i

Evidence Report/Technology Assessment 

Number 180 

Outcomes of Genetic Testing in Adults with a History 

of Venous Thromboembolism 

Prepared for: 

Agency for Healthcare Research and Quality 

U.S. Department of Health and Human Services 

http://www.ahrq.gov

Contract No. HHSA 290-2007-10061-I 

Prepared by: 

The Johns Hopkins University Evidence-based Practice Center 

Investigators 

Jodi B. Segal, M.D., M.P.H. 

Daniel J. Brotman, M.D. 

Ashkan Emadi, M.D., Ph.D. 

Alejandro J. Necochea, M.D., M.P.H. 

Lipika Samal, M.D. 

Lisa M. Wilson, M.S. 

Matthew T. Crim, M.Sc., M.A. 

Eric B. Bass, M.D., M.P.H. 

AHRQ Publication No. 09-E011 

June 2009 
ii

 This report is based on research conducted by the Johns Hopkins University Evidence-based 

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

(AHRQ), Rockville, MD (Contract No. HHSA 290-2007-10061-I). 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. 
iii

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: 

Segal JB, Brotman DJ, Emadi A, Necochea AJ, Samal L, Wilson LM, Crim MT, Bass EB.

Outcomes of Genetic Testing in Adults with a History of Venous Thromboembolism. Evidence 

Report/Technology Assessment No. 180. (Prepared by Johns Hopkins University Evidencebased Practice Center under contract no. HHSA 290-2007-10061-I). AHRQ Publication No. 09-

E011. Rockville, MD. Agency for Healthcare Research and Quality.
iv

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. This report was requested and funded by the Centers 

for Disease Control and Prevention (CDC), Office of Public Health Genomics (OPHG). 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 

Beth A. Collins Sharp, Ph.D., R.N. 

Acting Director, EPC Program 

Agency for Healthcare Research and Quality 

Gurvaneet Randhawa, M.D., M.P.H. 

Task Order Officer, Center for Outcomes and 

Evidence 

Agency for Healthcare Research and Quality 

Thomas R. Frieden, M.D., M.P.H. 

Director 

Centers for Disease Control and Prevention 
v

Acknowledgments 

The EPC thanks Renee F. Wilson, Dr. Olaide Odelola, and Ritu Sharma for their assistance 

with the final assembly and formatting of this report, and Dr. Gurvaneet Randhawa for his 

valuable insight. 
v 

Structured Abstract 

Objective: To address whether Factor V Leiden (FVL) testing alone, or in combination with 

prothrombin G20210A testing, leads to improved clinical outcomes in adults with a personal 

history of venous thromboembolism (VTE) or to improved clinical outcomes in adult family 

members of mutation-positive individuals. 

Data sources: Searches of MEDLINE®, EMBASE®, The Cochrane Library, the Cumulative 

Index to Nursing & Allied Health Literature, and PsycInfo© through December 2008. 

Review methods: We focused on the analytic validity, clinical validity, and clinical utility of 

these tests. Each included article underwent double review for data abstraction and assessment of 

study quality. We pooled the results of studies addressing the clinical validity of these tests when 

there were sufficient data. Other evidence was summarized in evidence tables. We graded the 

evidence by adapting a scheme recommended by the Grading of Recommendations Assessment, 

Development and Evaluation (GRADE) Working Group by assessing the limitations affecting 

individual study quality, the certainty regarding the directness of the observed effects in the 

studies, the precision and strength of the findings, and the availability (or lack) of data to answer 

the relevant key question. Evidence for each sub-question was graded as high, moderate, or low. 

Results: We reviewed 7,777 titles and included 124 articles. No direct evidence addressed the 

primary objective. However, high-grade evidence supported the conclusion that tests for the 

detection of FVL and prothrombin G20210A have excellent analytic validity. Most clinical 

laboratories test for these mutations accurately. Heterozygosity [odds ratio (OR) =1.56 (95 

percent confidence interval (CI) 1.14 to 2.12)] and homozygosity [OR=2.65 (95 percent C.I. 1.2 

to 6.0)] for FVL in probands are predictive of recurrent VTE. Heterozygosity for FVL predicts 

VTE in family members [OR=3.5 (95 percent C.I. 2.5 to 5.0)] as does homozygosity for FVL 

[OR=18 (95 percent C.I. 7.8 to 40)]. Heterozygosity for prothrombin G20210A is not predictive 

of recurrence in probands [OR=1.45 (95 percent C.I. 0.96-2.2)]. Evidence is insufficient about 

heterozygosity for prothrombin G20210A in family members and insufficient about 

homozygosity for prothrombin G20210A. A single study supported the hypothesis that clinicians 

might change management based on test results. There was high-grade evidence that 

anticoagulation reduces recurrent events in probands with FVL or prothrombin G20210A; 

however, there was low-grade evidence that the relative reduction with treatment is comparable 

to that seen in individuals without mutations. There was moderate evidence to support the 

conclusion that neither harms nor benefits of testing have been demonstrated conclusively. 

