, for TPMT 

normal activity

state NR if not reported

Cut of, or range, for TPMT 

intermediate activity

state NR if not reported

Cut of, or range, for TPMT 

low/absent activity

state NR if not reported

Study design 1 = cohort (retrospective or prospective), 2 = 

case-control, 3 = cross-sectional, 4 = other 

(specify) 

In this report, was there clearly 

no biased assessment of 

outcomes and/or phenotyping 

results based on a prior 

knowledge of each other? 

1 = yes, 2 = no, 3 = unclear

Were the groups similar at 

baseline? 

1 = yes, 2 = no, 3 = unclear

Were outcomes described 

adequately? 

1 = yes, 2 = no, 3 = unclear

Was there an intention to treat 

analysis?

0= no, 1 = yes, 2 = can't tell

Is there a potential for financial

conflict of interest?

1 = yes, 2 = no, 3 = unclear

Was there a high loss to followup or important differential loss 

to follow-up between groups?

1 = yes, 2 = no, 3 = unclear

Is the sample size adequate? 1 = yes, 2 = no, 3 = unclear

Was the selection of 

participants unlikely to be 

biased? (e.g. inception cohort 

or method to avoid selection 

bias)

1 = yes, 2 = no, 3 = unclear

Were methods to control 

confounding (e.g. matching, 

restriction to subgroups, 

analytic methods of 

stratification, propensity 

scores etc.) employed? 

1 = yes, 2 = no, 3 = unclear

Were the method of TPMT 

testing (as applicable, e.g. 

genotyping, or phenotyping or 

both) appropriate?

1 = yes, 2 = no, 3 = unclear

Reviewers overall risk of bias 

assessment for the study

1 = Good; 2 = Fair, 3 = Poor

Reason for overall risk of bias 

assessment (explain why good 

if good, explain why not good if 

fair or poor)

specify

Applicability based on 

population characteristics and 

testing methodology 

1 = applicable, 2 = not applicable (explain why), 

3= questionable applicability (explain why)

N total recruited (i.e. included 

in the study)
Key question 3c – phenotyping (continued)

B - 14

Data element Comments, coding for data extraction

N total analyzed

Dichotomous Outcome 1 = mortality, 2 = any infection (including 

sepsis), 3 = sepsis, 4 = hospitalization, 5 = ICU 

admission, 6 = WDAE, 7 = SAE, 8 = HQoL 

(describe instrument), 9 = hepatitis, 10 = 

pancreatitis, 11 = myelotoxicity, 12 = 

leukopenia, 13 = neutropenia, 14 = anemia, 15 

= thrombocytopenia, 16 = number in need of 

thiopurine dose reduction, 17 = number in need 

of switching to another immunomodulating 

treatment 

Definition (or details) of 

dichotomous outcomes where 

applicable

Continuous outcomes 1 = any infection (including sepsis), 2 = sepsis, 

3 = HQoL (describe instrument), 4 = hepatitis, 4 

= pancreatitis, 6 = myelotoxicity, 7 = leukopenia, 

8 = neutropenia, 9 = anemia, 10 = 

thrombocytopenia, 11 = number in need of 

thiopurine dose reduction, 12 = number in need 

of switching to another immunomodulating 

treatment 

Definition (or details) of 

continuous outcomes where 

applicable

Low or absent activity: n with 

events

Low or absent activity: n 

without events

Intermediate activity: n with 

events

Intermediate activity: n without 

events

Normal activity: n with events

Normal activity: n without 

events

Continuous outcome mean 

(specify whether its is mean 

change, post treatment mean, 

or percentage mean change) 

with unit

Continuous outcome --

applicable group

e.g. all homozygotes or all heterozygotes etc.

Measure of dispersion (SD or 

SE)

N analyzed for the group

If not eligible for extraction, 

provide reasons here

Abbreviations: HPLC = high performance liquid chromatography; HQoL = health related quality of life; ICU = 

intensive care unit; n = number with characteristic of interest; N = total number in group or trial or study; NR = not 

reported; RA = radiolabel assay; Ref id = unique reference identification number; SD = standard deviation; SE = 

standard error; SEA = serious adverse events; TPMT = thiopurine methyltransferase; WDAE = withdrawal due to 

adverse events.
B - 15

Key question 3c - genotyping

Association between TPMT status as determined by genotyping, and adverse events

Data element Comments, coding for data extraction

Ref id 

First Author, year

Thiopurine treatment tailored 

according to TPMT genotype?

0 = no, 1 = yes (if yes do not data extract the 

study on this sheet. That is, the study does not 

qualify for Q3c

Companion ref id (if applicable)

Author contacted? 1 = yes, 0 = no

Full or partial industry funding 0 = no, 1= yes, 2 = not reported 

Region of participants 

residence

1= North America; 2 = Europe, 3 = central and 

South America, 4 = sub Saharan Africa, 5 = 

Middle East and north Africa, 6 = South Asia, 7 

= Asia, 9 = Oceania, 10 = 1+ 2, 11 = mixed 

others, 12 = not reported 

Setting 1= inpatient, 2 = outpatient primary care, 3 = 

outpatient specialty clinic, 4 = NR or can't tell

Participants (a few words to describe the study population)

Participants, summary 

inclusion/exclusion criteria 

summary 

Underlying disease (s) including severity 

Type of thiopurine treatment? 0= no; 1 = AZA, 2 = 6-MP, 3 = mixed 

thiopurines, 4 = other, 5 = NR

Drug dosage e.g. x mg/day

Concomitant treatment 

potentially affecting TPMT 

activity 

1 = no, 2 = allopurinol, 3 = 5-ASA 

(mesalazine/mesalamine/sulphasalazine, 

olsalazine), 4 = furosemide/frusemide, 5 = 

NSAID, 6 = other (specify), 7 = NR 

Age group 1 = adults (>=16) only, 2 = children only (<16), 3 

= adolescence only, 4 = mixed, 5 = not sure/ NR

Age in years mean or median or range

Percentage females 

Caucasians %

African descent %

Hispanic %

Asians % 

Other % specify race and % of participants 

This report also has relevant 

subgroup data for

specify subgroup

Study duration, or duration of 

follow-up for outcome 

assessment

weeks
Key question 3c – genotyping (continued)

B - 16

Describe genotyping assay 1 pyrosequencing; 2 = PCR, 3 = other (specify)

Number of TPMT alleles tested

Specify TPMT Alleles tested

Study design 1 = cohort (retrospective or prospective), 2 = 

case-control, 3 = cross-sectional, 4 = other 

(specify) 

Were genotype groups 

comparable?

1 = yes, 0 = no, 2 = unclear (Subjects should 

have been matched by ethno-geographic and 

ancestral origin and confounding variables (e.g. 

social indicators) or by clearly describing 

techniques of matching by genetic background 

to avoid confounding by population 

stratifications. If not, genomic control to test for 

patterns in unlinked markers should be 

undertaken (i.e. results were adjusted for 

additional genetic markers associated with 

ancestry in the population). Also, subjects 

should be matched for characteristics that are 

also likely to be genetically determined and also 

associated with the outcome of interest (e.g. 

weight). Consider control for clinical and other 

modifiers (e.g. diet and smoking) between 

genotypes)

Were participants enrolled in 

studies without prior 

knowledge of genotype?

1 = yes, 0 = no, 2 = unclear

In this report, was there clearly 

no biased assessment of 

outcomes and/or genotyping 

results based on prior 

knowledge of each other? (i.e. 

blinded for outcomes and/or 

genotyping results)

1 = yes, 0 = no, 2 = unclear

Was genotyping method 

described adequately? 

1 = yes, 0 = no, 2 = unclear (how samples were 

handled, what genotyping methods was used, 

any quality checks, rules set to say when 

genotyping results would be considered valid)

Was reliability of genotyping 

method established?

1 = yes, 0 = no, 2 = unclear (i.e. reference to a 

paper where genotyping assay reliability was 

assessed or re-genotyping of all/random subset 

of samples)

Is the extent of ambiguous 

results due to genotyping error 

reported? 

1 = yes, 0 = no, 2 = unclear (i.e. reference to a 

paper where genotyping assay reliability was 

assessed or re-genotyping of all/random subset 

of samples)

Was Hardy-Weinberg 

equilibrium tested?

1 = yes, 0 = no, 2 = unclear (in cohort studies, 

HWE should have been tested in the whole 

study population, but in the case-control in the 

controls only, which represent the general 

population)

Was gene-gene interaction 

assessed? 

1 = yes, 0 = no, 2 = unclear
Key question 3c – genotyping (continued)

B - 17

Did studies assess compliance 

with thiopurine treatment (and 

correct for differences where 

applicable)? 

1 = yes, 0 = no, 2 = unclear

Did studies utilize different 

sources of DNA for all patients, 

introducing potential 

measurement bias?

1 = yes, 0 = no, 2 = unclear

Was lost to followup reported? 1 = yes, 0 = no, 2 = unclear

Was there possibility of 

survival bias? For example, 

hyper-normal population (older 

people, free of disease) can 

introduce survival bias

1 = yes, 0 = no, 2 = unclear

Is there a potential for financial

conflict of interest?

1 = yes, 0 = no, 2 = unclear

Reviewers overall risk of bias 

assessment for the study

1 = Good; 2 = Fair, 3 = Poor

Reason for overall risk of bias 

assessment (explain why good 

if good, explain why not good if 

fair or poor)

specify

Applicability based on 

population characteristics and 

testing methodology 

1 = applicable, 2 = not applicable (explain why), 

3= questionable applicability (explain why)

N total recruited (i.e. included 

in the study)

N total analyzed

Dichotomous Outcome 1 = mortality, 2 = any infection (including 

sepsis), 3 = sepsis, 4 = hospitalization, 5 = ICU 

admission, 6 = WDAE, 7 = SAE, 8 = HQoL 

(describe instrument), 9 = hepatitis, 10 = 

pancreatitis, 11 = myelotoxicity, 12 = 

leukopenia, 13 = neutropenia, 14 = anemia, 15 

= thrombocytopenia, 16 = number in need of 

thiopurine dose reduction, 17 = number in need 

of switching to another immunomodulating 

treatment 

Definition (or details) of 

outcomes where applicable

TPMT*2 homozygous: n with 

events

TPMT*2 homozygous: n 

without events

TPMT*2 heterozygous: n with 

events

TPMT*2 heterozygous: n 

without events

TPMT*3A homozygous: n with 

events

TPMT*3A homozygous: n 

without events

TPMT*3A heterozygous: n with 

events
Key question 3c – genotyping (continued)

B - 18

TPMT*3A heterozygous: n 

without events

TPMT*3C homozygous: n with 

events

TPMT*3C homozygous: n 

without events

TPMT*3C heterozygous: n with 

events

TPMT*3C heterozygous: n 

without events

TPMT wild type: n with events 

TPMT wild type: n without 

events 

All homozygotes for TPMT 

allelic variant: n with events

All homozygotes for TPMT 

allelic variant: n without events

All heterozygotes for TPMT 

allelic variant: n with events

All heterozygotes for TPMT 

allelic variant: n without events

If not eligible for extraction, 

provide reasons here

Allele specific subgroups 

Across alleles

Abbreviations: HPLC = high performance liquid chromatography; HQoL = health related quality of life; ICU = intensive 

care unit; n = number with characteristic of interest; N = total number in group or trial or study; NR = not reported; 

PCR = polymerase chain reaction; Ref id = unique reference identification number; SEA = serious adverse events; 

TPMT = thiopurine methyltransferase; WDAE = withdrawal due to adverse event
B - 19

Key Question 4

Costs of TPMT testing, costs of care, and costs of treating drug-associated complications

Data element Comments, coding for data extraction

Ref id 

First author, year

Companion ref id (if applicable)

Author contacted? 1 = yes, 0 = no

Country 

Type of Study E.g. Cost-effectiveness analysis, cost analysis 

Population Characteristics 

E.g. type and severity of chronic autoimmune

disease, ethnic and age distribution

Perspective

Costs of adverse events and care

Currency

Year of costing data

Source of cost data 

Costing item E.g. myelosuppression, ICU stay, etc. 

Type of costing data E.g. one time annual cost 

Cost 

Converted to USD 2008 (inflation and 

conversion rate) Must use the PPP for this

Costs of testing

Currency E.g. USD

Year of costing data

Source of cost data

Type of test

Costing details

Cost 

Converted to USD 2008 (inflation and 

conversion rate) 

Cost of treating drug-associated complications

Currency

Year of costing data

Source of cost data 

Type of drug e.g., AZA, 6-MP, AZA & 6-MP

Costing item e.g., myelosuppression due to AZA

Type of costing data E.g. one time annual cost 

Cost Denomination and year

Converted to USD 2008 (inflation and 

conversion rate) Must use the PPP for this

Other costs reported in study?

Abbreviations: AZA = azathioprine; 6-MP = 6-mercaptopurine; ICU = intensive care unit; PPP = purchasing power 

parities and the consumer price index, as described in the methods; Ref id = unique reference identification number; 

TPMT = thiopurine methyltransferase; USD = United States dollars
C - 1

Appendix C: Evidence Tables

Additional data are presented here for Key Questions 1b, 1c, 2, 3a, 3b, 3c and 4

Reference list is provided at the end of the document

Tables

Table C-1. KQ 1b: Precision and reproducibility of TPMT status determinations............ 3

Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – patient and 

study characteristics, part 1................................................................................................. 6

Table C-3. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – patient and 

study characteristics, part 2............................................................................................... 11

Table C-4. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias 

assessment of included studies, part 1 .............................................................................. 14

Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias 

assessment of included studies, part 2 .............................................................................. 16

Table C-6. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – summary 

data.................................................................................................................................... 23

Table C-7 KQ 2: TPMT status and change in management – characteristics and results of 

included study ................................................................................................................... 28

Table C-8 KQ 2: TPMT status and change in management – risk of bias assessment of included 

study, part 1....................................................................................................................... 29

Table C-9 KQ 2: TPMT status and change in management – risk of bias assessment of included 

study, part 1....................................................................................................................... 29

Table C-10 KQ 3a: TPMT status to guide therapy, clinical outcomes – characteristics and results 

of included study............................................................................................................... 30

Table C-11 KQ 3a: TPMT status to guide therapy, clinical outcomes – Risk of bias assessment of 

included study, part 1........................................................................................................ 30

Table C-12 KQ 3a: TPMT status to guide therapy, clinical outcomes – Risk of bias assessment of 

included study, part 2........................................................................................................ 31

Table C-13. KQ 3b: TPMT status to guide therapy, intermediate outcomes – characteristics of 

included studies................................................................................................................. 32

Table C-14. KQ 3b: TPMT status to guide therapy – risk of bias assessment of included study, 

part 1 ................................................................................................................................. 34

Table C-15. KQ 3b: TPMT status to guide therapy – risk of bias assessment of included study, 

part 2 ................................................................................................................................. 34

Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity –

patient and study characteristics, part 1 ............................................................................ 35

Table C-17. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity –

patient and study characteristics, part 2 ............................................................................ 42

Table C-18. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk 

of bias assessment, part 1.................................................................................................. 44

Table C-19. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk 

of bias assessment, part 2.................................................................................................. 46

Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity –

summary data .................................................................................................................... 49
C - 2

Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and 

study characteristics, part 1............................................................................................... 56

Table C-22. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and 

study characteristics, part 2............................................................................................... 66

Table C-23. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias 

assessment, part 1.............................................................................................................. 68

Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias 

assessment, part 2.............................................................................................................. 71

Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary 

data.................................................................................................................................... 81

Table C-26. KQ 4: Costs of TPMT testing....................................................................... 98

Table C-27. KQ 4: Costs of treating azathioprine-associated complications................. 100
3

Key Question 1b: Within and between laboratory precision and 

reproducibility of enzymatic measurement of TPMT and 

determination of TPMT allelic polymorphisms

Table C-1. KQ 1b: Precision and reproducibility of TPMT status determinations

Study Full or 

partial 

industry 

funding 

Study 

region

TPMT activity 

assay method 

particulars*

Inter-assay 

coefficient of 

variance 

(%)

Intra-assay 

coefficient of 

variance (%)

HPLC TPMT assays

Unless otherwise noted, TPMT is measured as the formation of 6-MMP from 6-MP, using 

nonradio-labelled SAM for the methyl donor, in RBC lysates.1,2

Lennard

20013

No Europe HPLC TPMT 

assay 

previously 

described2,4

5-9% 6.3%

JacqzAigrain, 

19941

NR Europe HPLC TPMT 

assay

6.2% (n=5) 8.9% (n=5)

Indjova

20035

NR Europe HPLC TPMT 

assay

4.4-4.9% 6.8%

Shipkova

20046

NR Europe HPLC TPMT 

assay

Pooled RBC: 5%; 

Control: 2.4% 

Pooled RBC: 

7.6%; 

Control: 2.4%

Zhang 

20077

No Asia HPLC TPMT 

assay

Control: 4.29%; 

Author control: 

3.29%

Control: 4.29%; 

Author control: 

3.25%

Indjova

20038

NR Europe HPLC TPMT 

assay

previously 

described5. 

Pooled RBC: <5% 

Control: <2.4%

Pooled RBC: 

<7.6% 

Control: <2.4%

Anglichea

u

20029

NR Europe HPLC TPMT 

assay 

- No extraction 

step. 

6.0% (n=5) 9.5% (n=5)

Dervieux

200110

No Europe HPLC TPMT 

assay 

previously 

described1.