Decision-analysis models suggest that testing may be cost-effective in select individuals. 

Conclusions: There is no direct evidence that testing for these mutations leads to improved 

clinical outcomes in adults with a history of VTE or their adult family members. The literature 

supports the conclusion that while these assays have high analytic validity, the test results have 

variable clinical validity for predicting VTE in these populations and have only weak clinical 

utility. 
vi 

Contents 

 

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

Evidence Report...........................................................................................................................15 

Chapter 1. Introduction .................................................................................................................17 

Venous Thromboembolism........................................................................................................3

Diagnosis and Treatment ...........................................................................................................4 

Genetic Mutations Associated with VTE: FVL and Prothrombin G20210A............................4 

Methods for Identifying Mutations............................................................................................5 

Objectives of this Evidence Report............................................................................................5 

Chapter 2. Methods..........................................................................................................................7 

Establishing a Technical Expert Panel.......................................................................................7 

Analytic Framework ..................................................................................................................7 

Key Questions............................................................................................................................8 

Search Strategy ..........................................................................................................................9 

Study Selection ........................................................................................................................10 

Data Abstraction ......................................................................................................................11 

Study Quality Assessment .......................................................................................................12 

Applicability ............................................................................................................................13 

Data Synthesis..........................................................................................................................13 

Data Entry and Quality Control ...............................................................................................14

Rating the Body of Evidence ...................................................................................................14 

Chapter 3. Results ..........................................................................................................................15 

Results of the Search................................................................................................................15 

 Description of types of studies retrieved ...........................................................................15 

Key Questions 1. Association of FVL testing alone, or in combination with prothrombin 

G20210A testing, with improved clinical outcomes in adults with a personal history of 

VTE, or in adult family members of mutation-positive individuals..................................15 

Key Question 2: Analytic validity of tests to identify FVL and prothrombin G20210A 

mutations............................................................................................................................17 

Detection of FVL ...............................................................................................................24 

Detection of prothrombin G20210A..................................................................................25 

Simultaneous detection of FVL and prothrombin G20210A mutations............................25 

External quality assurance ................................................................................................25 

Summary of evidence for Key Question 2.........................................................................31 

Key Question 3. Clinical validity of testing for FVL and/or prothrombin G202010A ...........32 

FVL and recurrent venous thrombosis in probands...........................................................32 

Summary of evidence for Key Question 3 (probands) ......................................................39 

FVL and prothrombin G20210A and venous thrombosis in family members ..................40 

Association of FVL and prothrombin G20210A with venous thrombosis during 

pregnancy and the post-partum period.........................................................................46 

Age and VTE among family members with mutations .....................................................47 
vii 

Other high-risk periods ......................................................................................................48 

Summary of evidence for Key Question 3 (family members)...........................................48 

Key Question 4. Clinical utility of testing for FVL and/or prothrombin G202010A..............49 

Effect of testing on clinicians’ management (KQ 4a) .......................................................49 

Effect of testing and resultant management on VTE-related outcomes (KQ 4b) ..............50 

Effect of testing and results on other outcomes (KQ 4c)...................................................53 

Cost-effectiveness of FVL and prothrombin G20210A testing in the case of probands 

 and their relatives (KQ4d) ...........................................................................................57 

Summary of evidence for Key Question 4.........................................................................63 

Chapter 4. Discussion ....................................................................................................................65 

Key Question 1. Association of FVL testing alone, or in combination with prothrombin 

G20210A testing lead to improved clinical outcomes in adults with a personal history of 

VTE, or in adult family members of mutation-positive individuals..................................65 

Key Question 2. Analytic validity of tests to identify FVL and prothrombin G20210A 

 mutations ...........................................................................................................................65 

Key Question 3. Clinical validity of testing for FVL and prothrombin G20210A 

 mutations............................................................................................................................66 

Testing probands................................................................................................................66 

Testing family members ....................................................................................................67 

Key Question 4. Clinical utility of testing for FVL and prothrombin G20210A mutations....68 

Effect of testing on clinicians’ management......................................................................68 

Effect of testing and resultant management on VTE-related outcomes ............................68 

Effect of testing and results on other outcomes.................................................................69 

Cost effectiveness of FVL and prothrombin G20210A testing in the care of 

 probands and their relatives .........................................................................................69 

Summary of the Evidence........................................................................................................71 

Limitations of this Report ........................................................................................................73 

Implications for Future Research.............................................................................................74

Analytic validity.................................................................................................................75 

Clinical validity..................................................................................................................75 

Clinical utility ....................................................................................................................76 

References......................................................................................................................................77 

Acronyms.......................................................................................................................................99 

Figures 

Figure 1. FVL and prothrombin testing for prevention of venous thromboembolic 

 events: analytic framework..................................................................................................8 

Figure 2. Summary of literature search (number of articles)...................................................16 

Figure 3. Odds ratios for VTE for probands with heterozygous FVL mutation, as 

 compared to probands without the mutation......................................................................34 

Figure 4. Odds ratios for VTE for probands with homozygous FVL mutation, as 

 compared to probands without the mutation......................................................................35 

Figure 5. Odds ratios for VTE for probands with mixed FVL mutation, as 
viii 

 compared to probands without the mutation......................................................................36 

Figure 6. Odds ratios for VTE for probands who were double heterozygous for the 

 FVL and prothrombin G20210A mutations, as compared to probands without 

 the mutation .......................................................................................................................37 

Figure 7. Odds ratios for VTE for probands who were heterozygous for prothrombin 

 G20210A, as compared with probands without the mutation............................................38 

Figure 8. Odds ratios for VTE for probands with unspecified prothrombin G20210A 

 Mutation, as compared with probands without the mutation.............................................39 

Figure 9. Odds ratios for VTE for family members with heterozygous FVL mutation, as 

compared to family members without the mutation ..........................................................42 

Figure 10. Odds ratios for VTE for family members with homozygous FVL , as compared 

 to family members without the mutation...........................................................................43 

Figure 11. Odds ratios for VTE for family members with unspecified FVL , as compared 

 to family members without the mutation...........................................................................44 

Figure 12. Odds ratios for VTE for family members with double heterozygous mutations, 

 as compared to family members without the mutation......................................................45 

Figure 13. Odds ratios for VTE for family members with unspecified prothrombin 

 G20210A mutation, as compared to family members without the mutation.....................46 

Summary Tables 

Table 1. List of additional exclusion criteria by Key Question ...............................................11 

Table 2. Analytic validity of testing for the FVL mutation .....................................................18 

Table 3. Details of studies on analytic validity of testing for FVL mutation ..........................22 

Table 4. Analytic validity of testing for prothrombin G20210A.............................................26 

Table 5. Details of studies on analytic validity of testing for prothrombin G20210A ............28 

Table 6. Analytic validity of simultaneous detection (multiplex) of FVL and prothrombin 

 G20210A mutations ..........................................................................................................29

Table 7. Details of studies on analytic validity of simultaneous detection (multiplex) of 

 FVL and prothrombin G20210A mutations ......................................................................30 

Table 8. Pooled results describing risk of recurrent VTE for probands with VTE .................33 

Table 9. Pooled results describing the risk of VTE for family members of probands with 

 VTE comparing those with a mutation to those without a mutation .................................40 

Table 10. Four studies examining the effect of a VTE-related outcomes of changes in 

 therapy based on testing for FVL or prothrombin G20210A.............................................51 

Table 11. Studies examining the effect of FVL and prothrombin G20210A 

 testing on non-VTE-related outcomes.................................................................................54 

Table 12. Summary of studies evaluating the cost-effectiveness of testing for FVL and/or 

prothrombin G20210A mutations .......................................................................................58 

Table 13. Strength of the evidence regarding Key Questions 1 and 2: overarching question 

 and analytic validity ............................................................................................................71 

Table 14. Strength of the evidence regarding Key Question 3: clinical validity.....................72 

Table 15: Strength of the evidence regarding Key Question 4: clinical utility .......................73 

Appendixes 

Appendix A: Technical Experts and Peer Reviewers 

Appendix B: Hand-searched Journals 
ix 

Appendix C: Detailed Search Strategies 

Appendix D: Excluded Articles 

Appendix E: Data Review Forms 

Appendix F: Evidence Tables 

Appendixes and Evidence Tables for this report are provided electronically at 

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

Executive Summary 

Introduction 

Venous thromboembolism (VTE) refers to pathologic thrombosis in the venous circulation. 