2.6% (n=5) 5.2% (n=8)

Dervieux

199911

No Europe HPLC TPMT 

assay 

previously 

described1,12

2.6% (n=5) 5.2% (n=8)

Menor

200113

No Europe HPLC TPMT 

assay 

2.4-2.8% 4.2-6.3%

GaniereMonteil

199914

NR Europe HPLC TPMT 

assay with liquid–

liquid extraction 

using a pH 9.5 

NH4Cl buffer 

6.1% (n=56) 9,5% (n=56)
Table C-1. Precision and reproducibility of TPMT status determinations

C - 4

Study Full or 

partial 

industry 

funding 

Study 

region

TPMT activity 

assay method 

particulars*

Inter-assay 

coefficient of 

variance 

(%)

Intra-assay 

coefficient of 

variance (%)

Escousse

199815

NR Europe HPLC assay 

previously 

described1,16

8.4-9.4% (n=3) 0–8.2% (n=5)

Medard

199712

NR Europe HPLC TPMT 

assay 

previously 

described1

. 

- Following 

incubation, liquidliquid extraction 

replaced liquidsolid extraction.

- Improved 

chromatographic

conditions. 

6.7-10.2% 

(n=21)

2.7-6.9% 

(n=5)

JohnsonDavis

200817

NR North 

America

HPLC TPMT 

assay 

0.2-0.8% 

(n=20)

3.2-8.7% 

(n=20)

Khalil

200518 Europe HPLC TPMT assay 

- Simplified by 

eliminating time 

consuming 

extraction steps 

with organic 

solvents, a 

heating step and 

gradient elution.

1.4-4.9%

(n=10)

1% 

(n=12)

GaniereMonteil

200419

NR Europe HPLC TPMT 

assay

previously 

described1,14.

1.7–4.3% (n=6) 0.8–6.0% (n=6)

Zhang

200620

NR Asia HPLC TPMT 

assay

previously 

described.1,14

4.29% 4.29%
Table C-1. Precision and reproducibility of TPMT status determinations

C - 5

Study Full or 

partial 

industry 

funding 

Study 

region

TPMT activity 

assay method 

particulars*

Inter-assay 

coefficient of 

variance 

(%)

Intra-assay 

coefficient of 

variance (%)

KeizerGarritsen

200321

NR Europe HPLC TPMT 

assay

Between day 

(n=5): 

Supernatants 

were stored at -

23°C. 

Initial activities: 

17.2±1.6, 38.5±2.4, 

17.6±2.1, 15.9±1.4, 

12.2±2.1 

pmol/h/107 RBCs. 

After 7 days: 

19.2±1.4, 36.4±2.1, 

17.0±1.8, 

19.1±2.5, 12.8±2.4 

pmol/h/107 RBCs. 

After 25 days 

stored at -80°C: 

17.0±2.2, 34.0±2.6, 

15.5±2.4, 16.6±1.8, 

10.6±1.6 

pmol/h/107 RBCs. 

15.0±1.4 

and 

22.4±2.0 

pmol/h/107 RBCs 

(n=2).

Oselin

200622

No Europe HPLC TPMT 

assay 

Precision 4% Accuracy 

96–103%.

Kroplin

199823

NR Europe HPLC TPMT 

assay to measure 

6-MTG in RBC 

lysates. 

5% (n=20) 6.7% (n=36)

Lentz

200024

NR North 

America

HPLC TPMT 

assay to measure 

6-MTG formed 

from 6-TG in 

whole blood and 

RBC lysates. 

2–3% 4–10%

Ford

200625

No Europe HPLC TPMT 

assay to measure 

6-MTG formed 

from 6-TG in 

whole blood and 

RBC lysates. 

Standard RBCs: 

3.6% (n=10) 

Whole blood: 

2.7% 

(n=10)

Standard RBCs: 

8.0% (n=20); 

Whole blood: 

7.6% (n=20)

Ford

200426

Yes Europe HPLC TPMT 

assay to measure 

6-MTG in RBC 

lysates. 

3.4% 

(n=20)

9.1% (n=37)

Decaux

200127

Yes Europe HPLC TPMT 

assay to measure 

6-MTG in RBC 

lysates. 

4.1-4.5% 

(n=6)

6.9-8% (n = 17)

Abbreviations: 6-MP = 6-mercaptopurine; 6-MMP = 6-methyl mercaptopurine; 6-MTG = 6-methylthioguanine; HPLC 

= high pressure liquid chromatography; RBCs = red blood cells; SAM = S-adenosyl-L-methionine; SEM = standard 

error of means; TPMT = thiopurine methyl transferase.
Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – study characteristics, part 1 (continued)

C - 6

Key Question 1c: Diagnostic sensitivity and specificity of TPMT allelic polymorphism 

measurement, compared with the measurement of TPMT enzymatic activity

Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – patient and study characteristics, part 1

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria 

Thiopurine 

treatment 

Concomitant 

treatments

TPMT 

activity 

testing 

method

TPMT activity range 

and cut-off values

Genotyping 

method

SNPs 

Genotyped

Ansari, 

200828

Europe

Outpatient 

specialty 

clinic

Nonrandomized 

intervention 

study

Exclusion: 18-80 

years of age; 

previous history of 

thiopurine or 

biologics use; very 

low TPMT activity

AZA

5-ASA, 

steroids

Mass 

spectrometr

y

Low 

0-10 U/g Hb

Intermediate 

11-25 U/g Hb

Normal 

26-50 U/g Hb

NR TPMT*3A, 

*3B, *3C

Ansari, 

200229

NR

Outpatient 

specialty 

clinic 

Crosssectional

Inclusion: Diagnosis 

of IBD; current or 

past treatment with 

AZA.

Exclusion: Reliable 

data on clinical 

response or adverse 

effects not available. 

AZA

5-ASA, 

steroids

Radioassay Low 

<2.5 U/mL RBC

Intermediate 

2.5- 7.5 U/mL RBC

High 

>7.5 U/mL RBC

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3C

Gardiner, 

200830

Oceania

Inpatient and 

outpatient 

specialty 

clinics

Prospective 

observational

Inclusion: Diagnosis 

of IBD; normal or 

intermediate TPMT 

activity; first time 

treatment with AZA 

or 6-MP. 

Exclusion: Preexisting neutropenia

AZA, 6-MP

5-ASA

Radioassay Normal 

9.3- 17.6 U/mL RBC

Intermediate 

5- 9.2 U/mL RBC

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3C

Haglund, 

200431

Europe

NR

NR Mixed 

thiopurines

Radioassay Low 

<5.0 U/mL RBC

Intermediate 

PyrosequencingTPMT*2, 

*3A, *3B, 

*3C, *3D, 
Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – study characteristics, part 1 (continued)

C - 7

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria 

Thiopurine 

treatment 

Concomitant 

treatments

TPMT 

activity 

testing 

method

TPMT activity range 

and cut-off values

Genotyping 

method

SNPs 

Genotyped

Crosssectional

5-ASA, 

steroids

5.0- 9.0 U/mL RBC

Normal 

>9.0 U/mL RBC

*4, *5, *6, 

*7, *8

Hindorf, 

200632

Europe

NR

Crosssectional

Inclusion: Diagnosis 

of IBD; history of 

Metabolite or TPMT 

measurements 

between 2003 and 

2007; current or past 

treatment with 

thiopurines.

AZA

5-ASA, 

steroids

Radioassay Low 

<2.5 U/mL RBC

Intermediate

2-5-8.9 U/mL RBC

Normal 

>9.0 U/mL RBC

PyrosequencingTPMT*2, 

*3A, *3B, 

*3C, *3D, 

*4, *5, *6, 

*7, *8, 10, 

*14, *15

Hindorf, 

200433 Europe

NR

Prospective 

observational

Inclusion: Diagnosis 

of IBD; current 

treatment with AZA 

or 6-MP for at least 4 

months and 

unchanged dose for 

at least 3 months.

Exclusion: 

Repeatedly low or 

immeasurable 

metabolite levels.

AZA, 6-MP

5-ASA

Radioassay NR Polymerase 

chain 

reaction

TPMT*3A, 

*3B, *3C, 

*3D

Langley, 

200234

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis 

of autoimmune 

hepatitis; 

hypergammaglobulie

mia, elevated serum 

aminotransferase 

and liver biopsies.

Exclusion: Possible 

causes of liver 

disease.

AZA

steroids

Radioassay Low 

<5.0 U/mL RBC

Intermediate 

5.0-13.7 U/mL RBC

High

>13.7 U/mL RBC

Polymerase 

chain 

reaction

TPMT*3A, 

*3B, *3C

Lindqvist, 

200635 Europe

Outpatient 

Exclusion: Recent 

thiopurine therapy; 

history of 

AZA, 6-MP

5-ASA, 

Radioassay Low 

<3 U/mL RBC

Intermediate 

PyrosequencingTPMT*2, 

*3A, *3B, 

*3C, *3D, 
Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – study characteristics, part 1 (continued)

C - 8

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria 

Thiopurine 

treatment 

Concomitant 

treatments

TPMT 

activity 

testing 

method

TPMT activity range 

and cut-off values

Genotyping 

method

SNPs 

Genotyped

specialty 

clinic

Prospective 

observational

pancreatitis with 

thiopurines; 

pregnancy; 

breastfeeding 

steroids 3-8.9 U/mL RBC

Normal/high 

>8.9 U/mL RBC

*4, *5, *6, 

*7, *8, 10, 

*14, *15

Marinaki, 

200336

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis 

of IBD; East Indian or 

Pakistani heritage

AZA

NR

Radioassay Low 

>2.5 U/g Hb

Intermediate

2.5–7.5 U/g Hb

Normal 

7.6–14.5 U/g Hb

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3C

Okada, 

200537

Asia

NR

Crosssectional

Inclusion: Diagnosis 

of SLE; current or 

past treatment with 

AZA

AZA

NR

High 

performance 

liquid 

chromatogra

phy

NR Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3B, 

*3C

Schwab, 

200238 Europe

Outpatient 

specialty 

clinic

Retrospective 

review of 

records

Inclusion Diagnosis 

of IBD; current or 

past treatment with 

AZA.

AZA

5-ASA, 

steroids, 

other

High 

performance 

liquid 

chromatogra

phy

Low

<5 U/mL RBC

Intermediate 

5-10 U/mL RBC

Normal/high: 

>10 U/mL RBC

Denatured 

high 

performance 

liquid 

chromatogra

phy

TPMT*2, 

*3A, *3B, 

*3C, *3D

Snow, 

199539

North 

America

NR

Crosssectional

NR AZA

NR

Radioassay Low 

<5 U/mL RBC

Intermediate 

5-13.7 U/mL RBC

High 

13.8 to 25.1 U/mL RBC

NR NR
Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – study characteristics, part 1 (continued)

C - 9

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria 

Thiopurine 

treatment 

Concomitant 

treatments

TPMT 

activity 

testing 

method

TPMT activity range 

and cut-off values

Genotyping 

method

SNPs 

Genotyped

Stassen, 

200940

NR

NR

Retrospective 

review of 

records

NR AZA

NR

High 

performance 

liquid 

chromatogra

phy

Low 

<2 U/g Hb

Intermediate 

2-23.5 U/g Hb

Normal/high 

>23.5 U/g Hb

Polymerase 

chain

reaction

TPMT*2, 

*3A, *3B, 

*3C

Stocco, 

200541

Europe

Outpatient 

specialty 

clinic

Prospective 

observational

Inclusion: Diagnosis 

of IBD; current 

treatment with a 

thiopurine for at least 

3 months or having 

experienced an 

adverse effect during 

thiopurine treatment

Mixed 

thiopurines

NR

High 

performance 

liquid 

chromatogra

phy

Low 

<4 U/mL RBC

Intermediate 

4-8U/mL RBC

Normal 

8-12 U/mL RBC

High 

>12U/mL RBCs 

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3B, 

*3C

Stocco, 

200442

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis 

of IBD; child or 

young adult; current 

or past treatment 

with thiopurines 

AZA

NR

High 

performance 

liquid 

chromatogra

phy 

Intermediate 

<8.5 U/mL RBC

High 

>8.5 U/mL RBC

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3B, 

*3C

von Ahsen, 

200543

Europe

NR

Randomized 

controlled 

trial

Inclusion: Diagnosis 

of active Crohn’s 

disease; >18 years; 

prednisone treatment 

>300mg during last 4 

weeks or relapse 

within 6 months after 

steroid pulse 

therapy. 

Exclusion: History of 

cancer; preexisting 

renal or hepatic 

AZA

5-ASA, other

Radioassay Low 

<5 U/mL RBC

Intermediate 

5-9.9 nmol/mL

Normal/high >=10 

U/mL RBC

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3B, 

*3C
Table C-2. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – study characteristics, part 1 (continued)

C - 10

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria 

Thiopurine 

treatment 

Concomitant 

treatments

TPMT 

activity 

testing 

method

TPMT activity range 

and cut-off values

Genotyping 

method

SNPs 

Genotyped

disease; pregnancy; 

breastfeeding

Winter, 

200744

Europe

NR

Crosssectional

Inclusion: Diagnosis 

of IBD; current or 

past treatment with 

thiopurines.

AZA

5-ASA

Mass 

spectrometr

y 

Low 

<10 U/g Hb

Intermediate 

10-25 U/g Hb

Normal 

26-50 U/g Hb

High 

>50 U/g Hb

Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3B, * 

3C

Newman, 

201045

Europe

Outpatient 

Specialty 

Clinic

Crosssectional 

Nursing and 

pregnant women, 

and those likely to 

experience adverse 

events were 

excluded

AZA

NR

High 

performance 

liquid 

chromatogra

phy 

NR Polymerase 

chain 

reaction

TPMT*2, 

*3A, *3B, * 

3C

Hindorf, 

201046 Europe

NR

Crosssectional

Inclusion: post 

treatment AIH scores 

> 11

Mixed 

thiopurines

NR

High 

performance 

liquid 

chromatogra

phy

Normal: equal or 

above 9.0 U/mL RBC; 

Intermediate: 2-5-8.9 

U/mL RBC; Low: 

below 2.5 U/mL RBC

PyrosequencingTPMT *2, 

*3A, *3B, 

*3C, *3D, 

*4, *5, *6, 

*7, *8, and 

10 Also *14 

and *15

Abbreviations: 5-ASA = 5-aminosalicylates; 6-MP = 6-mercaptopurine; AZA = azathioprine; IBD = inflammatory bowel disease; NR = not reported; RBCs = red blood 

cells; SLE = systemic lupus erythematosus; SNP = single nucleotide polymorphism; TPMT = thiopurine methyltransferase.
11

Table C-3. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – patient and study characteristics, part 2

Study Underlying 

disease(s)

Age group

Age (years)

mean (range)

(unless otherwise 

noted)

Females 

(%)

Number 

analyzed

Number of 

dropouts, 

withdrawals 

and untested 

participants

TPMT Activity: 

Number with 

noninterpretable 

test results

Genotyping: 

Number with 

noninterpretable 

test results

Ansari, 

200828

IBD

Adults

40.3 (18 - 80) 54 192 23 NR NR

Ansari, 

200229

IBD

Adults

44 46 40 0 NR NR

Gardiner, 

200830

IBD

Adult

39.2 (35.4- 42.9) 51 68 9 NR NR

Haglund, 

200431

IBD

Mixed

38 (12-61) 43 30 0 0 0

Hindorf, 

200632

IBD

Adults

28 (18-42) (median) 47 52 NR NR NR

Hindorf, 

200433

IBD

Adults

36 (18-63) 47 55 6 0 0

Langley, 

200234

AIH 

Mixed

45 (13-72) (median) 74 53 19 NR NR

Lindqvist, 

200635

IBD

Adults

40.5 (18-76) 47 60 NA NR NR

Marinaki, 

200336

IBD

NR

NR NR 85 NR NR NR

Okada, 

200537

SLE

NR

NR NR 55 NR NR NR
Table C-3. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – patient and study characteristics, part 2 (continued)

C - 12

Study Underlying 

disease(s)

Age group

Age (years)

mean (range)

(unless otherwise 

noted)

Females 

(%)

Number 

analyzed

Number of 

dropouts, 

withdrawals 

and untested 

participants

TPMT Activity: 

Number with 

noninterpretable 

test results

Genotyping: 

Number with 

noninterpretable 

test results

Schwab, 

200238

IBD

Adults

41 (17-71) (median) 52 93 NR NR NR

Snow, 

199539

Autoimmune 

dermatologic 

conditions

Adults

NR (26-88) 39 26 2 NR NR

Stassen, 

200940

Anti-neutrophil 

cytoplasmic 

antibody 

associated 

vasculitis

NR

NR NR 108 0 NR NR

Stocco, 

200541

IBD

Mixed

14.2 (0.8-38.8) 

(median)

51 28 NR NR NR

Stocco, 

200442

IBD

Mixed

16.4 (4-38) 45 27 NR NR NR

von Ahsen, 

200543

IBD

Adults

36 (SD 11.6) 56 71 0 0 0

Winter, 

200744

IBD

NR

45 46 130 14 14 14

Newman, 

201045

Majority of IBD 

patients

Adults

42 50 333 11 NR NR
Table C-3. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – patient and study characteristics, part 2 (continued)

C - 13

Study Underlying 

disease(s)

Age group

Age (years)

mean (range)

(unless otherwise 

noted)

Females 

(%)

Number 

analyzed

Number of 

dropouts, 

withdrawals 

and untested 

participants

TPMT Activity: 

Number with 

noninterpretable 

test results

Genotyping: 

Number with 

noninterpretable 

test results

Hindorf, 

201046

Autoimmune 

hepatitis

Adults

55 (35-67) 80 229 9 NR 0

Abbreviations: AIH = autoimmune hepatitis; IBD = inflammatory bowel disease; NR = not reported; SD = standard deviation; SLE = systemic lupus erythematosus; TPMT = 

thiopurine methyltransferase.
14

Table C-4. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 1

Study Representative 

spectrum of 

patients

Clear 

Description of 

selection 

criteria

Activity 

assay 

correctly 

measures 

enzyme 

activities 

Whole or 

random 

sample 

administ

ered 

activity 

test

TPMT activity 

test 

administered 

independent 

of genotype 

results

Independence 

of TPMT 

activity and 

genotyping 

tests

Genotyping 

method 

description 

clear for 

replication 

TPMT 

activity test 

method 

description 

clear for 

replication

Ansari, 

200828

Yes Clear Yes Unclear Unclear Yes Unclear Clear

Ansari, 

200229

Unclear Clear Yes Yes Yes Yes Clear Clear

Gardiner, 

200830

No Clear Yes No Yes Yes Clear Clear

Haglund, 

200431

No Partially clear Yes Yes Yes Yes Clear Clear

Hindorf, 

200632

Unclear Clear Yes Yes Yes Yes Clear Clear

Hindorf, 

200433

No Clear Yes Yes Yes Yes Clear Clear

Langley, 

200234

Unclear Clear Yes Unclear Yes Yes Clear Clear

Lindqvist, 

200635

Unclear Clear Yes Yes Yes Yes Clear Clear

Marinaki, 

200336

Unclear Unclear Yes Yes Yes Yes Clear Clear

Okada, 

200537

Unclear Unclear Yes Unclear Unclear Yes Unclear Clear

Schwab, 

200238

Unclear Partially clear Yes Yes Yes Yes Clear Clear

Snow, 

199539

Unclear Unclear Yes Yes Yes Yes Unclear Clear

Stassen, 

200940

Unclear Unclear Yes Yes Yes Yes Clear Clear
Table C-4. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 1 (continued)

C - 15

Stocco, 

200541

Unclear Clear Yes Unclear Unclear Yes Clear Clear

Stocco, 

200442

Unclear Partially clear Yes Unclear Unclear Yes Clear Clear

von Ahsen, 

200543

Unclear Clear Yes Yes Yes Yes Clear Clear

Winter, 

200744

Unclear Unclear Yes Yes Yes Yes Clear Clear

Newman, 

201045

Unclear Clear Yes Yes Yes Yes Yes Yes

Hindorf, 

201046

Unclear Unclear Yes Yes Yes Yes Yes Yes

Abbreviations: TPMT = thiopurine methyltransferase.
16

Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Ansari, 

200828

Unclear Unclear Unclear No Yes Yes Applicable Fair - Unclear if 

TPMT testing 

was blinded. 