Although the most frequent venous thromboembolic event is deep venous thrombosis in the 

veins of the legs, thromboses can also occur in the veins of the upper extremities, pelvis, 

abdomen, and cerebral venous sinuses. Pulmonary embolism is the main life-threatening 

complication of deep vein thrombosis, in which a portion of the venous thrombus is carried to 

the pulmonary arteries by blood flow, potentially obstructing the pulmonary vasculature. 

Treatment begins with short-term use of a parenteral anticoagulant (and sometimes thrombolytic 

therapy) and then usually continues with a vitamin K antagonist, most commonly warfarin. The 

duration of therapy depends on whether the patient is considered to have continuing risk factors 

for recurrence. 

Much effort has been devoted to quantifying the risk of recurrent thrombosis. If the patient 

has a persistent risk factor for thrombosis, anticoagulant therapy is often continued, sometimes 

for the life of the patient. The clinician and patient also try to reduce exposure to any modifiable 

risk factors and may use mechanical or pharmacological means of preventing thrombosis at highrisk times, such as during hospitalization or pregnancy. 

The question of whether a patient has ongoing risk leads to the subject of this report, the 

value of testing individuals who have had a venous thromboembolic event for Factor V Leiden 

(FVL) and prothrombin G20210A. The FVL mutation is a base change (from G to A at position 

1691) in the gene coding for the Factor V protein; the resulting amino acid substitution 

eliminates one of three activated protein C cleavage sites in Factor V. As a result, Factor V is 

inactivated to a lesser extent and persists longer in the circulation, leading to more thrombin 

generation. In the United States, a single FVL allele is present in about 5, 2.2, and 1.2 percent of 

the Caucasian, Hispanic, and African American populations, respectively. The prothrombin 

(Factor II) mutation is the second most common inherited risk factor for thrombosis. The 

mutation in the prothrombin G20210A gene is associated with an elevation of prothrombin levels 

to about 30 percent above normal in heterozygotes and to 70 percent above normal in 

homozygotes. In the United States, the prevalence of this allele is 1.1 percent in Caucasians and 

Hispanics and 0.3 percent in African Americans. 

The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) initiative was 

established by the Office of Public Health Genomics at the Centers for Disease Control and 

Prevention (CDC) to address the increasingly urgent need for timely and objective information 

that would allow health care providers and payers, policymakers, and consumers to identify 

genetic tests that are safe and useful and to provide guidance on their appropriate use in practice, 

based on available evidence. At their request, we reviewed the evidence regarding the value of 

testing for these mutations in two specific populations: (1) individuals who have had a venous 

thromboembolic event (probands), and (2) their family members. 
2

Methods 

The overarching question we were asked to address (Key Question [KQ] 1) was: Does FVL 

testing, alone or in combination with prothrombin G20210A testing, lead to improved clinical 

outcomes (e.g., avoidance of a recurrent VTE) in adults with a personal history of VTE or to 

improved clinical outcomes (e.g., avoidance of an initial VTE) in adult family members of 

mutation-positive individuals? Are testing results useful in medical, personal, or public health 

decision making? To address this question, we reviewed the literature regarding these tests’ 

analytic validity, clinical validity, and clinical utility when used in probands with VTE and in 

their family members. 

The other KQs were as follows: 

KQ2 What is the evidence regarding the analytic validity of existing diagnostic tests for the 

FVL mutation and the prothrombin G20210A mutation, specifically their analytic 

sensitivity and specificity, reproducibility, and robustness (sources of variability)? 

KQ3a What is the evidence that the presence of FVL alone, prothrombin G20210A alone, or the 

two in combination predicts the risk of recurrent VTE in individuals (probands) who have 

had VTE and predicts the risk of VTE in the probands’ family members who have been 

tested? Does the testing add predictive information beyond clinical data? 

KQ3b What is the evidence that demographic or clinical factors modify the relationship between 

the presence of FVL or prothrombin G20210A and the risk of VTE? 

KQ4a What is the evidence that clinicians manage patients differently based on the results of 

testing for FVL or prothrombin G20210A? How do clinicians manage anticoagulation of 

individuals who have had testing, as compared to those who have not had testing? What 

other diagnostic tests do clinicians order or not order, based on testing results? What 

recommendations do clinicians make regarding other therapies and exposures, based on 

testing results? 

KQ4b What is the evidence that testing, and the resultant management, reduces VTE relatedoutcomes or has other benefits in individuals who have had VTE or in the probands’ 

family members who have been tested? 

KQ4c What is the evidence of harms to individuals with VTE or to the probands’ family 

members who are tested for FVL or prothrombin G20210A as a result of testing or as a 

result of changed management based on the test results? 