Ansari, 

200229

Unclear Unclear Unclear No No No Unclear -

Uncertain 

representativ

eness of 

adult IBD 

patients 

presenting to 

a specialty 

clinic.

Excluded 

patients with 

low TPMT 

enzymatic 

activity

Fair - Unclear if 

TPMT testing 

was blinded. 

HWE not 

tested.
Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

C - 17

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Gardiner, 

200830

Unclear Unclear Unclear No Yes No Unclear - Well 

representativ

e of adult IBD 

patients 

presenting to 

a 

gastroenterol

ogy clinic, 

but excluded 

patients with 

low TPMT 

enzymatic 

activity

Poor - HWE 

not tested. 

Sample not 

representative. 

Unclear if 

phenotyping 

influenced by 

genotyping 

results, and 

vice versa.

Haglund, 

200431

Unclear Unclear Unclear No Yes No Unclear -

Uncertain 

representativ

eness of 

adolescent 

and adult IBD 

patients. 

Patients were 

pre-selected 

based on 

their TPMT 

activity.

Poor - HWE 

not tested. 

Exclusion 

criteria not 

reported. 

Samples 

selected based 

on TPMT 

activity. TPMT 

testing not 

blinded. 
Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

C - 18

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Hindorf, 

200632

Unclear Unclear Unclear No No No Unclear - Not 

representativ

e of IBD 

patients, 

including 

only those 

currently or 

previously on 

thiopurines

Fair - HWE not 

reported. 

Unclear if 

genotyping 

was blinded.

Hindorf, 

200433

Unclear Unclear Unclear Unclear Yes No Unclear - Not 

representativ

e of adult IBD 

patients, 

including 

only adult 

IBD patients 

on long-term 

thiopurine 

treatment

Fair - HWE not 

reported. 

Unclear if 

TPMT testing 

was blinded. 

Langley, 

200234

Yes Yes Yes Unclear No No Unclear - Not 

representativ

e of AIH 

patients, 

including 

only those in 

a tertiary care 

setting

Poor - HWE 

not reported. 

Unclear 

representative

ness of sample 

and unclear 

random 

genotyping of 

a subset of 

patients .
Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

C - 19

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Lindqvist, 

200635

Unclear Unclear Yes Unclear Yes No Unclear - Not 

representativ

e of adult IBD 

patients, 

excluding 

those likely to 

experience 

adverse 

events.

Fair - Sample 

not 

representative. 

Unclear if 

TPMT testing 

was blinded.

Marinaki, 

200336

Unclear Unclear Unclear No No No Unclear -

Uncertain 

representativ

eness of IBD 

patients. 

Includes only 

those of 

South Asian 

ancestry.

Fair - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded.

Okada, 

200537

Unclear Unclear Unclear No No No Unclear - Not 

representativ

e of Japanese 

SLE patients, 

including 

only those in 

a tertiary care 

setting.

Poor - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded 

and whether 

testing was 

non-selective. 
Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

C - 20

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Schwab, 

200238

Unclear Unclear Unclear No No No Unclear - Not 

representativ

e of IBD 

patients, 

including 

only those in 

a tertiary care 

setting.

Fair - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded. 

Snow, 

199539

Unclear Unclear Unclear No Yes No Unclear -

Uncertain 

representativ

eness of 

autoimmune 

dermatologic 

patients. No 

description of 

testing 

methodology.

Poor - Poorly 

reported 

testing 

methodology 

and inclusion 

criteria.

Stassen, 

200940

Unclear Unclear Unclear Unclear No No Unclear -

Little to no 

reporting of 

population 

characteristic

s.

Fair – Unclear 

if TPMT testing 

was blinded.
Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

C - 21

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Stocco, 

200541

Unclear Unclear Yes No Yes No Unclear -

Uncertain 

representativ

eness of IBD 

patients.

Poor - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded. 

Unclear if 

phenotyping 

influenced by 

genotyping 

results or 

other factors. 

Stocco, 

200442

Unclear Unclear Unclear No No No Unclear - Not 

representativ

e of IBD 

patients, 

including 

only those in 

a tertiary care 

setting.

Poor - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded.

von Ahsen, 

200543

Unclear Unclear Unclear Unclear Yes No Unclear -

Unrepresenta

tive of active 

Crohn's 

disease 

patients.

Fair - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded.
Table C-5. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – risk of bias assessment of included studies, part 2

C - 22

Study TPMT 

activity test 

results 

assessed 

independent 

of 

genotyping 

results 

Genotyping 

results 

assessed 

independent 

of TPMT 

activity test 

results

Clinical data 

availability 

before 

assessing 

activity or 

genotyping 

was routine 

Reporting of 

uninterpretable 

test results

Explanation 

provided for 

study 

withdrawals

HWE 

Tested

Applicability Summary risk 

of bias 

assessment

Winter, 

200744

Unclear Unclear Unclear Yes Yes No Unclear -

Uncertain 

representativ

eness of 

patients with 

IBD. All had 

previously 

received 

thiopurines

Fair - Unclear 

representative

ness of 

sample. 

Unclear if 

TPMT testing 

was blinded.

Newman, 

201045

Unclear Unclear Yes No Yes No Unclear -

Uncertain 

representativ

eness of 

patients with 

IBD. excluded 

patients with 

important 

adverse 

events due to 

previous 

thiopurine 

exposure

Fair - Unclear 

blinded 

assessment, 

and exclusion 

of participants 

likely to 

experience 

SAE

Hindorf, 

201046

Unclear Unclear Unclear No Yes No Little 

reporting of 

patient 

characteristic

s. Unclear 

representativ

eness of AIH 

patients

Fair - Unclear 

blinded 

assessment

Abbreviations: HWE = Hardy-Weinberg equilibrium; IBD = inflammatory bowel disease; TPMT = thiopurine methyltransferase.
23

Table C-6. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – summary data

Study Genotypes Number of 

participants

Genotypes Number of 

participants

Normal and 

hyperactivity

Normal, hyperactivity 

or intermediate activity

Normal 

or 

hyperactivity

Intermediate 

or low 

activity

Normal, 

hyperactivity or 

intermediate 

activity

Low 

activity

Sensitivity Specificity Sensitivity Specificity

Ansari, 

200828 Heterozygous or 

homozygous 

for variant(s) 

1 17 Homozygous 

for variant(s)

0 0 73.9 (52.8, 

87.8)

99.4 (95.9, 

99.9)

0/0 99.7 (96, 

100)

Non-carriers 168 6 Non-carriers 

or 

heterozygous 

for variant(s) 

192 0

Ansari, 

200229

Heterozygous 

or 

homozygous 

for variant(s) 

0 10 Homozygous 

for variant(s)

0 0 95.5 (55.2, 

99.7)

98.4 (78.9, 

99.9)

0/0 98.8 (83.3, 

99.9)

Non-carriers 30 0 Non-carriers 

or 

heterozygous 

for variants

40 0

Gardiner, 

200830

Heterozygous 

or 

homozygous 

for variant(s) 

2 5 Homozygous 

for variant(s)

0 0 55.6 (25.1, 

82.3)

96.6 (87.4, 

99.2)

0/0 99.3 (89.5, 

100)

Non-carriers 57 4 Non-carriers 

or 

heterozygous 

for variants

68 0

Haglund, 

200431

Heterozygous 

or 

homozygous 

for variant(s) 

0 21 Homozygous 

for variant(s)

0 6 97.7 (72.3, 

99.9)

95 (52.5, 

99.7)

75.0 (37.7, 

93.7)

97.8 (73.2, 

99.9)

Non-carriers 9 0 Non-carriers 

or 

heterozygous 

for variant(s) 

22 2
Table C-6. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – summary data (continued)

C - 24

Study Genotypes Number of 

participants

Genotypes Number of 

participants

Normal and 

hyperactivity

Normal, hyperactivity 

or intermediate activity

Normal 

or 

hyperactivity

Intermediate 

or low 

activity

Normal, 

hyperactivity or 

intermediate 

activity

Low 

activity

Sensitivity Specificity Sensitivity Specificity

Hindorf, 

200632

Heterozygous 

or 

homozygous 

for variant(s) 

1 11 Homozygous 

for variant(s)

0 6 91.7 (58.7, 

98.8)

97.5 (84.3, 

99.6)

92.9 (42.3, 

99.6)

98.9 (85.1, 

99.9)

Non-carriers 39 1 Non-carriers 

or 

heterozygous 

for variant(s) 

46 0

Hindorf, 

200433 Heterozygous or 

homozygous 

for variant(s) 

0 5 Homozygous 

for variant(s)

0 1 91.7 (37.8, 

99.5)

99 (86.2, 

99.9)

100% 99.1 (87.1, 

99.9)

Non-carriers 50 0 Non-carriers 

or 

heterozygous 

for variant(s) 

54 0

Langley, 

200234

Heterozygous 

or 

homozygous 

for variant(s) 

4 6 Homozygous 

for variant(s)

0 0 66.7 (33.3, 

88.9)

90.9 (78.2, 

96.5)

0% 99.1 (86.6, 

99.9)

Non-carriers 40 3 Non-carriers 

or 

heterozygous 

for variant(s) 

52 1

Lindqvist, 

200635

Heterozygous 

or 

homozygous 

for variant(s) 

1 7 Homozygous 

for variant(s)

0 1 63.6 (33.9, 

85.7)

98 (86.9, 

99.7)

1/1 99.2 (88, 

99.9)

Non-carriers 48 4 Non-carriers 

or 

heterozygous 

for variant(s) 

59 0
Table C-6. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – summary data (continued)

C - 25

Study Genotypes Number of 

participants

Genotypes Number of 

participants

Normal and 

hyperactivity

Normal, hyperactivity 

or intermediate activity

Normal 

or 

hyperactivity

Intermediate 

or low 

activity

Normal, 

hyperactivity or 

intermediate 

activity

Low 

activity

Sensitivity Specificity Sensitivity Specificity

Marinaki, 

200336

Heterozygous 

or 

homozygous 

for variant(s) 

0 5 Homozygous 

for variant(s)

0 0 91.7 (37.8, 

99.5)

99.4 (90.9, 

100)

0/0 99.4 (91.4, 

100)

Non-carriers 80 0 Non-carriers 

or 

heterozygous 

for variant(s) 

85 0

Okada, 

200537 Heterozygous or 

homozygous 

for variant(s) 

1 3 Homozygous 

for variant(s)

0 0 23.1 (7.6, 

52.2)

97.6 (84.9, 

99.7)

0/0 99.1 (87.3, 

99.9)

Non-carriers 41 10 Non-carriers 

or 

heterozygous 

for variant(s) 

55 0

Schwab, 

200238

Heterozygous 

or 

homozygous 

for variant(s) 

3 5 Homozygous 

for variant(s)

0 1 91.7 (37.8, 

99.5)

96.6 (90, 

98.9)

1/1 99.5 (92, 

100)

Non-carriers 85 0 Non-carriers 

or 

heterozygous 

for variant(s) 

92 0

Snow, 

199339

Heterozygous 

or 

homozygous 

for variant(s) 

0 5 Homozygous 

for variant(s)

0 0 91.7 (37.8, 

99.5)

97.7 (72.3, 

99.9)

0/0 98.1 (76.4, 

99.9)

Non-carriers 21 0 Non-carriers 

or 

heterozygous 

for variant(s) 

26 0
Table C-6. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – summary data (continued)

C - 26

Study Genotypes Number of 

participants

Genotypes Number of 

participants

Normal and 

hyperactivity

Normal, hyperactivity 

or intermediate activity

Normal 

or 

hyperactivity

Intermediate 

or low 

activity

Normal, 

hyperactivity or 

intermediate 

activity

Low 

activity

Sensitivity Specificity Sensitivity Specificity

Stassen, 

200940

Heterozygous 

or 

homozygous 

for variant(s) 

0 7 Homozygous 

for variant(s)

0 0 93.8 (46.1, 

99.6)

99.5 (92.7, 

100)

0/0 99.5 (93.1, 

100)

Non-carriers 101 0 Non-carriers 

or 

heterozygous 

for variant(s) 

108 0

Stocco, 

200541 Heterozygous or 

homozygous 

for variant(s) 

0 3 Homozygous 

for variant(s)

0 0 87.5 (26.6, 

99.3)

98.1 (75.6, 

99.9)

0/0 98.3 (77.7, 

99.9)

Non-carriers 25 0 Non-carriers 

or 

heterozygous 

for variant(s) 

28 0

Stocco, 

200442

Heterozygous 

or 

homozygous 

for variant(s) 

0 4 Homozygous 

for variant(s)

0 0 90 (32.6, 

99.4)

97.9 (74.1, 

99.9)

0/0 98.2 (77, 

99.9)

Non-carriers 23 0 Non-carriers 

or 

heterozygous 

for variant(s) 

27 0

von 

Ahsen, 

200543

Heterozygous 

or 

homozygous 

for variant(s) 

0 5 Homozygous 

for variant(s)

0 0 23.8 (10.3, 

46)

99 (86.2, 

99.9)

0/0 99.3 (89.9, 

100)

Non-carriers 50 16 Non-carriers 

or 

heterozygous 

for variant(s) 

71 0
Table C-6. KQ1c: Diagnostic sensitivity and specificity of TPMT determinations – summary data (continued)

C - 27

Study Genotypes Number of 

participants

Genotypes Number of 

participants

Normal and 

hyperactivity

Normal, hyperactivity 

or intermediate activity

Normal 

or 

hyperactivity

Intermediate 

or low 

activity

Normal, 

hyperactivity or 

intermediate 

activity

Low 

activity

Sensitivity Specificity Sensitivity Specificity

Winter, 

200744

Heterozygous 

or 

homozygous 

for variant(s) 

0 11 Homozygous 

for variant(s)

0 0 64.7 (40.4, 

83.2)

99.6 (93.4, 

100)

0/0 99.6 (94.2, 

100)

Non-carriers 113 6 Non-carriers 

or 

heterozygous 

for variant(s) 

129 1

Newman, 

201045 Heterozygous or 

homozygous 

for variant(s) 

0 35 Homozygous 

for variant(s)

0 1 94.6 (80.8, 

98.6)

99.8 (97.0, 

100)

1/1 99.8 (97.6, 

100)

Non-carriers 296 2 Non-carriers 

or 

heterozygous 

for variant(s) 

332 0

Hindorf, 

201046

Heterozygous 

or 

homozygous 

for variant(s) 

2 20 Homozygous 

for variant(s)

0 0 87.0 (66.5, 

95.7)

99.0 

(96.2, 

99.8)

0/0 99.8 (96.6, 

100)

Non-carriers 204 3 Non-carriers 

or 

heterozygous 

for variant(s) 

229 0

Abbreviations: TPMT = thiopurine methyltransferase.
28

Key question 2: Pre-testing for TPMT status, and change in management of patients with chronic 

inflammatory diseases 

Table C-7 KQ 2: TPMT status and change in management – characteristics and results of included study

Study Study 

Design 

Study 

Region

Study 

Setting

Inclusion and 

exclusion 

criteria 

Thiopurine 

treatment in 

group that was 

genotyped 

apriori 

(intervention 

group)

N=167

Thiopurine 

treatment in 

group that was 

not genotyped 

apriori (control 

group)

N=166

Intervention 

group 

description 

Control group 

description 

Number of 

participants

Results 

Newman, 

201045

RCT

Europe

Outpatient 

specialty 

clinics at 

19 study 

centers 

Inclusion: 

Adults with 

chronic 

inflammatory 

diseases, 

mostly IBD, 

eligible for 

azathioprine 

therapy

Exclusion:

Nursing and 

pregnant 

women and 

those likely to 

experience 

adverse 

events were 

excluded

AZA

Mean (SD) 

starting dose 

was 0.92 (0.61) 

in non-carriers 

and 0.67 (0.35) 

in 

heterozygous 

carriers.

There were no 

homozygous 

carriers in this 

group

AZA

Mean (SD) 

starting dose 

0.88 (0.54) in 

those later found 

out to be noncarriers and 0.94 

(0.68) in 

heterozygous 

carriers. There 

was one 

homozygous 

carrier in this 

group

Therapy was 

advised to be 

guided by 

genotyping 

results, 

however, all 

treatment 

decisions 

were at the 

discretion of 

treating 

physicians 

Patient 

management 

was at the 

discretion of 

the treating 

physician 

Outcome of interest 

(i.e. number of 

patients requiring 

dose reduction) was 

not reported. 

However, over a 4 

month period, 

genotyping did not 

significantly alter 

prescribing practice. 

Most patients in 

both groups were 

given starting doses 

lower than 

2mg/kg/day. 7.3% of 

those in whom noncarrier genotype was 

predisclosed 

received AZA doses 

>=2mg/kg/day as 

compared to 8.4% of 

those in whom noncarrier genotype was 

found out after the 

fact. Furthermore, 

there was no 
C - 29

significant 

difference between 

the two groups in 

terms of mean AZA 

prescribed dose at 

the end of the study 

period. 

Table C-8 KQ 2: TPMT status and change in management – risk of bias assessment of included study, part 1

Study Was the 

method of 

randomization 

appropriate?

Adequacy of 

allocation 

concealment?

Were 

Investigators 

and participants 

blinded to 

interventions?

Was there 

blinded 

assessment 

of 

outcomes?

Were 

groups 

similar at 

baseline? 

Were 

outcomes 

described 

adequately? 

Was there 

intentionto-treat 

analysis?

Was there 

a potential 

for 

financial 

conflict of 

interest?

Newman, 

201045

Yes Unclear No Not 

applicable

Yes Yes Yes No

Table C-9 KQ 2: TPMT status and change in management – risk of bias assessment of included study, part 2

Was there a high loss to follow-up 

or important differential loss to 

follow-up between groups? 

Was treatment 

adherence 

assessed?

Reviewers’ overall risk of bias 

assessment for the study

Applicability based on population 

characteristics and testing methodology 

No Yes Fair – concealment of 

allocation is not certain, 

potential for selection bias 

Limited applicability because there was just 

one homozygous carrier in the whole 

sample of mostly IBD patients 
C - 30

Key question 3a: TPMT status to guide therapy, clinical outcomes

Table C-10 KQ 3a: TPMT status to guide therapy, clinical outcomes – characteristics and results of included study

Study Study 

Design 

Study 

Region

Study 

Setting

Inclusion and 

exclusion 

criteria 

Thiopurine 

treatment in 

group that was 

genotyped 

apriori 

(intervention 

group)

N=167

Thiopurine 

treatment in group 

that was not 

genotyped apriori 

(control group)

N=166

Intervention 

group 

description 

Control group 

description 

Results 

(n/N)

Newman, 

201045

RCT

Europe

Outpatient 

specialty 

clinics at 

19 study 

centers 

Inclusion: 

Adults with 

chronic 

inflammatory 

diseases, 

mostly IBD, 

eligible for 

azathioprine 

therapy

Exclusion:

Nursing and 

pregnant 

women and 

those likely to 

experience 

adverse events 

were excluded

AZA

Mean (SD) 

starting dose 

was 0.92 (0.61) 

in non-carriers 

and 0.67 (0.35) 

in heterozygous 

carriers.

There were no 

homozygous 

carriers in this 

group

AZA

Mean (SD) starting 

dose 0.88 (0.54) in 

those later found 

out to be noncarriers and 0.94 

(0.68) in 

heterozygous 

carriers. There 

was one 

homozygous 

carrier in this 

group

Therapy was 

advised to be 

guided by 

genotyping 

results, 

however, all 

treatment 

decisions were 

at the 

discretion of 

treating 

physicians 

Patient 

management 

was at the 

discretion of 

the treating 

physician 

Over a 4 month 

duration, there 

were no 

significant 

differences in the 

outcomes of 

mortality (1/167 

vs.3/166); SAE 

(4/167 vs. 8/166); 

and WDAE (0/167 

vs. 0/166). Odds 

ratios of 

0.33 (0.03, 3.18), 

0.48 (0.14, 1.64) 

and nonestimibale, 

respectively

Table C-11 KQ 3a: TPMT status to guide therapy, clinical outcomes – Risk of bias assessment of included study, part 1

Study Was the 

method of 

randomization 

appropriate?

Adequacy of 

allocation 

concealment?

Were 

Investigators 

and participants 

blinded to 

interventions?

Was there 

blinded 

assessment of 

outcomes?

Were 

groups 

similar at 

baseline? 

Were 

outcomes 

described 

adequately? 

Was there 

intentionto-treat 

analysis?

Was there 

a potential 

for 

financial 

conflict of 

interest?
C - 31

Newman, 

201045

Yes Unclear No Yes for 

mortality and 

SAE, no for 

WDAE and 

other 

intermediate 

outcomes 

Yes Yes Yes No

Table C-12 KQ 3a: TPMT status to guide therapy, clinical outcomes – Risk of bias assessment of included study, part 2

Was there a high loss to follow-up 

or important differential loss to 

follow-up between groups? 

Was treatment 

adherence 

assessed?

Reviewers’ overall risk of 

bias assessment for the 

study

Applicability based on population 

characteristics and testing methodology 

No Yes Fair –

Concealment of allocation is 

not certain, potential for 

selection bias 

Limited applicability because there was just 

one homozygous carrier in the whole 

sample of mostly IBD patients 
C - 32

Key Question 3b: TPMT status to guide therapy, intermediate outcomes

Table C-13. KQ 3b: TPMT status to guide therapy, intermediate outcomes – characteristics of included studies

Study Study 

Region

Study 

Design

Study 

Setting

Inclusion 

and 

exclusion 

criteria 

Thiopurine 

treatment in 

group that 

was pretested for 

TPM status 

(intervention 

group)

Thiopurine 

treatment in 

group that 

was not pretested 

(control 

group)

Intervention group 

description 

Number of 

participants

Control group 

description 

Number of 

participants

Outcomes 

assessed

Newman, 

201045

RCT

Europe

Outpatient 

specialty 

clinics at 19 

study 

centers 

Inclusion: 

Adults with 

chronic 

inflammator

y diseases, 

mostly IBD, 

eligible for 

azathioprine 

therapy

Exclusion:

Nursing and 

pregnant 

women and 

those likely 

to 

experience 

adverse 

events were 

excluded

AZA

Mean (SD) 

starting 

dose was 

0.92 (0.61) in 

non-carriers 

and 0.67 

(0.35) in 

heterozygou

s carriers.

There were 

no 

homozygou

s carriers in 

this group

AZA

Mean (SD) 

starting 

dose 0.88 

(0.54) in 

those later 

found out to 

be noncarriers and 

0.94 (0.68) in 

heterozygou

s carriers. 

There was 

one 

homozygou

s carrier in 

this group

Therapy was 

advised to be 

guided by 

genotyping results, 

however, all 

treatment decisions 

were at the 

discretion of 

treating physicians 

167

Patient 

management was at 

the discretion of the 

treating physician 

166

A 

statistically 

significant 

difference 

was not 

observed for 

the 

outcomes 

neutropenia 

(2/167 vs. 

1/166); 

hepatitis 

(19/167 vs. 

8/166); and 

pancreatitis 

(1/167 vs. 

4/166). Odds 

ratios of 

2 (0.18, 

22.27); 

2.54 (1.08, 

5.97); and 

0.24 (0.03, 

2.21), 

respectively
C - 33

Banerjee, 

200647

Retrospectiv

e cohort

North 

America

Tertiary care 

academic 

centre

Inclusion: 

Diagnosis of 

IBD; 

pediatric 

patients 

(1.7-20 

years, mean 

14 years)

Mixed 

thiopurines 

AZA mean 

dose 1.7 

mg/kg/day; 

about 10% 

received 6-

MP dose NR

Length of 

followup, 68 

weeks

Mixed 

thiopurines

AZA mean 

dose 1.2 

mg/kg/day; 

about 10% 

received 6-

MP dose NR

Length of 

followup, 

108 weeks

TPMT status by 

enzymatic activity. 

Normal patients 

given normal 

starting dose. 

Intermediate activity 

patients given lower 

starting dose. 

Additional dose 

change based on 6-

TGN levels. 

64

TPMT not tested. 

Dose titrated based 

on clinical response 

or drug toxicity. 

7

A 

statistically 

significant 

difference 

was not 

observed for 

the outcome 

of 

leukopenia 

(study 

versus 

control 

group; 4.7 

percent 

versus 0 

percent) and 

hepatotoxici

ty (study 

versus 

control 

group; 9.4 

percent 

versus 16.2 

percent)

Abbreviations: 6-MP = 6-mercaptopurine; 6-TGN = 6-thioguanine nucleotide; AZA = azathioprine; IBD = inflammatory bowel disease; NR = not reported; TPMT = thiopurine 

methyltransferase.

.
C - 34

Table C-14. KQ 3b: TPMT status to guide therapy – risk of bias assessment of included study, part 1

Study Blinded 

participants 

and 

investigators

Blinded 

outcome 

assessment

Similarity of 

groups at 

baseline

Outcomes 

adequately 

described

Intention to 

treat analysis

Potential for 

financial 

conflict of 

interest

High loss to 

follow-up or 

differential 

loss to 

followup 

between 

groups

Newman, 

201045

See Table C-10. KQ 3a above

Banerjee, 

200647

Unclear Unclear Yes Yes Yes Unlikely No

Abbreviation: TPMT = thiopurine methyltransferase.

Table C-15. KQ 3b: TPMT status to guide therapy – risk of bias assessment of included study, part 2

Study Adequate 

sample 

size

Avoidance of 

selection bias

Methods to 

control 

confounding 

Appropriate 

TPMT test

Applicability Summary risk of bias 

assessment

Banerjee, 

200647

Unclear Unclear Unclear Yes Unclear - Uncertain of 

representativeness of 

pediatric IBD patients. 

Included on those 

previously treated with a 

stable thiopurine dose.

Poor - Only intervention 

group received regular 

metabolite monitoring with 

accordingly regular 

thiopurine adjustment -- an 

important unaccounted for 

confounding in this study 

that limits validity of 

inference drawn about the 

comparative effectiveness 

of testing from this study. 

Abbreviations: IBD = inflammatory bowel disease; TPMT = thiopurine methyltransferase
35

Key Question 3c: Association between TPMT status and thiopurine toxicity

Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

Ansari, 200229 NR

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Current or 

past treatment with 

AZA. 

Exclusion: Reliable 

data on clinical 

response or adverse 

effects not obtained. 

AZA (1.69 (NR))

5-ASA, steroids

Radioassay Low

<2.5 U/mL RBC

Intermediate

2.5-7.5 U/mL 

RBC

High

>7.5 U/mL RBC

Neutropenia

Czaja, 200648 Europe

NR

Crosssectional

Inclusion: Diagnosis 

of autoimmune 

hepatitis; current AZA 

treatment 

AZA (50 mg/day 

(median); (50-

150)

steroids

Radioassay Low

<6.3 U/mL RBC

Intermediate

6.3-15 U/mL 

RBC

Normal 

15.1-26.4 U/mL 

RBC

High

>26.4 U/mL RBC

Withdrawal due to 

adverse events
Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 36

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

Firooz, 200849 Middle East 

and North 

Africa

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Current or 

past treatment with 

AZA and 

prednisolone. 

Exclusion: Reliable 

data on clinical 

response or adverse 

effects not available; 

other variants of 

pemphigus

AZA (NR (2-3))

steroids

High 

performance 

liquid 

chromatography

Low

undetectable

Intermediate

<20 ng/mL/h 

RBC

Normal 

20-130 ng/mL/h 

RBC

High

>130 ng/mL/h 

RBC

Anemia, hepatitis, 

leukopenia, 

pancreatitis, 

withdrawal due to 

adverse events 

Gisbert, 

200650

Europe

NR

Prospective 

cohort

Inclusion: Diagnosis 

of IBD; adult; first time 

treatment with AZA

AZA (2.3 (SD: 

0.5))

5-ASA, steroids

Radioassay Low

<5 U/mL RBC

Intermediate

5-13.7 U/mL 

RBC

High

>13.8 U/mL RBC

Hepatitis, 

myelotoxicity, 

pancreatitis

Hindorf, 

200632

Europe

NR

Crosssectional

Inclusion: Diagnosis 

of IBD; history of 

metabolite or TPMT 

measurements 

between 1997 and 

2003; current or past 

treatment with 

thiopurine drugs; not 

yet started therapy

AZA (NR (NR))

5-ASA, steroids

Radioassay Low

<2.5 U/mL RBC

Intermediate

2.5–8.9 U/mL 

RBC

Normal 

>=9.0 U/mL RBC

Hepatitis, 

myelotoxicity, 

pancreatitis
Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 37

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

Kader, 200051 North 

America

NR

Crosssectional

Inclusion: <21 years of 

age; current or past 

treatment with 6-MP or 

AZA 

AZA (1.8 (1.1-

2.3)); 

6-MP (0.9 (0.42-

1.11))

NR

NR Low

<5.0 U/mL RBC

Intermediate

5.0-13.7 U/mL 

RBC

Normal 

13.8-25.1 U/mL 

RBC

Hepatitis, 

leukopenia

Lennard, 

198952

Europe and 

North 

America

NR

Case-control

Inclusion (cases): 

diagnosis of 

autoimmune disorder; 

current AZA 

treatment; acute and 

prolonged 

immunosupression 

during short course 

(<3 months) of lowdose (<3 mg/kg) AZA.

Inclusion (controls): 

current treatment with 

<3 mg/kg/day AZA for 

more than 6 months at 

unchanging dose; 

normal renal and 

hepatic function; 

recorded white blood 

cell counts above 5.0 

x 109/L. 

Exclusion: History of 

myelosuppression. 

AZA (Controls 

1.45 (median) 

(1.1-2.0); Cases 

1.9 (median) (1-

2.6))

steroids

Radioassay Low

<5 U/mL RBC

Intermediate

5-10 U/mL RBC

Normal 

>10 U/mL RBC

Anemia, 

leukopenia, 

myelotoxicity, 

thrombocytopenia
Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 38

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

Okada, 200537 Asia

NR

Crosssectional

Inclusion: Diagnosis 

of SLE; current or 

past treatment with 

AZA

AZA (NR (NR))

NR

High 

performance 

liquid 

chromatography

NR Leukopenia

Schedel, 

200653

Europe

NR

Cohort

Inclusion: Diagnosis 

of chronic 

inflammatory disease

AZA (NR (NR))

NR

Radioassay NR Leukopenia, 

myelotoxicity, 

Shah, 200854 Europe

NR

Crosssectional

Inclusion: Diagnosis 

of IBD

AZA (NR (NR))

5-ASA

NR NR Withdrawal due to 

adverse events

Snow, 199539 North 

America

NR

Crosssectional

NR AZA (NR (0.4-2.6))

None

Radioassay Low

<5 U/mL RBC

Intermediate

5.0-13.7 U/mL 

RBC

Normal 

13.8-25.1 U/mL 

RBC

Hepatitis, 

leukopenia

Stassen, 

200940

NR

NR

Chart review

NR AZA (NR (NR))

NR

High 

performance 

liquid 

chromatography

Low

<2.0 U/g Hb

Intermediate

2-23.5 U/g Hb

Normal/high 

>23.5 U/g Hb

Leukopenia
Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 39

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

Stocco, 200541 Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis 

of IBD; thiopurine 

treatment for at least 3 

months or having 

experienced an 

adverse effect during 

thiopurine treatment

AZA (2.0 (median) 

(1-4);

6-MP (NR (NR))

5-ASA

High 

performance 

liquid 

chromatography

Low

<4 U/mL RBC

Intermediate

4-8 U/mL RBC

Normal 

8-12 U/mL RBC

High

>12 U/mL RBC

Hepatitis, 

myelotoxicity, 

pancreatitis
Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 40

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

Stolk, 199855 Europe

Outpatient 

specialty 

clinic

Nonrandomized 

intervention 

study

Inclusion: Diagnosis of 

RA; 18-75 years of age; 

active disease with at 

least 3 of the following 

features: ≥3 swollen 

joints, ≥6 joints painful 

on motion or pressure, 

morning stiffness ≥45 

minutes, and erythrocyte 

sedimentation rate, ≥28 

mm/hour; no prior 

treatment with AZA. 

Exclusion: blood 

transfusion within 

previous 4 months; 

hematologic disease or 

abnormal hematologic 

parameters; kidney 

disease; liver disease; 

malignancy; acute or 

chronic infection; 

insulin-dependent 

diabetes mellitus; 

pregnancy; alcohol 

abuse; ACR functional 

class IV; use of 

medication that may 

interact with AZA or 

affect purine 

metabolism; history of 

poor compliance with 

medication regimens.

AZA (1.45 (0.6-

2.9))

None

Radioassay Low

<8.3 pmol/106 

erythrocytes/h

Intermediate

8.3-18.0 

pmol/106 

erythrocytes/h

High

18.1-39.4 

pmol/106 

erythrocytes/h

Any infection, 

hepatitis, 

myelotoxicity
Table C-16. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 41

Study Study Region

Study Setting

Study Design

Inclusion and 

exclusion criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day unless 

otherwise noted)

Concomitant 

treatments

TPMT activity 

testing method

TPMT activity 

range and cutoff values

Outcomes 

assessed

von Ahsen, 

200543

Europe

NR

Randomized 

controlled 

trial

Inclusion: Diagnosis 

of active Crohn's 

disease; >18 years of 

age; prednisone 

treatment >300 mg 

during last 4 weeks or 

a relapse within 6 

months after steroid 

pulse therapy. 

Exclusion: 

malignancy; preexisting renal or 

hepatic disease; 

pregnancy or 

breastfeeding.