KQ4d What is the evidence that testing for FVL alone, prothrombin G20210A alone, or the two 

tests in combination is a cost-effective strategy when caring for a patient with VTE or a 

family member of a proband? 

Our comprehensive search included electronic and hand searching. We searched five 

databases, MEDLINE® (1950 through May 2008), EMBASE® (1974 through December 2008), 
3

The Cochrane Library (Issue 2, 2008), the Cumulative Index to Nursing & Allied Health 

Literature (CINAHL®; 1982 through December 2008) and PsycInfo©, to identify primary 

literature on the analytic validity, clinical validity, and clinical utility of testing for FVL and 

prothrombin G20210A. 

Two independent reviewers, from among six study team members, conducted title scans in 

parallel. The title review was designed to capture as many studies as possible that reported on the 

analytic validity, clinical validity, and clinical utility of testing for FVL and prothrombin 

G20210A. All titles potentially addressing these issues were promoted to the abstract review 

phase. 

Abstracts were reviewed independently by two investigators. Abstracts were excluded if the 

investigators agreed that the article: (1) was not relevant to any of the key questions; (2) did not 

include any human data; (3) contained no original data; (4) was not conducted in adults; and (5) 

was not published in English. Differences of opinion were resolved through consensus 

adjudication. 

Full articles selected for review underwent another independent parallel review by two 

investigators. In addition to the exclusion criteria used for the abstract review, there were 

additional exclusion criteria for each KQ. For the question about clinical validity of the 

mutations (KQ 3), we included only prospective studies of probands, although we permitted 

retrospective studies of family members because we anticipated few prospective studies. Each 

article underwent double review by study investigators for full data abstraction and assessment 

of study quality. We used a sequential review process in which the primary reviewer completed 

all data abstraction forms, and the second reviewer checked the first reviewer’s data abstraction 

forms for completeness and accuracy. Reviewer pairs were formed to include personnel with 

both clinical and methodological expertise. All information from the article review process was 

entered directly into the SRS 4.0 database. 

The primary outcome extracted from the studies of analytic validity was concordance 

between the test and the reference test, as that was most often reported. When there were 

sufficient data (three or more studies) and the studies were qualitatively homogeneous with 

respect to key variables (population characteristics, study duration, mutation status, and length of 

follow-up), we conducted meta-analyses for the studies addressing the clinical validity of the 

tests. When it was inappropriate to combine studies quantitatively, we qualitatively summarized 

the results. For pooling, we used the number of events and count of the patients under 

observation in each group. We calculated a pooled estimate of the odds ratio for VTE in 

probands and separately in family members. We used a random effects model with the 

DerSimonian and Laird method for calculating between-study variance. 

At the completion of our review, we graded the quantity, quality, and consistency of the best 

available evidence addressing the KQs by adapting an evidence-grading scheme recommended 

by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) 

Working Group. To assess the quantity of evidence, we focused on the number of studies with 

the strongest design. We also assessed the quality and consistency of the best available evidence, 

including assessment of the limitations affecting individual study quality (using the individual 

study quality assessments), certainty regarding the directness of the observed effects in the 

studies, the precision and strength of the findings, and the availability (or lack) of data to answer 

the KQ. We classified evidence bodies as: (1) “high” grade, indicating confidence that further 

research is very unlikely to change our confidence in the estimated effect in the abstracted 

literature; (2) “moderate” grade, indicating that further research is likely to have an important 
4

impact on our confidence in the estimates of effects and may change the estimates in the 

abstracted literature; (3) “low” grade, indicating the further research is very likely to have an 

important impact on confidence in the estimates of effects and is likely to change the estimates in 

the abstracted literature. 

Results 

 We reviewed 7,777 titles and included 124 articles in the review of one or more of the KQs. 

Key Question 1 

We identified no evidence to directly address KQ1, which required studies that directly tested 

the impact of testing on patient outcomes. The articles addressing the questions below, 

particularly KQ4, provided indirect evidence to answer KQ1. 

Key Question 2 

 The conventional “gold standard” method for FVL and prothrombin G20210A detection is 

the sequencing of the specific genetic region of the gene of interest. However, a number of other 

reference methods are used instead because of the complexity and high costs associated with 

sequencing. Many studies that we reviewed used polymerase chain reaction and restriction 

fragment length polymorphism (PCR-RFLP) or allele-specific polymerase chain reaction (ASPCR) assays as their reference standards, both of which are considered by the Food and Drug 

Administration to be acceptable reference standards. 