AZA (2.5 for first 

two weeks; 

subsequently two 

randomized 

groups: one 

remaining on 2.5, 

the other with 6-

TGN 

concentration 

dose adjustment)

5-ASA, other

Radioassay Low

< 5 U/mL RBC

Intermediate

<10 U/mL RBC

Normal 

>=10 U/mL RBC

Hepatitis, 

myelotoxicity, 

pancreatitis, 

withdrawal due to 

adverse events

Winter, 200744 Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis 

of IBD; current or past 

treatment with 

thiopurine drugs.

AZA (1.6 (median) 

(NR))

5-ASA

Mass 

spectrometry

Low

<10 U/g Hb 

Intermediate

10-25 U/g Hb

Normal 

26-50 U/g Hb 

High

>50 U/g Hb

Hepatitis, 

leucopenia, 

pancreatitis

Abbreviations: 5-ASA = 5-aminosalicylates; 6-MP = 6-mercaptopurine; 6-TGN = 6-thioguanine nucleotide; AZA = azathioprine; IBD = inflammatory bowel disease; NR = not 

reported; RA = rheumatoid arthritis; RBCs = red blood cells; SD = standard deviation; SLE = systemic lupus erythematosus; TPMT = thiopurine methyltransferase.
42

Table C-17. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 2

Study Underlying disease(s)

Age group

Age (years)

mean (range)

(unless 

otherwise 

noted)

Females 

(%)

Number 

analyzed

Duration of 

observation on 

treatment 

mean (range) in 

months unless 

otherwise noted

Ansari, 

200229

IBD

NR

44 46 106 6 (1–108) 

Czaja, 

200648

AIH

Adults

44 (SD 2) 71 86 26 (1-180) 

Firooz, 

200849 Pemphigus vulgaris

NR

40.8 73.1 138 NR

Gisbert, 

200650

IBD

Adults

43 (SD: 14) 50 394 38.3 (95% CI: 

10.3-66.3)

Hindorf, 

200632

IBD

Adults

28 (18-42) 

(median) 

47.2 364 18 (6-36) 

(median)

Kader, 

200051

IBD

Mixed

13.7 (6-21) 63.6 22 7.5 (1-24)

Lennard, 

198952

Autoimmune disorders

Adults

Controls: 52 

(20-78) Cases: 

55 (38-63)

42.9 21 Controls: 12 

Cases: <3 

Okada, 

200537

SLE

Mixed

SLE: 39.8 (14-

76) Healthy 

volunteers 25.4 

(21-57)

NR 18 NR

Schedel, 

200653

SLE, RA, IBD, AIH and 

others

Adults

NR NR 96 NR

Shah, 

200854

IBD

NR

NR NR 131 19 (6-96)
Table C-17. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – patient and study characteristics, part 2 (continued)

C - 43

Study Underlying disease(s)

Age group

Age (years)

mean (range)

(unless 

otherwise 

noted)

Females 

(%)

Number 

analyzed

Duration of 

observation on 

treatment 

mean (range) in 

months unless 

otherwise noted

Snow, 

199539

Autoimmune 

dermatologic 

conditions

Adults

NR (26-88) 38.5 26 NR

Stassen, 

200940

Anti-neutrophil 

cytoplasmic antibody 

associated vasculitis 

NR

NR NR 108 47

Stocco, 

200541

IBD

Mixed

14.2 (0.8-38.8) 

(median)

51.4 28 19.6 (0.5–85.0) 

Stolk, 

199855

RA

Adults

56.2 (33-74) 75 32 6

von 

Ahsen, 

200543

IBD

Adults

36 (SD: 11.6) 56.3 71 6

Winter, 

200744

IBD

NR

45 51.4 130 Adverse effects: 

1.4 (median) No 

adverse effects: 

30 (median)

Abbreviations: AIH = autoimmune hepatitis; CI = confidence interval; IBD = inflammatory bowel disease; 

NR = not reported; RA = rheumatoid arthritis; SD = standard deviation; SLE = systemic lupus erythematosus; 

TPMT = thiopurine methyltransferase.
44

Table C-18. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk of bias assessment, part 1

Study Unbiased 

outcomes 

assessment 

and 

phenotyping 

Similarity of 

groups at 

baseline 

Outcomes 

adequately 

described 

Intention to 

treat analysis

Potential for 

financial 

conflict of 

interest

High loss to 

follow-up or 

differential 

loss to 

followup 

between 

groups 

Adequate 

sample size

Ansari, 200229 Unclear Unclear Yes Yes Unlikely No Unclear

Czaja, 200648 Unclear Unclear Yes Yes Unlikely No Unclear

Firooz, 200849 Unclear Unclear No Yes Unlikely No Unclear

Gisbert, 

200650

Unclear Unclear Yes Yes Unlikely No Unclear

Hindorf, 

200632

Unclear Not similar Yes Unclear Unlikely No Unclear

Kader, 200051 Unclear Unclear Yes Yes Unlikely No Unclear

Lennard, 

198952

Unclear Unclear Yes No Unlikely No Unclear

Okada, 200537 Unclear Unclear Yes Unclear Unlikely Unclear Unclear

Schedel, 

200653

Unclear Unclear No Yes Unlikely Unclear Unclear

Shah, 200854 Unclear Unclear Yes Yes Unlikely Yes Unclear

Snow, 199539 Unclear Unclear Yes No Unlikely No Unclear

Stassen, 

200940

Unclear Unclear Unclear Yes Unlikely No Unclear
Table C-18. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk of bias assessment, part 1 (continued)

C - 45

Stocco, 200541 Unclear Unclear Yes No Unlikely Yes Unclear

Stolk, 199855 Unclear Unclear No No Unlikely No Unclear

von Ahsen, 

200543

Unclear Unclear Yes Unclear Likely Yes Unclear

Winter, 200744 Unclear Unclear No No Unlikely No Unclear

Abbreviations: TPMT = thiopurine methyltransferase.
46

Table C-19. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk of bias assessment, part 2

Study Avoidance 

of selection 

bias

Methods to 

control 

confounding 

Appropriateness 

of TPMT activity 

test

Applicability Summary risk of bias 

assessment

Ansari, 

200229

Unclear No Yes Unclear - Uncertain 

representativeness of patients 

with IBD. 

Fair – Unclear if outcome 

assessment is biased. Methods 

to control confounding not 

described. Avoidance of 

selection bias is unclear.

Czaja, 

200648

Unclear No Yes Unclear - Uncertain 

representativeness of patients 

with autoimmune hepatitis

Fair - Unclear if outcome 

assessment is biased. Avoidance 

of selection bias is unclear. 

Unclear reliability and suitability 

of TPMT activity assay. 

Firooz, 

200849

Unclear No Yes Unclear - Uncertain 

representativeness of pemphigus 

vulgaris patients. Excluded 

patients for whom adverse event 

of clinical data were not 

available.

Fair - Unclear if outcome 

assessment is biased. No 

methods to control confounding. 

Unclear similarity of groups. 

Adequacy of sample size is 

unclear. 

Gisbert, 

200650

Unclear Unclear Yes Unclear - Uncertain 

representativeness of patients 

with IBD.

Fair - Unclear if outcome 

assessment is biased. Methods 

to control confounding not 

described. Unclear similarity of 

groups.

Hindorf, 

200632

Yes No Yes Unclear – Uncertain 

representativeness of patients 

with IBD. Excluded patients for 

whom TPMT metabolite or 

enzymatic assessment had not 

been previously performed. 

Fair - Unclear if outcome 

assessment is biased. Potential 

confounding by age, use of 

corticosteroids and 5-ASAs. 

Kader, 

200051

No No Unclear Unclear - Uncertain 

representativeness of patients 

with IBD.

Poor - Unclear reliability and 

suitability of TPMT activity assay. 

Uncertain representativeness of 

pediatric patients. Unclear 

similarity of groups.
Table C-19. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 47

Study Avoidance 

of selection 

bias

Methods to 

control 

confounding 

Appropriateness 

of TPMT activity 

test

Applicability Summary risk of bias 

assessment

Lennard, 

198952

No No Yes Unclear - Uncertain 

representativeness of patients 

with chronic autoimmune 

disease.

Poor - Unclear if outcome 

assessment is biased. Methods 

to control confounding not 

described. Avoidance of 

selection bias is unclear. Unclear 

similarity of groups. Uncertain 

representativeness of 

autoimmune disorder patients. 

Okada, 

200537

Unclear No Yes Unclear - Uncertain 

representativeness of Japanese 

patients with SLE.

Fair - . Avoidance of selection 

bias is unclear. Small sample 

size. Unclear if blinded outcome 

assessment and TPMT activity 

testing. 

Schedel, 

200653

Unclear No Unclear Unclear - Uncertain 

representativeness of patients 

with chronic autoimmune 

disease.

Fair - Unclear if outcome 

assessment is biased. Avoidance 

of selection bias is unclear. 

Uncertain representativeness of 

patients with autoimmune 

disease. Unclear reliability and 

suitability of TPMT activity assay.

Shah, 200854 Unclear No Unclear Unclear - Uncertain 

representativeness of patients 

with IBD.

Poor - No methods to control 

confounding. Unclear similarity 

of groups. Unclear reliability and 

suitability of TPMT activity assay. 

Adequacy of sample size is 

unclear. Unclear if blinded 

outcome assessment and TPMT 

activity testing.

Snow, 

199539

No No Yes Unclear – Uncertain 

representativeness of patients 

with autoimmune dermatologic 

conditions

Poor – Unclear similarity of 

groups. Small sample size. 

Unclear if blinded outcome 

assessment and TPMT activity 

testing. Methods to control 

confounding not described. 

Avoidance of selection bias is 

unclear.
Table C-19. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 48

Study Avoidance 

of selection 

bias

Methods to 

control 

confounding 

Appropriateness 

of TPMT activity 

test

Applicability Summary risk of bias 

assessment

Stassen, 

200940

Unclear No Yes Unclear – Uncertain 

representativeness of patients 

with anti-neutrophil cytoplasmic 

antibody associated vasculitis

Fair - Unclear if outcome 

assessment is biased. Unclear 

similarity of groups. Methods to 

control confounding not 

described.

Stocco, 

200541

Unclear No Yes Unclear - Uncertain 

representativeness of patients 

with IBD.

Fair - Unclear if outcome 

assessment is biased. Potential 

for selection bias.

Stolk, 199855 No Yes Yes Unclear - Uncertain 

representativeness of patients 

with rheumatoid arthritis. Strict 

inclusion criteria.

Fair - Unclear if outcome 

assessment is biased. Avoidance 

of selection bias is unclear. Small 

sample size.

von Ahsen, 

200543

Unclear No Yes Unclear - Uncertain 

representativeness of patients 

with IBD. Excluded those likely to 

experience adverse events.

Fair – Avoidance of selection 

bias is unclear. Uncertain 

representativeness of adult IBD 

patients. Unclear if blinded 

outcome assessment and TPMT 

activity testing. Potential for 

selective outcome reporting bias.

Winter, 

200744

Unclear No Yes Unclear - Uncertain 

representativeness of patients 

with IBD. Included only those 

patients with a history of 

thiopurine use. 

Fair - Unclear if outcome 

assessment is biased. Unclear 

similarity of groups.

Abbreviations: 5-ASA = 5-aminosalicylates; IBD = inflammatory bowel disease; SLE = systemic lupus erythematosus; TPMT = thiopurine methyltransferase.
49

Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

Ansari, 

200229

Neutropenia

Neutrophil count 

<2.0 X 109 cells

0 0 1 9 1 95 10.56 (0.61, 

183.38)

Czaja, 

200648

WDAE

Included a range 

of side effects 

(thrombocytopenia

, leukopenia, 

pancytopenia, 

nausea and 

vomiting, malaise 

and opportunistic 

infection) that 

treating physician 

deemed severe 

enough to end 

treatment

0 1 2 10 9 64 1.42 (0.27, 

7.56)

1.4 (0.04, 

45.68)

2.26 

(0.09, 

59.69)

Firooz, 

200849

WDAE

NR

0 0 1 10 13 114 0.88 (0.1, 

7.41)

Hepatitis

>3 fold increase of 

the upper limit of 

normal liver 

enzymes after 10 

days

0 0 1 10 10 117 1.17 (0.14, 

10.09)

Pancreatitis

NR

0 0 0 11 1 126 3.67 (0.14, 

95.24)
Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data (continued)

C - 50

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

Leukopenia

NR

0 0 0 11 1 126 3.67 (0.14, 

95.24)

Anemia

NR

0 0 0 11 1 126 3.67 (0.14, 

95.24)

Gisbert, 

200650

Pancreatitis

Abdominal pain 

present and serum 

amylase >3 times 

upper normal limit

0 0 0 28 11 355 0.54 (0.03, 

9.44)

Myelotoxicity

NR

0 0 4 24 13 353 4.53 (1.37, 

14.94)

Hepatitis

Transaminases 

two times higher 

than normal values

0 0 0 0 4 362

Hindorf, 

200632

Hepatitis

Included all types 

of hepatotoxic 

reactions

0 6 4 41 15 298 1.94 (0.61, 

6.12)

0.71 (0.03, 

14.78)

1.48 

(0.08, 

27.51)

Pancreatitis

NR

0 6 2 43 13 300 1.07 (0.23, 

4.92)

1.34 (0.06, 

31.12)

1.71 

(0.09, 

31.99)
Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data (continued)

C - 51

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

Myelotoxicity

Included all types 

of haematological 

toxicity (anemia, 

leukopenia, 

neutropenia, 

thrombocytopenia)

3 3 3 42 22 291 0.94 (0.27, 

3.29)

14 (1.93, 

101.72)

13.23 

(2.52, 

69.43)

Kader, 

200051

Leukopenia

WBC <4000/mm3

0 0 1 1 1 19 19 (0.62, 

583.42)

Hepatitis

Aminotransferases 

>2 times normal

0 0 1 1 2 18 9 (0.39, 

206.54)

Lennard, 

198952

Leukopenia

NR

4 1 0 5 0 11 33 (1.06, 

1023.62)

69 

(2.35, 

2028.86

)

Thrombocytopenia

NR

3 2 0 5 0 11 15.4 (0.56, 

425.55)

32.2 

(1.23, 

841.87)

Anemia

NR

4 1 0 5 0 11 33 (1.06, 

1023.62)

69 

(2.35, 

2028.86

)

Myelotoxicity

NR

3 2 0 5 0 11 15.4 (0.56, 

425.55)

32.2 

(1.23, 

841.87)

Okada, 

200537

Leukopenia

WBC <2300/mm3

0 0 1 2 2 13 3.25 (0.19, 

54.78)
Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data (continued)

C - 52

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

Schedel, 

200653 Myelotoxicity

Pancytopenia (not 

defined)

0 3 1 27 1 64 2.37 (0.14, 

39.3)

2.62 (0.09, 

77.58)

6.14 

(0.21, 

179.81)

Leukopenia

NR

2 1 2 26 0 65 12.36 (0.57, 

266.16)

26 (1.58, 

426.87)

218.33 

(6.99, 

6815.05

)

Shah, 

200854

WDAE

NR

NR NR 1 11 18 101 0.51 (0.06, 

4.2)

Snow, 

199539 Hepatitis 

Raised aspartate 

transaminase and 

alanine 

transaminase

0 0 0 5 1 20 1.24 (0.04, 

34.93)

Leukopenia

NR

0 0 2 3 1 20 13.33 (0.91, 

196.38)

Stassen, 

200940

Leukopenia

NR

0 0 3 4 34 67 1.48 (0.31, 

6.98)

Thrombocytopenia

NR

0 0 0 7 8 93 0.73 (0.04, 

13.98)

Anemia

NR

0 0 3 4 30 71 1.78 (0.37, 

8.42)
Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data (continued)

C - 53

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

Stocco, 

200541 Hepatitis

Alanine 

aminotransferase, 

gamma-glutamyl 

transferase or 

alkaline 

phosphatase >2 

times normal 

levels

0 0 0 3 0 25

Pancreatitis

Severe abdominal 

pain accompanied 

by serum amylase 

level >2 times 

normal levels

0 0 0 3 1 24 2.33 (0.08, 

69.29)

Myelotoxicity

Leukopenia (WBC 

<3000/mm3

) and/or 

thrombocytopenia 

(platelets 

<100,000/mm3)

0 0 0 3 3 22 0.92 (0.04, 

21.86)

Stolk, 

199855

Any infection

Upper airway 

infection

0 0 1 7 0 24 9.8 (0.36, 

266.64)

Hepatitis

NR

0 0 1 7 1 23 3.29 (0.18, 

59.6)

Myelotoxicity

Pancytopenia

0 0 0 8 1 23 0.92 (0.03, 

24.87)
Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data (continued)

C - 54

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

von 

Ahsen, 

200543

WDAE

Withdrawal from 

study for whatever 

reason

0 0 14 7 18 32 3.56 (1.21, 

10.42)

Hepatitis

Aspartate or 

alanine 

aminotransferase 

>=2 times upper 

limit of reference 

interval

0 0 1 20 2 48 1.2 (0.1, 14)

Pancreatitis

Upper abdominal 

pain accompanied 

by pancreatic 

amylase or lipase 

>=2 times upper 

limit of reference 

interval

0 0 1 20 1 49 2.45 (0.15, 

41.11)

Myelotoxicity

Myelosuppression 

(leukocyte count 

<2.5x109

/L or 

platelet count 

<100x109/L)

0 0 0 21 0 40

Winter, 

200744 Hepatitis

Deranged liver 

function tests

0 1 1 15 8 105 0.88 (0.1, 

7.5)

3.44 (0.09, 

127.7)

4.14 

(0.16, 

109.52)
Table C-20. KQ 3c: Association between TPMT enzymatic activity and thiopurine toxicity – summary data (continued)

C - 55

Study Outcomes and 

definition

Low activity Intermediate 

activity

Normal activity Intermediate 

vs. normal

Low vs. 

intermediate

Low vs. 

normal

n with 

events

n 

without 

events

n with 

events

n 

without 

events

n with 

events

n 

without 

events

Odds ratio (95% CI)

Pancreatitis

NR

0 1 1 15 0 113 21.97 (0.86, 

563.38)

3.44 (0.09, 

127.7)

Leukopenia 

Mild leukopenia 

(WBC 2-3 x109

/L) 

and severe 

leukopenia (WBC < 

2 x109/L)

1 0 1 15 8 105 0.88 (0.1, 

7.5)

31 (0.84, 

1149.3)

37.24 

(1.41, 

985.68)

Abbreviations: CI = confidence interval; n = number; NR = not reported; WBC = white blood cell count; WDAE = withdrawal due to adverse events
56

Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Ansari, 

200828

Europe

NR

Nonrandomized 

intervention 

study

Inclusion: Diagnosis of IBD; 

18-80 years of age.

Exclusion: Past thiopurine 

treatment; history of use of 

biologics; very low TPMT 

activity (<10 pmol ⁄ h ⁄mg Hb).

AZA (2.0 (1.9-2.4) 

(median))

5-ASA, steroids

NR TPMT*3A, 

*3B, *3C

Hepatitis, 

leukopenia, 

pancreatitis, 

withdrawal due to 

adverse events

Bezier, 200856 Europe

Inpatient

Crosssectional

Inclusion: Diagnosis of 

bullous pemphigoid, 

cicatrical pemphigoid, 

pemphigus or epidermolysis 

bullosa acquista; 

hospitalized in a 

Dermatology department; 

history of TPMT genotyping.

Exclusion: IgA bullous 

dermatosis linear; 

dermatosis herpetiformis; 

pemphigoid gestationis.

AZA (2.2 (NR))

steroids, others

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Anemia, any 

infection, 

leukopenia, 

neutropenia, 

withdrawal due to 

adverse events

Black, 199857 Europe

Outpatient 

specialty 

clinic

Prospective 

cohort

Inclusion: Current or past 

AZA treatment

AZA (NR (2-3))

NSAIDs

Polymerase 

chain 

reaction

TPMT*2, *3A Hepatitis, 

leukopenia
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 57

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Corominas, 

200358

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of 

rheumatoid arthritis; current 

treatment with low-dose AZA

AZA (NR (0.5-

1.5))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C, *7, 

*8

Withdrawal due to 

adverse events

De Ridder, 

200659 Europe

NR

Crosssectional

Inclusion: Diagnosis of 

pediatric-onset IBD; current 

or past AZA treatment of at 

least 3 months unless 

stopped due to adverse 

effects; <19 years of age

AZA (NR (2-2.5))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Hepatitis, 

leukopenia, 

pancreatitis 
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 58

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Derijks, 

200460

Europe

Outpatient 

specialty 

clinic

Nonrandomized 

intervention 

study

Inclusion: Diagnosis of IBD; 

18-75 years of age; 6-MP 

indicated due to steroid 

tolerance, steroid resistance, 

or AZA intolerance. 

Exclusion: Pregnancy or 

expected pregnancy within 6 

months; inadequate 

contraception in women; 

lactation; active infection; 

history of tuberculosis; HIV; 

hepatitis B or C; severe 

pancreatitis; malignancy; 

current treatment with other 

immunosuppressive drugs; 

impaired renal function; 

elevated liver function tests; 

bone marrow suppression.

6-MP (50 mg/day 

(NR))

5-ASA

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Hepatitis, 

leukopenia, 

pancreatitis

Dubinsky, 

200061

North 

America

Outpatient 

specialty 

clinic

Prospective 

cohort

Exclusion: Leukopenia; 

increased serum hepatic or 

pancreatic enzyme activity

AZA (NR (NR)), 

6-MP (1.25 (0.4-

2.4))

5-ASA

Polymerase 

chain 

reaction

TPMT*3A, 

*3B, *3C

Leukopenia
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 59

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Gearry, 

200462

Oceania

Outpatient 

specialty 

clinic

Case-control

Inclusion: Past treatment 

with thiopurine drugs

Mixed 

thiopurines

NR

Polymerase 

chain 

reaction

TPMT*1, *2, 

*3A, *3C

Hepatitis, 

leukopenia, 

pancreatitis

Hibi, 200363 Asia

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of IBD; 

past AZA or 6-MP treatment

AZA (NR (25-100 

mg/day)), 6-MP 

(NR (30-50 

mg/day)

NR

Polymerase 

chain 

reaction

TPMT*2, *3B, 

*3A, *3C

Leukopenia

Hindorf, 

200632

Europe

NR

Crosssectional

Inclusion: Diagnosis of IBD; 

history of metabolite or 

TPMT measurements; 

current or past treatment 

with thiopurine drugs. 

Exclusion: Patients who had 

not yet started therapy

AZA (NR (NR))

5-ASA, steroids

Pyrosequenc

ing

TPMT*2, *3A, 

*3B, *3C, *3D, 

*4, *5, *6, *7, 

*8, *10, *14, 

*15

Hepatitis, 

myelotoxicity, 

pancreatitis

Ishioka, 

199964

Asia

NR

Unclear if 

prospective 

or 

retrospective 

design

Included: Diagnosis of a 

rheumatic disease; past AZA 

treatment; Japanese

AZA (50 mg/day 

(NR))

NR

Polymerase 

chain 

reaction

TPMT*1, *2, 

*3A, *3B, *3C

Leukopenia
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 60

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Jae Hak, 

200865

Asia

Outpatient 

specialty 

clinic

Case-control

Inclusion: Diagnosis of IBD; 

current or past thiopurine 

treatment for at least 6 

months 

AZA (NR (NR)), 

6-MP (NR (NR))

NR

High 

performance 

liquid 

chromatogra

phy

TPMT*1, *2, 

*3A, *3B, *3C

Leukopenia

Joji, 200366 Europe

NR

Prospective 

cohort

NR AZA (NR (NR))

5-ASA

Polymerase 

chain 

reaction

TPMT*1, *2, 

*3A, *3B, *3C

Leukopenia, 

myelotoxicity

Jun, 200567 Asia

NR

Crosssectional

Inclusion: Diagnosis of SLE; 

Korean 

AZA (22.1 

mg/day (NR))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C, *3D, 

*6

Hepatitis, 

leukopenia

Lopez, 200668 NR

NR

Case-control

NR AZA (NR (NR))

NR

NR TPMT*2, *3A, 

*3B, *3C, *3D

Pancreatitis

Marinaki, 

200469

Europe

Outpatient 

specialty 

clinic

Case-control

Cases: Diagnosis of IBD; 

Caucasian; history of AZArelated adverse events. 

Controls: Caucasian; past 

AZA treatment for at least 3 

months without adverse 

events.

AZA (Cases 1.81 

(0.39-2.59) 

Controls 1.92 

(0.91-3.26))

5-ASA, steroids, 

others

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3C

Hepatitis, 

neutropenia, 

pancreatitis
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 61

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Okada, 200537 Asia

NR

Crosssectional

Inclusion: Diagnosis of SLE AZA (NR (NR))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Leukopenia

Palmieri, 

200770

Europe

NR

Prospective

cohort

Inclusion: Diagnosis of IBD; 

21-58 years; current AZA or 

6-MP treatment. Exclusion: 

Past AZA or 6-MP treatment 

of less than 6 months; no 

history of adverse events.

AZA (NR (2-2.5)), 

6-MP (NR (1-

1.25))

5-ASA

Polymerase 

chain 

reaction

TPMT*3A, 

*3B, *3C

Hepatitis, 

leukopenia, 

pancreatitis

Reuther, 

200371

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of 

Crohn's disease; current 

AZA maintenance treatment; 

no history of adverse effects

AZA (1.57 (0.58-

2.24))

5-ASA, steroids

Polymerase 

chain 

reaction

TPMT*3A, 

*3B, *3C

Hepatitis, 

leukopenia

Schmeling, 

200772

NR

NR

Crosssectional

Inclusion: Diagnosis of SLE; 

current AZA treatment; 

pediatric

AZA (NR (NR))

NR

NR TPMT*2, *3A, 

*3B, *3C

Hepatitis, 

leukopenia
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 62

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Seddik, 

200373

NR

NR

Prospective 

cohort

NR AZA (NR (100-

200mg/day)), 6-

MP (NR (50-

100mg/day))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Leukopenia

Snow, 199539 North 

America

NR

Crosssectional

NR AZA (NR (0.4-

2.6))

NR

NR NR Hepatitis, 

leukopenia

Stassen, 

200940

NR

NR

Chart review

NR AZA (NR (NR))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Anemia, 

leukopenia, 

thrombocytopenia

Stocco, 

200774

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of IBD; 

current AZA treatment

AZA (2 (1-5))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Any infection, 

hepatitis, 

myelotoxicity, 

pancreatitis
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 63

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Stocco, 

200541

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of IBD; 

past thiopurine treatment for 

at least 3 months or having 

experienced an adverse 

effect during thiopurine 

treatment

AZA (2.0 (1-4) 

(median)); 6-MP 

(NR (NR))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Hepatitis, 

myelotoxicity, 

pancreatitis

Stocco, 

200442 Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of IBD; 

current AZA treatment 

AZA (2 (1-3) 

(median))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Hepatitis, 

leukopenia, 

pancreatitis, 

thrombocytopenia

Tamori, 

200775

Asia

NR

Crosssectional

Inclusion: Diagnosis of AIH; 

Japanese 

AZA (50 (NR))

NR

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Myelotoxicity

Tani, 200976 Europe

NR

Crosssectional

Included: Diagnosis of 

rheumatic disease; current 

AZA treatment; Italian; 

Caucasian

AZA (1.42 (0.5-

2))

5-ASA, steroids, 

others

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Any infection, 

leukopenia, 

thrombocytopenia, 

withdrawal due to 

adverse events
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 64

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

van Dieren, 

200577

NR

NR

Prospective 

cohort

Inclusion: Diagnosis of IBD; 

first AZA treatment between 

January 2003 and November 

2004

AZA (NR (NR))

NR

Polymerase 

chain 

reaction

NR Hepatitis, 

leukopenia, 

thrombocytopenia

von Ahsen, 

200543

Europe

NR

Randomized 

controlled 

trial

Inclusion: Diagnosis of 

active Crohn's disease; >18 

years of age; prednisone 

treatment >300 mg during 

last 4 weeks or relapse 

within 6 months after steroid 

pulse therapy. 

Exclusion: History of cancer; 

preexisting renal or hepatic 

disease; pregnant; breast 

feeding.

AZA (2.5 for first 

two weeks; 

subsequently 

two randomized 

groups: one 

remaining on 

2.5, the other 

with 6-TGN 

concentration 

dose 

adjustment)

5-ASA, others

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, *3C

Hepatitis, 

myelotoxicity, 

pancreatitis

Winter, 

200744

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of IBD; 

current or past treatment 

with thiopurine drugs

AZA (1.6 (NR) 

(median))

5-ASA

Polymerase 

chain 

reaction

TPMT*2, *3A, 

*3B, * 3C

Hepatitis, 

leukopenia, 

pancreatitis, 
Table C-21. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 1 (continued)

C - 65

Study Study region

Study setting

Study design

Inclusion and exclusion 

criteria

Thiopurine 

treatment dose 

(mean (range) in 

mg/kg/day 

unless otherwise

noted)

Concomitant 

treatments

Genotyping 

method

SNPs 

genotyped

Outcomes 

assessed

Zelinkova, 

200678

Europe

Outpatient 

specialty 

clinic

Crosssectional

Inclusion: Diagnosis of IBD; 

past AZA treatment; reliable 

data available on AZA use 

and related side effects

AZA (132 (50-

250) mg/day)

5-ASA, steroids

Polymerase 

chain 

reaction

TPMT*1, *2, 

*3A, *3B, *3C

Hepatitis, 

leukopenia

Newman, 

201045 Europe

Outpatient 

specialty 

clinic

Prospective 

Cohort

Nursing and pregnant 

women and those likely to 

experience adverse events 

were excluded

AZA (on average 

approx. 

1mg/kg/day)

Polymerase 

chain 

reaction

TPMT *2, *3A, 

*3B, *3C

Mortality, WDAE, 

SAE, hepatitis, 

pancreatitis, and 

neutropenia 

Kolorz 200979 Europe

NR

Crosssectional

NR AZA, 

Dosage NR

Polymerase 

chain 

reaction

TPMT *2, *3A, 

*3B, *3C

Leukopenia

Abbreviations: 5-ASA = 5-aminosalicylates; 6-MP = 6-mercaptopurine; AIH = autoimmune hepatitis; AZA = azathioprine; HIV = human immunodeficiency virus; IBD = 

inflammatory bowel disease; NR = not reported; NSAID = Non-steroidal anti-inflammatory drug; SLE = systemic lupus erythematosus; SNP = single nucleotide polymorphism; 

TPMT = thiopurine methyltransferase; WDAE = withdrawal due to adverse events; SAE = serious adverse events; 
66

Table C-22. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 2

Study Underlying disease(s)

Age group

Age (years)

mean (range)

(unless 

otherwise 

noted)

Females 

(%)

Number analyzed Duration of observation on 

treatment 

mean (range) in months unless 

otherwise noted

Ansari, 

200229

IBD

NR

44 46 106 6 (1–108) 

Czaja, 

200648

AIH

Adults

44 (SD 2) 71 86 26 (1-180) 

Firooz, 

200849

Pemphigus vulgaris

NR

40.8 73.1 138 NR

Gisbert, 

200650

IBD

Adults

43 (SD: 14) 50 394 38.3 (95% CI: 10.3-66.3)

Hindorf, 

200632

IBD

Adults

28 (18-42) 

(median) 

47.2 364 18 (6-36) (median)

Kader, 

200051

IBD

Mixed

13.7 (6-21) 63.6 22 7.5 (1-24)

Lennard, 

198952

Autoimmune disorders

Adults

Controls: 52 

(20-78) Cases: 

55 (38-63)

42.9 21 Controls: 12 Cases: <3 

Okada, 

200537

SLE

Mixed

SLE: 39.8 (14-

76) Healthy 

volunteers 25.4 

(21-57)

NR 18 NR

Schedel, 

200653

SLE, RA, IBD, AIH and 

others

Adults

NR NR 96 NR

Shah, 

200854

IBD

NR

NR NR 131 19 (6-96)
Table C-22. KQ 3c: Association between TPMT genotype and thiopurine toxicity – patient and study characteristics, part 2 (continued)

C - 67

Study Underlying disease(s)

Age group

Age (years)

mean (range)

(unless 

otherwise 

noted)

Females 

(%)

Number analyzed Duration of observation on 

treatment 

mean (range) in months unless 

otherwise noted

Snow, 

199539

Autoimmune 

dermatologic 

conditions

Adults

NR (26-88) 38.5 26 NR

Stassen, 

200940

Anti-neutrophil 

cytoplasmic antibody 

associated vasculitis 

NR

NR NR 108 47

Stocco, 

200541

IBD

Mixed

14.2 (0.8-38.8) 

(median)

51.4 28 19.6 (0.5–85.0) 

Stolk, 

199855

RA

Adults

56.2 (33-74) 75 32 6

von 

Ahsen, 

200543

IBD

Adults

36 (SD: 11.6) 56.3 71 6

Winter, 

200744

IBD

NR

45 51.4 130 Adverse effects: 1.4 (median) 

No adverse effects: 30 

(median)

Newman, 

201045

Majority IBD patients 43 50.6 166 4

Kolorz 

200979

IBD 35 41 87 NR

Abbreviations: AIH = autoimmune hepatitis; CI = confidence interval; IBD = inflammatory bowel disease; NR = not reported; RA = rheumatoid arthritis; SD = standard deviation; 

SLE = systemic lupus erythematosus; TPMT = thiopurine methyltransferase.
68

Table C-23. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 1

Study Comparable 

genotype 

groups

Participants 

enrolled 

without prior 

knowledge of 

genotype

Unbiased 

outcome and 

genotype 

assessment

Clear 

genotyping 

method 

description

Reliable 

genotyping 

method

Reported 

ambiguous 

results due to 

genotyping 

error

HWE tested

Ansari, 

200828

Unclear Yes Yes No Unclear No Yes

Bezier, 

200856

Unclear Yes Unclear Yes Yes No No

Black, 

199857

Unclear Yes Yes Yes Yes Unclear No

Corominas, 

200358

Unclear Yes Unclear Yes Unclear Unclear No

De Ridder, 

200659

Unclear Yes Unclear No No No No

Derijks, 

200460

Unclear Yes Unclear Yes Yes Yes No

Dubinsky, 

200061

Unclear Yes Yes Yes Unclear No No

Gearry, 

200462

Unclear Yes Unclear Yes Unclear No No

Hibi, 200363 Unclear Yes Unclear Yes Yes No No

Hindorf, 

200632

Unclear Unclear Unclear Unclear Unclear No No

Ishioka, 

199964

Unclear Unclear Unclear Yes No No No

Jae Hak, 

200865

Unclear Unclear Unclear No Unclear No No
Table C-23. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 1 (continued)

C - 69

Study Comparable 

genotype 

groups

Participants 

enrolled 

without prior 

knowledge of 

genotype

Unbiased 

outcome and 

genotype 

assessment

Clear 

genotyping 

method 

description

Reliable 

genotyping 

method

Reported 

ambiguous 

results due to 

genotyping 

error

HWE tested

Joji, 200366 Unclear Yes Unclear Yes Unclear No No

Jun, 200567 Unclear Yes Unclear Yes Unclear No No

Lopez, 

200668

Unclear Unclear Unclear No No No No

Marinaki, 

200469

Unclear Yes Unclear No No No No

Okada, 

200537

Unclear Yes Unclear Yes Yes No No

Palmieri, 

200770

Yes Yes Unclear Yes Unclear No Yes

Reuther, 

200371

Unclear Yes Unclear No No Yes No

Schmeling, 

200772

Unclear Unclear Unclear Unclear Unclear Unclear Unclear

Seddik, 

200373

Unclear Unclear Unclear No Unclear No No

Snow, 

199539

Unclear Unclear Unclear No Unclear No No

Stassen, 

200940

Unclear Yes Unclear Yes Unclear No No

Stocco, 

200774

Unclear Yes Unclear Yes No No No

Stocco, 

200541

Unclear Yes Unclear No Unclear No No
Table C-23. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 1 (continued)

C - 70

Study Comparable 

genotype 

groups

Participants 

enrolled 

without prior 

knowledge of 

genotype

Unbiased 

outcome and 

genotype 

assessment

Clear 

genotyping 

method 

description

Reliable 

genotyping 

method

Reported 

ambiguous 

results due to 

genotyping 

error

HWE tested

Stocco, 

200442

Unclear Yes Yes Yes Unclear Unclear No

Tamori, 

200775

Unclear Yes Unclear Yes Unclear Unclear No

Tani, 

200976

Unclear Yes Unclear Yes No No No

van Dieren, 

200577

Unclear Yes Unclear No Unclear No No

von Ahsen, 

200543

Unclear Yes Unclear Yes Yes No No

Winter, 

200744

Unclear Yes Unclear Yes Unclear No No

Zelinkova, 

200678

Unclear Yes Unclear Yes Unclear No No

Newman, 

201045

Unclear Yes Yes Yes Unclear No No

Kolorz 

200979

Unclear Yes Unclear Yes Unclear No No

Abbreviations: HWE = Hardy-Weinberg equilibrium; TPMT = thiopurine methyltransferase.
71

Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Ansari, 

200828

No No Yes Yes No No Applicable Fair - Methods to 

control 

confounding not 

described. 