Detection of FVL. Forty-one studies compared at least two methods for FVL detection. The 

majority of studies demonstrated 100 percent concordance between the experimental method and 

the reference method. The least concordance was seen in a study using electrochemical 

genosensors, which had a concordance of only 93 percent. 

Detection of prothrombin G20210A. The concordance rates between the experimental methods 

and the reference standards for the detection of prothrombin G20210A were 100 percent in 

nearly all of the 23 studies. 

Simultaneous detection of FVL and prothrombin G20210A. All 12 studies that employed 

multiplex technologies for the simultaneous detection of the two mutations reported 100 percent 

concordance between this approach and the matched reference standard. 

Quality assurance. We identified three studies that addressed external quality assurance or 

laboratory performance relative to the gold standards. One described the results from the United 

Kingdom National External Quality Assessment Scheme (UK NEQAS) Thrombophilia 

Screening Program. Two hundred eighty centers participated in the thrombophilia screening 

exercises. For the centers performing genetic analysis for FVL and the prothrombin mutation, an 
5

error rate of 3 to 6 percent was identified, with both transcription-related and analytical errors 

observed. 

Another study described results from the Royal College of Pathologists of Australasia 

external quality assurance (QA) program. This program sent 133 DNA samples with known 

mutations to laboratories in 10 separate surveys. Of 3,799 responses, the overall successful 

identification rate was 98.6 percent. Success rates in identifying specific mutations were 98.1 

percent for FVL and 98.8 percent for prothrombin G20210A. 

Finally, a survey was organized by the Subcommittee on Hemostasis of the Italian 

Committee for Standardization of Laboratory Tests (CISMEL). The authors concluded that 

regular quality control programs are warranted to identify the causes of failures to correctly 

identify specimens. 

Key Question 3 

Probands. Twenty-two articles examined the rates of recurrent venous thromboembolism in 

individuals with a history of VTE (probands) and the FVL mutation. 

We pooled 13 studies comparing probands with heterozygous FVL to probands without this 

mutation, yielding an odds ratio for recurrence of 1.56 (95 percent confidence interval [CI], 1.14 

to 2.12). The annualized event rates among heterozygous individuals were 3.1 percent to 7.5 

percent in three studies. Seven studies described event rates in probands homozygous for FVL 

and those without mutations. The number of homozygous individuals ranged from 1 to 11 across 

studies. The odds ratio of recurrence was 2.65 (95 percent CI, 1.2 to 6.0). 

Eight studies did not specify if the participants were heterozygous or homozygous for FVL. 

The pooled odds ratio (from 3 studies) associated with this unspecified status was 1.56 (95 

percent CI, 0.75-3.2), very similar to the odds ratio for known heterozygous individuals. 

We identified 18 articles that examined rates of recurrent VTE in probands with the 

prothrombin G20210A mutation. Nine of these compared individuals heterozygous for the 

mutation to individuals without mutation. The pooled odds ratio was 1.45 (95 percent CI, 0.96-

2.2). Two studies described individuals homozygous for this mutation, but there were only 3 

individuals with homozygosity. None of the three had a recurrent thrombosis. 

Seven studies did not specify whether the individuals had homozygous or heterozygous 

prothrombin G20210A. The pooled odds ratio in the four studies with available data was 0.73 

(95 percent CI, 0.37-1.4). 

Doubly heterozygous individuals (with FVL and the prothrombin G20210A mutations) had 

an odds ratio of 4.8, with a wide confidence interval (95 percent CI, 0.5 to 46.) 

When we separately evaluated patients with idiopathic VTE as the index event, we found that 

the odds ratio associated with heterozygous FVL was close to one (1.17 [95 percent CI, 0.63-

2.18]). 

Family members. We identified 17 articles that evaluated the occurrence of venous thrombosis 

in family members of probands. The majority of these were retrospective studies, although three 

included a prospective component. Nine studies described results for family members who were 

heterozygous for FVL. Six studies contributed to the pooled odds ratio, which was 3.5 (95 

percent CI, 2.5 to 5.0). Homozygosity for FVL among family members had a pooled odds ratio 

of 18 (95 percent CI, 7.8 to 40) in the five studies pooled. The six studies that did not specify 
6

whether individuals were homozygous or heterozygous had a pooled odds ratio of 2.9 (95 

percent CI, 1.8-4.8). 

Only three studies evaluated heterozygosity for prothrombin G20210A in family members. 

The odds ratio was 1.9 (95 percent CI, 0.35-10). There was only a single study of family 

members with homozygosity for this mutation. 