Genotyping method 

unclear.

Bezier, 

200856

No No Yes Yes No No Unclear – Uncertain 

representativeness of 

patients with 

autoimmune bullous 

diseases. Included 

only hospitalized 

severe cases. 

Fair – HWE not 

reported. 

Compliance not 

clear. Unclear 

similarity of groups. 

Unclear if blinded 

outcome or 

genotype 

assessment. 

Black, 

199857

No Yes Yes Yes No Unclear Unclear - Uncertain 

representativeness of 

patients with several 

rheumatic diseases.

Fair – HWE not 

reported.

Corominas, 

200358

Unclear No Yes Yes Unclear Unclear Applicable Fair - HWE not 

reported. Methods 

to control 

confounding not 

described. Unclear 

if blinded outcome 

or genotype 

assessment.
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 72

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

De Ridder, 

200659

No No Yes No No No Unclear - Uncertain 

representativeness of 

pediatric patients 

with IBD.

Fair - Avoidance of 

selection bias is 

unclear. Unclear if 

blinded outcome or 

genotype 

assessment. 

Inadequate 

description of 

genotyping method. 

Compliance not 

clear. 

Derijks, 

200460

No No Yes No No Unclear Unclear - Uncertain 

representativeness of 

patients with IBD. 

Strict eligibility 

criteria.

Fair – Potential 

selection bias. 

Unclear if blinded 

outcome or 

genotype 

assessment.

Dubinsky, 

200061

No No Yes Yes No Yes Not applicable – Not 

representative of 

children with IBD. 

Excluded children 

with leukopenia and 

increased hepatic or 

pancreatic enzymes 

before treatment 

initiation.

Fair – HWE not 

reported. Unclear 

similarity of groups. 

Unclear reliability of 

genotyping method. 
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 73

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Gearry, 

200462

No No Yes No No No Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair – Avoidance of 

selection bias is 

unclear. Unclear 

similarity of groups. 

Unclear if blinded 

outcome or 

genotype 

assessment.

Hibi, 200363 No No Yes Yes No No Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - Potential 

selection bias. 

Unclear if blinded 

outcome or 

genotype 

assessment.

Hindorf, 

200632

Unclear No Unclear Yes Unclear No Unclear - Unclear 

representativeness of 

patients with IBD 

population. Included 

only patients with 

history of TPMT 

metabolite or 

enzymatic assay 

testing. 

Fair - Inadequate 

description of 

genotyping method. 

Unclear reliability of 

genotyping method. 

Unclear similarity of 

groups. Methods to 

control 

confounding not 

described.
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 74

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Ishioka, 

199964

No No Yes Yes No No Unclear – Unclear 

representativeness of 

Japanese patients 

with rheumatic 

diseases. 

Fair - HWE not 

reported. Unclear 

similarity of groups. 

Unclear if blinded 

outcome or 

genotype 

assessment. 

Unclear reliability of 

genotyping method.

Jae Hak, 

200865

No No Unclear No Unclear No Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - HWE not 

reported. Unclear if 

blinded outcome or 

genotype 

assessment. 

Unclear similarity of 

groups.

Joji, 200366 No No Yes No No Unclear Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - HWE not 

reported. Unclear if 

blinded outcome or 

genotype 

assessment. 

Unclear similarity of 

groups.

Jun, 200567 No No Yes No No No Unclear - Unclear 

representativeness of 

Korean patients with 

SLE.

Fair – Avoidance of 

selection bias is 

unclear. Unclear if 

blinded outcome or 

genotype 

assessment.
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 75

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Lopez, 

200668

No No Unclear No Unclear Unclear Unclear - Uncertain 

representativeness of 

patients with IBD.

Poor - Poor 

reporting of quality 

items (only abstract 

available)

Marinaki, 

200469

Yes No Yes No No No Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - Avoidance of 

selection bias is 

unclear. Unclear if 

blinded outcome or 

genotype 

assessment. 

Inadequate 

description of 

genotyping method.

Okada, 

200537

No No Yes No Unclear Unclear Unclear - Uncertain 

representativeness of 

Japanese patients 

with SLE.

Fair - Avoidance of 

selection bias is

unclear. Small 

sample size. 

Unclear if blinded 

outcome or 

genotype 

assessment.

Palmieri, 

200770

No No Yes No No No Applicable Fair - Compliance 

not clear. Unclear if 

blinded outcome or 

genotype 

assessment. 

Unclear reliability of 

genotyping method. 
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 76

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Reuther, 

200371

No No Yes Yes No No Unclear - Uncertain 

representativeness of 

patients with Crohn's 

disease.

Fair - Avoidance of 

selection bias is 

unclear. Inadequate 

description of 

genotyping method.

Schmeling, 

200772

Unclear Unclear Unclear Unclear Unclear Unclear Unclear Poor - Poor 

reporting of risk of 

bias items (only 

abstract available)

Seddik, 

200373

No No Unclear No Unclear Unclear Unclear - Uncertain 

representativeness of 

patients with IBD.

Poor - Poor 

reporting of risk of 

bias items (only 

abstract available)

Snow, 

199539

No No Unclear Yes No Unclear Unclear - Uncertain 

representativeness of 

patients with 

autoimmune 

dermatologic 

conditions.

Poor - Unclear if 

blinded outcome or 

genotype 

assessment. 

Inadequate 

description of 

genotyping method. 

Unclear reliability of 

genotyping method. 

HWE not reported.
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 77

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Stassen, 

200940

No Unclear Yes No Unclear No Unclear – Unclear 

representativeness of 

patients with antineutrophil 

cytoplasmic antibody 

associated vasculitis

Fair - HWE not 

reported. Unclear if 

blinded outcome or 

genotype 

assessment. 

Unclear similarity of 

groups. Unclear if 

genotype was 

determined 

prospectively or 

retrospectively.

Stocco, 

200774

No No Yes No No No Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair – Unclear if 

blinded outcome or 

genotype 

assessment. 

Inadequate 

description of 

genotyping method. 

Unclear reliability of 

genotyping method. 

HWE not reported. 

Incomplete 

outcomes data. 

Stocco, 

200541

No No Yes No Unclear No Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair – Potential 

selection bias. 
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 78

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Stocco, 

200442

Unclear Unclear Yes Yes Unclear Unclear Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - HWE not 

reported. 

Compliance not 

clear. Avoidance of 

selection bias not 

clear. 

Tamori, 

200775

No Unclear Yes Unclear Unclear No Unclear – Unclear 

representativeness of 

Japanese patients 

with autoimmune 

hepatitis. Small 

sample size. 

Fair - HWE not

reported. Unclear if 

blinded outcome or 

genotype 

assessment.

Tani, 200976 No No Yes Yes No No Unclear – Unclear 

representativeness of 

patients with 

rheumatic diseases. 

Included only those 

in a tertiary care 

setting.

Fair - HWE not 

reported. Unclear if 

blinded outcome or 

genotype 

assessment. 

Unclear similarity of 

groups. Unclear if 

genotype was 

determined 

prospectively or 

retrospectively.
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 79

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

van Dieren, 

200577

No Unclear Unclear No Unclear Unclear Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - Poor 

reporting of risk of 

bias items (only 

abstract available). 

Unclear if blinded 

outcome or 

genotype 

assessment. 

Inadequate 

description of 

genotyping method. 

Unclear reliability of 

genotyping method. 

Avoidance of 

selection bias is not 

clear.

von Ahsen, 

200543

No Unclear Unclear Yes Unclear Yes Unclear - Uncertain 

representativeness of 

patients with IBD. 

Excluded those likely 

to experience adverse 

event.

Fair – Avoidance of 

selection bias is 

unclear. Unclear if 

blinded outcome or 

genotype 

assessment. 

Potential outcome 

reporting bias. 

Winter, 

200744

No No Yes Yes No No Unclear - Uncertain 

representativeness of 

patients with IBD. 

Included only those 

who had previously 

received thiopurines. 

Fair - Unclear 

similarity of groups. 

Methods to control 

confounding not 

described.
Table C-24. KQ 3c: Association between TPMT genotype and thiopurine toxicity – risk of bias assessment, part 2 (continued)

C - 80

Study Assessed 

gene-gene 

interaction

Assessed 

compliance 

with 

thiopurine 

treatment 

(and 

corrected 

differences 

as required)

Consistent 

DNA 

source for 

all patients

Reported 

loss to 

followup 

Potential 

for 

survival 

bias

Potential 

for 

financial 

conflict 

of 

interest

Applicability Summary risk of 

bias assessment

Zelinkova, 

200678

No No Yes No Unclear Unclear Unclear - Uncertain 

representativeness of 

patients with IBD.

Fair - Unclear if 

blinded outcome or 

genotype 

assessment. 

Avoidance of 

selection bias is 

unclear. 

Newman, 

201045

No Yes Yes Yes Unclear No Unclear -- excluded 

those likely to 

experience adverse

event and no 

description provided 

of those who did not 

agree to participate

Good. The main 

study was an RCT. 

Kolorz 

200979

No No Yes No Unclear No Unclear -- little 

information is 

provided regarding 

sample selection and 

those who were 

included versus not

Fair – unclear 

blinded assessment 

of outcomes and 

comparability of 

groups 

Abbreviations: DNA = deoxyribonucleic acid; HWE = Hardy-Weinberg equilibrium; IBD = inflammatory bowel disease; SLE = systemic lupus erythematosus; TPMT = 

thiopurine methyltransferase.
81

Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Ansari, 

2008 28

TPMT 

*3A, 

*3B, 

*3C

WDAE

NA

66 122 NR NR 15 4 6.93 (2.21, 

21.74)

Hepatitis

NR

8 180 NR NR 0 19 0.54 (0.03, 

9.8)

Pancreatitis

NR

8 180 NR NR 0 19 0.54 (0.03, 

9.8)

Leukopenia

WBC < 

3500/mm3 or 

neutrophils < 

1500/mm3

2 186 NR NR 5 14 33.21 (5.9, 

186.88)

Hindorf, 

2006 32

TPMT 

*2, *3A, 

*3B, 

*3C, 

*3D, *4, 

*5, *6, 

*7, *8, 

10, *14, 

*15

Hepatitis

All types of 

hepatotoxic 

reactions

15 25 4 2 0 6 3.33 (0.54, 

20.45)

0.13 (0.01, 

2.41)

23.4 (0.89, 

613.02)

Pancreatitis

NR

13 27 2 4 0 6 1.04 (0.17, 

6.42)

0.16 (0.01, 

2.99)

7.22 (0.28, 

189.2)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 82

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Myelotoxicity

Included all 

types of 

haematological 

toxicity 

(anaemia, 

leucopoenia, 

neutropenia, 

thrombocytopen

ia)

22 18 3 3 3 3 0.82 (0.15, 

4.56)

0.82 (0.15, 

4.56)

1 (0.1, 9.61)

Winter, 

2007

44

TPMT 

*2, *3A, 

*3B, * 

3C

Hepatitis

deranged liver 

function tests

8 111 0 0 1 10 1.39 (0.16, 

12.24)

Pancreatitis

NR

1 118 0 0 0 11 3.43 (0.13, 

89.24)

Leukopenia

WBC < 

3000/mm3

9 110 0 0 1 10 1.22 (0.14, 

10.65)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 83

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

De 

Ridder, 

2006

59

TPMT*2

, *3A, 

*3B, 

*3C

Hepatitis

Hepatotoxicity 

by serum 

alanine 

transaminase 

levels greater 

than twice the 

upper normal 

limit (50 IU/L) 

and resolution 

after withdrawal 

of AZA

0 67 0 3 0 2

Pancreatitis

Severe 

abdominal pain 

and 

hyperamylasemi

a and resolution 

after withdrawal 

of AZA

4 63 0 3 0 2 2.02 (0.09, 

45.36)

2.82 (0.12, 

68.1)

Leukopenia

Leucocyte count 

< 2.5x109 cells

1 66 0 3 1 1 6.33 (0.22, 

185.33)

66 (2.2, 

1984.33)

0.14 (0, 5.95)

Black, 

1998

57

TPMT

*3A, 

TPMT*2

Leukopenia

Low leukocyte 

count

0 61 0 0 5 1 451 (16.35, 

12442.54)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 84

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Hepatitis

Abnormal liver 

function test

6 55 0 0 1 5 1.83 (0.18, 

18.41)

Ishioka, 

1999

64

TPMT*1

, *2, 

*3A, 

*3B, * 

3C

Leukopenia

WBC<4000/mm3

& 75% before 

administration 

4 29 0 0 3 0 45.89 (2.02, 

1044.27)

Derijks, 

2004

60

TPMT 

*2, *3A, 

*3B, * 

3C

Hepatitis

Hepatotoxicity -

ALT>80 U/L, 

AST>80 U/L, 

bilirubins > 40 

μmol/L

1 19 0 1 0 4 4.33 (0.12, 

159.53)

1.44 (0.05, 

41.62)

Pancreatitis

Pancreatitis -

amylase > 220 

U/L, lipase > 120 

U/L 

(elevations>2tim

es normal upper 

limit)

3 17 0 1 0 4 1.67 (0.06, 

49.95)

0.56 (0.02, 

12.82)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 85

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Leukopenia

Myelotoxicity 

(leukocyte count 

< 4.0 × 109/L, 

platelet count < 

100 × 109/L)

1 19 1 0 2 2 39 (1.06, 

1435.74)

19 (1.15, 

314.99)

3 (0.08, 

115.35)

Gearry, 

2004

62

TPMT*1

, *2, 

*3A, 3C

Hepatitis

Hepatitis: 

elevation of 

serum liver 

transaminases 

greater than 

twice the upper 

limit of normal

18 115 0 1 0 13 2.08 (0.08, 

53.04)

0.23 (0.01, 

4.06)

Pancreatitis

Pancreatitis: 

severe 

abdominal pain 

associated with 

an elevation of 

serum amylase 

greater than 

three times the 

upper limit of 

normal

5 128 0 1 2 11 7.79 (0.28, 

213.81)

4.65 (0.81, 

26.83)

1.53 (0.05, 

49.8)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 86

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Leukopenia

WBC < 

3000/mm3 or 

neutrophils < 

2000/mm3

2 131 1 0 1 12 157.8 (5.08, 

4904.53)

5.46 (0.46, 

64.67)

25 (0.67, 

934.5)

Joji, 2003

66

TPMT 

*1, *2, 

*3A, 

*3B, * 

3C

Leukopenia

WBC<3000/mm3

12 13 0 0 2 0 5.4 (0.24, 

123.81)

Myelotoxicity

Pancytopenia

1 24 0 0 0 2 3.27 (0.1, 

103.43)

Hibi, 2003

63

TPMT 

*2, 3B, 

*3A, 

*3C

Leukopenia

WBC < 

3000/mm3 

5 69 1 0 6 1 37.91 (1.38, 

1044.73)

82.8 (8.27, 

828.71)

0.69 (0.02, 

26.91)

Coromina

s, 2003

58

TPMT 

*2, *3A, 

*3B, 

*3C, *7, 

*8

WDAE

NA

3 32 0 0 3 2 16 (1.87, 

136.7)

Dubinsky, 

2000

61

TPMT 

*3A, 

*3B, 

*3C

Leukopenia

WBC<4000/mm3

12 72 0 0 1 7 0.86 (0.1, 7.6)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 87

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Bezier, 

2008

56

TPMT*2

,

*3A, 

*3B, 

*3C

Any infection 

(including 

sepsis)

Grade 1 (minor) 

to grade 4 

(sepsis with 

hypotension)

9 22 0 0 0 2 0.47 (0.02, 

10.83)

WDAE

NA

15 16 0 0 1 1 1.07 (0.06, 

18.62)

Leukopenia

WBC <3.9 x 109/L

8 23 0 0 0 2 0.55 (0.02, 

12.73)

Neutropenia

WMC <1.9 x 

109/L

4 27 0 0 0 2 1.22 (0.05, 

29.86)

Anemia

<109 g/L

7 24 0 0 0 2 0.65 (0.03, 

15.16)

Reuther, 

2003

71

TPMT 

*3A, 

*3B, 

*3C

Hepatitis

Alanine 

transaminase 

levels twice the 

upper normal 

level (40 U/L)

0 62 0 0 0 4
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 88

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Leukopenia

WBC less than 

3x109/L

0 62 0 0 0 4

Stocco, 

2005

41

TPMT 

*2, *3A, 

*3B, 

*3C

Hepatitis

ALT, GGT or 

alkaline 

phosphatase 

more than twice 

normal levels

6 59 0 0 0 5 0.83 (0.04, 

16.82)