Doubly heterozygous family members had a pooled odds ratio of 6.7 (95 percent CI, 2.9 to 

15) in the three studies with data to pool. 

A sizeable subgroup of family members had VTE associated with pregnancies. Four articles 

exclusively addressed the risk of VTE attributable to FVL and prothrombin G20210A during 

pregnancies of family members, and one additional study of family members had useable 

pregnancy data. 

Two of these studies, which included few women, evaluated the risk associated with 

homozygous FVL. The odds ratios for venous thrombosis were 16 (95 percent CI, 0.9 - 278) and 

41(95 percent CI, 5.5-419). The odds ratios associated with heterozygous FVL in two studies 

were 5.4 (95 percent CI, 0.65-46) and 3.4 (95 percent CI, 0.35 to 33). The rates of events were 

low, at 2.5 percent per pregnancy (95 percent CI, 0.9-5.4) and 1.5 percent per pregnancy (95 

percent CI, 0.5-4.3), respectively. Two studies evaluated the risk associated with heterozygosity 

for the prothrombin G20210A mutation. The rates of VTE were low, at 0.3 percent per 

pregnancy (95 percent CI, 0.1 to 1.6) and 1 percent per pregnancy (95 percent CI, 0.2-3.6), 

respectively. The odds ratios in these two studies were close to 1. 

Individuals who were doubly heterozygous and pregnant were evaluated in two studies. The 

larger of the two studies reported an odds ratio similar to the odds ratios associated with 

heterozygosity for either mutation (odds ratio, 4.1; 95 percent CI, 0.37-46). The smaller study 

reported an odds ratio that was identical to that for homozygous FVL (odds ratio, 16; 95 percent 

CI, 0.9-278). 

Key Question 4 

Effect of testing on clinicians’ management. We found a single study addressing how 

physicians’ management decisions are affected by FVL testing. Canadian obstetrical care 

providers (N=662) were asked about management recommendations in response to four clinical 

scenarios involving pregnant women with FVL. For scenarios involving asymptomatic women, 

if the patient was described as having a family history of VTE, the percentage of doctors 

recommending thromboprophylaxis was twice the percentage recommending it for women 

lacking a family history (58 versus 26-34 percent). 

Effect of management, stratified by test results, on VTE-related outcomes. No studies 

directly addressed the effect of testing on outcomes. We therefore also included the four studies 

that described VTE recurrence rates during anticoagulation among probands with FVL or 

prothrombin G20210A. Two studies investigated the effect of warfarin on recurrence rates, and 

one the effect of ximelagatran; one did not specify the treatment that patients received. 

One study assessed thromboembolism recurrence rates among individuals with FVL or 

prothrombin G20210A who received either a low-intensity warfarin regimen or placebo. Lowintensity warfarin reduced the rate of recurrence among thrombophilic patients by 75 percent. 

This risk reduction, however, was not significantly different than the 58 percent reduction seen 

among patients without either mutation. 
7

In one study, the recurrence rate for patients with FVL while receiving low- or conventionalintensity warfarin therapy was 0.8 percent per year (95 percent CI, 0.2 to 2.2). This rate was not 

statistically different from the rate among those participants without FVL (hazard ratio 0.7; 95 

percent CI, 0.2 to 2.6). 

In another study, 2 of 111 FVL carriers receiving ximelagatran had recurrences of VTE, as 

compared to 16 of the 125 with mutations who were assigned to receive the placebo; this 

difference in recurrence rates was described as statistically significant. Recurrence rates with 

treatment were very similar for individuals without FVL or prothrombin G20210A, There was no 

interaction between FVL or prothrombin G20210A and the effect of ximelagatran in preventing 

recurrent VTE (p-value for the interaction, 0.92 for FVL and 0.98 for prothrombin G20210A). 

Finally, one group studied the rate of recurrence of VTE in a cohort of 304 patients in 

thrombophilic families, according to whether they were or were not receiving long-term 

anticoagulation. Long-term anticoagulation decreased the rate of VTE recurrence among these 

probands. A quantitative estimate of the risk reduction was not provided, nor were the details of 

the anticoagulation regimens available 

Effect of testing and results on other outcomes. Four studies addressed how probands’ and 

family members’ knowledge, behaviors, and healthcare experiences were affected by their being 

tested for FVL or prothrombin G20210A. Two studies employed cross-sectional surveys of 

convenience samples of probands and family members to assess risk perception and behavioral 

effects following genetic testing. The other two studies used qualitative, structured interviews of 

probands and relatives to describe their experience during the process of testing as well as their 

interpretation of the results. 