Pancreatitis

Severe 

abdominal pain, 

accompanied by 

serum amylase 

level of greater 

than twice 

normal levels

4 61 0 0 0 5 1.24 (0.06, 

26.21)

Myelotoxicity

Leucopoenia 

(WBC < 

3000mm3

) and/or 

thrombocytopen

ia (platelets < 

100,000mm3)

7 58 0 0 0 5 0.71 (0.04, 

14.15)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 89

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Zelinkova

, 2006

78

TPMT 

*1, *2, 

*3A, 

*3B, 

*3C

Leukopenia

WBC < 3x10^9/L 

resolving after 

discontinuation 

or dose 

reduction

8 230 1 0 3 20 81.35 (3.08, 

2147.05)

4.31 (1.06, 

17.55)

17.57 (0.59, 

524.14)

Hepatitis

Hepatotoxicity -

serum alanine 

transaminase 

levels greater 

than twice the 

upper normal 

limit (45 U/L) and 

resolution after 

withdrawal or 

dose reduction

9 229 0 0 2 22 2.31 (0.47, 

11.38)

Okada, 

2005

37

TPMT 

*2, *3A, 

*3B, 

*3C

Leukopenia

WBC < 

2300/mm3

2 14 0 0 1 1 7 (0.3, 162.21)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 90

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Stocco, 

2007

74

TPMT 

*2, *3A, 

*3B, 

*3C

Pancreatitis

Severe 

abdominal pain 

accompanied by 

serum amylase 

level more than 

twice the normal 

limit

6 59 0 0 1 4 2.46 (0.24, 

25.69)

Hepatitis

NR

3 64 0 0 0 3 2.63 (0.11, 

61.59)

Leukopenia

NR

2 65 0 0 0 3 3.74 (0.15, 

93.79)

Thrombocytope

nia

NR

1 66 0 0 0 3 6.33 (0.22, 

185.33)

Tamori, 

2007

75

TPMT 

*2, *3A, 

*3B, 

*3C

Myelotoxicity

Thrombocytope

nia, 

granulocytopeni

a and 

neutropenia 

1 7 1 0 0 0 15 (0.39, 

576.73)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 91

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Marinaki, 

2004

69

TPMT 

*2, *3A, 

*3C

Hepatitis

Serum alanine 

transaminase 

levels greater 

than twice upper 

normal limit (50 

IU/L) and 

resolution after 

withdrawal of 

AZA

3 110 0 0 1 16 2.29 (0.22, 

23.39)

Pancreatitis

Severe 

abdominal pain 

and serum 

amylase > 800 

U/L

7 106 0 0 1 16 0.95 (0.11, 

8.21)

Neutropenia

Neutrophil count 

of <2x109 cells

10 103 0 0 1 16 0.64 (0.08, 

5.37)

von 

Ahsen, 

2005

43

TPMT 

*2, *3A, 

*3B, 

*3C

Hepatitis

Aspartate or 

alanine 

aminotransferas

e 2 times the 

upper limit of 

reference 

interval

3 63 0 0 0 5 1.65 (0.08, 

36.2)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 92

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Pancreatitis

Upper 

abdominal pain 

with pancreatic 

amylase or 

lipase 2 times 

the upper limit of 

the reference 

interval

1 65 0 0 1 4 16.25 (0.85, 

310.48)

Myelotoxicity

Myelosuppressi

on - leukocyte 

counts < 

2.5x109

/L or 

platelet counts 

<100x109/L

0 66 0 0 0 5

Jun, 2005

67

TPMT 

*2, *3A, 

*3B, 

*3C, 

3D, *6

Hepatitis

>= Two fold 

elevation of 

upper normal 

limit

1 86 0 0 0 7 3.84 (0.14, 

102.83)

Leukopenia

WBC < 4000 

mm3 and <75% 

before 

administration

16 71 0 0 1 6 0.74 (0.08, 

6.58)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 93

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Stocco, 

2004

42

TPMT 

*2, *3A, 

*3B, * 

3C

Any infection 

(including 

sepsis)

Infection 

documented 

clinically or 

microbiologicall

y

3 37 0 0 0 4 1.19 (0.05, 

26.97)

Hepatitis

ALT, GGT or 

alkaline 

phosphatase 

more than twice 

their normal 

levels

2 38 0 0 0 4 1.71 (0.07, 

41.54)

Pancreatitis

Severe 

abdominal pain,

accompanied by 

a serum amylase 

level of greater

than twice

their normal 

levels

2 38 0 0 0 4 1.71 (0.07, 

41.54)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 94

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Myelotoxicity

WBC count < 

3000 cells / 

mm3, and 

platelets < 

100,000 / mm3

13 27 0 0 0 4 0.23 (0.01, 

4.52)

Snow, 

1995

39

NR Hepatitis

9 (Raised AST 

and ALT)

1 20 0 0 0 5 1.24 (0.04,

34.93)

Leukopenia

NR

1 20 0 0 2 3 13.33 (0.91, 

196.38)

Tani, 2009

76

TPMT*2

, *3A, 

*3B, 

*3C

WDAE

NA

18 58 0 1 0 1 1.05 (0.04, 

27)

1.05 (0.04, 27)

Leukopenia

<3000/mm3

7 69 0 1 0 1 3.09 (0.12, 

82.76)

3.09 (0.12, 

82.76)

Thrombocytope

nia

<100000/ mm3

1 75 0 1 0 1 16.78 (0.47, 

605.23)

16.78 (0.47, 

605.23)

Any infection 

(including 

sepsis)

NR

4 72 0 1 0 1 5.37 (0.19, 

151.44)

5.37 (0.19, 

151.44)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 95

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Palmieri, 

2007

70

TPMT

*3A, 

*3B, 

*3C

Pancreatitis

Upper 

abdominal pain 

with pancreatic 

amylase and 

lipase greater 

than twice the 

normal upper 

limit

16 377 0 2 0 27 4.58 (0.21, 

99.18)

0.42 (0.02, 

7.12)

Leukopenia

WBC <3.0 x 109/L

17 376 2 0 4 23 107.57 (4.97, 

2326.61)

3.85 (1.2, 

12.37)

26.11 (1.06, 

640.27)

Hepatitis

Serum alanine 

transaminase 

increase greater 

than twice the 

upper normal 

limit and 

resolution after 

withdrawal or 

dose reduction

11 382 0 2 1 26 6.65 (0.3, 

146.57)

1.34 (0.17, 

10.75)

3.53 (0.11, 

111.68)

Jae Hak, 

2008

65

TPMT 

*1, *2, 

*3A, 

*3B, 

*3C

Leukopenia

WBC<3.0 x 109

/L 

113 166 0 0 5 2 3.67 (0.7, 

19.26)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 96

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

Schmelin

g, 2007

72

TPMT 

*2, *3A, 

*3B, 

*3C

Leukopenia

WBC <3.0x 

10^9/L

4 48 0 0 2 2 12 (1.32, 

109.34)

Hepatitis

LFT >100U/L

12 40 0 0 0 4 0.36 (0.02, 

7.16)

Lopez, 

2006

68

TPMT*2

, *3A, 

*3B, 

*3C, 

*3D

Pancreatitis

NR

52 50 0 0 1 4 0.24 (0.03, 

2.23)

Seddik, 

2003

73

TPMT 

*2, *3A, 

*3B, 

*3C

Leukopenia

WBC <3.0 x 109

/L

6 63 0 0 1 5 2.1 (0.21, 

21.04)

van 

Dieren, 

2005

77

NR Hepatitis

NR

8 77 0 1 0 10 3.04 (0.11, 

80.61)

0.43 (0.02, 

8.08)

Leukopenia

NR

6 79 1 0 2 8 36.69 (1.35, 

993.86)

3.29 (0.57, 

19.09)

10.2 (0.31, 

336.95)

Thrombocytope

nia

NR

0 85 1 0 0 10 513 (7.4, 

35584.39)

63 (0.87, 

4537.84)

Stassen, 

2009

40

TPMT 

*2, *3A, 

*3B, 

Leukopenia

NR

34 67 0 0 3 4 1.48 (0.31, 

6.98)
Table C-25. KQ 3c: Association between TPMT genotype and thiopurine toxicity – summary data (continued)

C - 97

First 

Author, 

year

TPMT 

alleles 

tested

Outcomes and 

definition

Non-carriers Homozygotes Heterozygote

s

Homozygote

s vs. noncarriers

Heterozygote

s vs. noncarriers

Homozygote

s vs. 

heterozygote

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

n 

with 

event

s

n 

witho

ut 

event

s

Odds Ratio (95% CI)

*3C Thrombocytope

nia

NR

8 93 0 0 0 7 0.73 (0.04, 

13.98)

Anemia

NR

30 70 0 0 3 4 1.75 (0.37, 

8.30)

Newman, 

201045

TPMT 

*2, *3A, 

*3B, 

*3C

Mortality 3 147 0 1 0 15 14.05 (0.48, 

409.13)

1.36 (0.07, 

27.55)

WDAE 0 150 0 1 0 15

SAE 7 143 1 0 0 15 57.4 (2.15, 

1531.14)

0.62 (0.03, 

11.33)

93 (1.31, 

6606.26)

Hepatitis 8 142 0 1 0 15 5.59 (0.21, 

147.71)

0.54 (0.03, 

9.83)

Pancreatitis 4 146 0 1 0 15 10.85 (0.39, 

304.77)

1.05 (0.05, 

20.43)

Neutropenia 0 150 1 0 0 15 903 (13.05, 

62488.75)

93 (1.31, 

6606.26)

Kolorz 

200979

TPMT 

*2, *3A, 

*3B, 

*3C

Leukopenia 16 64 0 0 5 2 10 (1.77, 

56.35)

Abbreviations: ALT = Alanine transaminase; AZA = azathioprine; GGT = Gamma-glutamyl transferase; L = liter; LFT = liver function test; mm = millimetre; NR = not reported; 

TPMT = thiopurine methyltransferase; WBC = white blood cell count; U = Unit of enzymatic activity = nanomole of product per hour; WDAE = withdrawal due to adverse events; 

SAE = serious adverse event
C - 98

Key Question 4: For patients with chronic autoimmune disease, costs 

of TPMT testing, and treating drug-associated complications.

Table C-26. KQ 4: Costs of TPMT testing

Study Currency Year of 

costing 

data

Source of 

costing data

Type of test Costing 

details

Cost 

reported 

in study

Converted 

to

USD 2009*

Hagaman 

201080

NZ 2007 Not reported Phenotype Not reported $300.00 $320.98

Gurwitz 

200981

GBP 2006 Estimated 

from a RCT

Genotype Cost of test 

per patient

$30.00 $61.67

Gurwitz 

200981

Euro 2006 UCB Pharma 

(Spain)

Phenotype Cost of test 

per patient

$40.00 $58.57

Gurwitz 

200981

GBP 2006 Average 

from the 

Guy's 

Hospital and 

the London 

City Hospital

Phenotype Cost of test 

per patient

$29.00 $59.61

Compagni 

200882

Euro 2006 Survey of 

European 

labs

Genotype 

TPMT*1, *2, 

*3A, *3B, *3C

Pharmacogenetic kits, 

cost per 

patient

$33.00 $46.25

Compagni 

200882

Euro 2006 Survey of 

European 

labs

Genotype 

TPMT*2, 

*3A, *3B, *3C

Pharmacogenetic kits, 

cost per 

patient

$21.00 $29.43

Compagni 

200882

Euro 2006 Survey of 

European 

labs

Genotype DNA 

extraction, 

PCR, 

technician 

time total 

costs per 

patient

$20.00 to 

$100.00 

$28.03 to 

$140.14

Sayani 

200583

CAD 2005 Alberta’s 

provincial 

laboratory 

fees

Phenotype Cost of test 

per patient

$50.00 $47.95

Priest 

200684

USD 2004 Cost at local 

lab

Genotype Cost of test 

per patient

$78.00 $94.48

Priest 

200684

USD 2004 Cost at local 

lab

Phenotype Cost of test 

per patient

$57.75 $46.36

Dubinsky 

200585

USD 2004 Author's 

estimate 

based on 

literature 

search 

(private lab 

costs)

Genotype Cost of test 

per patient

$510.06 $617.80

Winter 

200486

GBP 2003 Cost at local 

lab

Genotype Cost of test 

per patient

$30.00 $61.92

Oh 

200487

USD 2002 Hanyang 

University 

Hospital, 

Genotype Cost of test 

per patient

$100.00 $131.51
Table C-26. KQ 4: Costs of TPMT testing

C - 99

Study Currency Year of 

costing 

data

Source of 

costing data

Type of test Costing 

details

Cost 

reported 

in study

Converted 

to

USD 2009*

Seoul

Marra

200288

CAD 1999 Based on 

other PCR 

tests

Genotype Cost of test 

per patient

$100.00 $100.88

Tavadia 

200089

CAD 1999 Cost at local 

lab and 

other PCR 

tests

Genotype Cost of test 

per patient

$100.00 $100.88

Note: * inflation and conversion rate.

Abbreviations: CAD = Canadian dollar; DNA = deoxyribonucleic acid; Euro = European Union dollar; GBP = British pound; 

PCR = polymerase chain reaction; RCT=randomized controlled trial; TPMT = thiopurine methyltransferase; USD = United 

States dollar.
C - 100

Table C-27. KQ 4: Costs of treating azathioprine-associated complications

Study Currency, 

year of 

data

Source of 

costing data

Costing item Costing details Cost 

reported 

in study

Converted 

to

USD 2009*

Prakshar 

199590

USD, 

1995

Accounting 

data of 3 US 

hospitals

One-time cost 

of adverse drug 

events (low 

range)

Derived by 

assuming that 10% 

of patients 

developed sideeffect that is 

resolved in 6 

months

$802.41 $1366.82

Prakshar 

199590

USD,

1995

Accounting 

data of 3 US 

hospitals

One-time cost 

of adverse drug 

events (high 

range)

Derived by 

assuming that 30% 

patients developed 

side-effect that is 

resolved in 6 

months

$2407.22 $4100.45

Tavadia 

200089

CAD,

1999

Sunnybrook 

and Women's 

College 

Health Centre

One-time cost 

of adverse drug 

events

$7048.00 $7110.02

Marra

200288

CAD,

1999

Canadian 

provincial 

guide to 

medical fees

One-time cost 

of adverse drug 

events

Derived by 

assuming 50% will 

require outpatient 

and 50% will 

require inpatient 

care

$1734.50 $1749.76

Winter 

200486

GBP,

2003

Information 

and statistics 

division of 

Common 

Services 

Agency

One-time cost 

of adverse drug 

events

Derived by 

assuming 32% will 

develop 

leukopenia

$1367.00 $2821.66

Oh 

200487

USD,

2002

Hanyang 

University 

Hospital, 

Seoul

One-time cost 

of adverse drug 

events

Average of 4 cases 

with AZA-induced 

neutropenia

$2501.00 $3289.13

Priest 

200684

USD,

2004

Local public 

hospital in 

New Zealand

One-time cost 

of adverse drug 

events

Average costs of 

life-threatening, 

severe, and 

moderate 

leukopenia

$5009.67 $6067.82

Hagaman 

201080

USD, 

2007

DRG 420, 

Professional 

costs

One-time cost 

of adverse drug 

events

Average cost of 

complicated 

leukopenia and 

uncomplicated 

leukopenia

$5279.50 $5648.65

Hagaman 

201080

USD, 

2007

DRG 420, 

Professional 

costs

One-time cost 

of adverse drug 

events

Cost of 

complicated 

leukopenia leading 

to death

$14666.0

0

$15691.46

Note: * inflation and conversion rate.

Abbreviation: AZA = azathiopurine; CAD = Canadian dollar; GBP = British pound; USD = United States dollar. 
C - 101

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Appendix D. Excluded Studies

Level II Exclusions by reasons (for all 334 excluded records):

Exclude on record type (editorial, review, commentary, letter, news, report or 

case report), n=44:

17th IFCC-FESCC European Congress of Clilnical 

Chemistry and Laboratory Medicine/60th National 

Congress of the Netherlands-Society-for-ClinicalChemistry-and-Laboratory-Medicine 

(EUROMEDLAB 2007), Amsterdam, 

NETHERLANDS, June 03 -07, 2007. Clinical 

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S):S5,S7,S9-S5,SS448. 

43rd Annual Meeting of the Academy-of-ClinicalLaboratory-Physicians-and-Scientists, Philadelphia, 

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E-1

Appendix E. Additional Acknowledgements

The UO-EPC gratefully acknowledges the following individuals who served on our 

Technical Expert Panel (TEP). Acknowledgment does not reflect endorsement of this 

report.

Dr. Thomas Trikalinos, MD, PhD

Co-Director

Evidence-Based Practice Centre

Tufts University

Boston, MA

Dr. Marc S. Williams, MD

Director

Intermountain Healthcare Clinical Genetics 

Institute

Salt Lake City, UT

Dr. Jonathan Berg, PhD

Director, Divison of Pathology

Clinical Biochemistry

City Hospital

Birmingham, UK

Dr. Peter Bunting, PhD

Associate Professor / Clinical Biochemist

University of Ottawa / The Ottawa Hospital

Ottawa, ON

The UO-EPC gratefully acknowledges the following individuals who peer reviewed the 

initial draft of this report. Acknowledgment does not reflect endorsement of this report.

Dr. Marta Gwinn, MD, M.P.H.

Associate Director for Epidemiology 

National Office of Public Health Genomics

Center for Disease Control and Prevention

Atlanta, GA

Dr. Marc S. Williams, MD

Director

Intermountain Healthcare Clinical Genetics 

Institute

Salt Lake City, UT

Dr. Loralie Langman, PhD

Associate Professor

Department of Laboratory Medicine & 

Pathology

Mayo Clinic

Rochester, MN

Dr. Nicolas von Ahsen, PhD

Department of Clinical Chemistry

University of Göttingen

Göttingen, Germany