One group surveyed the perception of VTE risk and changes in behavior following testing for 

FVL or prothrombin G20210A among first-degree relatives of probands. More mutation carriers 

recognized trauma as a risk factor for VTE than did non-carriers, but otherwise there were no 

statistically significant differences between the two groups regarding their recognition of risk 

factors for VTE. Behavior changes following testing were uncommon in both groups. 

One group investigated whether the type of thrombophilic mutation and history of VTE 

affect the perception of risk and level of worry among probands or their relatives with FVL, as 

compared to other thrombophilic mutations. Individuals with a history of VTE had an increased 

perception of risk and worried more about VTE than did individuals without prior VTE. 

However, worry and risk perception were not measured in non-carriers for comparison. 

One study involved a qualitative study of asymptomatic relatives of probands with FVL to 

assess their overall experience with the testing process and how the results affected their daily 

lives. Among the 17 participants, most found that the testing process itself was not stressful; all 

had received written information about the test prior to testing. Although the majority of 

participants indicated that the testing had not altered their daily lives, many wanted to screen 

their children to decrease their risk of VTE from pregnancy or oral contraceptive use. 

One group (in two studies) assessed the level of understanding of the testing process and the 

implications of the results among probands and relatives referred for FVL testing by their 

primary care doctor or specialist. Most participants did not consider thrombophilia testing to be 

different from other tests ordered by their providers. Most participants did not incorporate 

behaviors to reduce their risk for VTE into their daily routines as a result of the testing, although 

most participants who were aware of their positive status stated they had undergone testing to 
8

inform their decision about whether to take hormonal therapies, or to advise relatives on the 

matter. 

Cost-effectiveness of FVL and prothrombin G20210A testing in the care of probands and 

their relatives. We identified six studies that assessed the cost-effectiveness of genetic testing 

and resultant changes in management and one study that assessed only effectiveness. The costeffectiveness studies all used decision analytic models, which can provide support for further 

investigation of the utility of an intervention if the assumptions in the models are compatible 

with actual practice. The data ranges explored in the sensitivity analyses demonstrated the 

variables to which the cost-effectiveness of the interventions were most sensitive. 

One group used decision analysis to assess the cost-effectiveness of testing for FVL and 

extending warfarin anticoagulation for 3 years or for life in carriers following a first VTE in a 

hypothetical cohort of 35-year-old women. If the rate of recurrence remained constant (7.3 

percent/year), lifelong anticoagulation was the more cost-effective strategy (incremental costeffectiveness ratio [ICER] = $16,823/quality-adjusted life year [QALY]) when compared to no 

testing and 6 months of anticoagulation). Lifelong anticoagulation was less cost-effective in 

patient populations with low FVL prevalence, low risk of recurrent VTE, or risk factors for 

bleeding on anticoagulant therapy. 

Another group used decision analysis to assess the cost-effectiveness of testing for FVL and 

2 years of warfarin anticoagulation in carriers following a VTE in a hypothetical cohort of 60-

year-old men. FVL testing with 2 years of anticoagulation for carriers was a cost-effective 

strategy (ICER = $12,833/QALY) when compared to no testing and 6 months of anticoagulation. 

However, this intervention was not cost-effective for individuals with a high risk of fatal 

bleeding on warfarin, low VTE recurrence rate, low anticoagulation efficacy, or low 

anticoagulation compliance. 

Building on the previous study, the author employed the same model to assess the costeffectiveness of testing for double heterozygosity, followed by 2 years of warfarin 

anticoagulation for doubly heterozygous individuals. Testing for both mutations was costeffective (ICER = $13,624/QALY) when compared to no testing and 6 months of 

anticoagulation. Testing was not cost-effective for patient populations with a high bleeding risk, 

low double-heterozygote prevalence, low levels of pulmonary embolism or mortality, or low 

anticoagulation efficacy. 

Another group assessed the cost-effectiveness of a hypercoagulability testing panel and 

warfarin anticoagulation for 6, 12, 18, 24, or 36 months, or for life, following an apparently 

idiopathic deep venous thrombosis (DVT) in a hypothetical cohort of 40 year- olds. In the base 

case analysis, extending anticoagulation for 24 months following a positive test was the most 

cost-effective option (ICER = $11,100/ QALY) when compared to the least costly option of not 

testing and treating for 24 months. The authors concluded that tests detecting disorders present in 

at least 5 percent of the population that confer a relative risk exceeding 1.25, including FVL and 

prothrombin G20210A, should be included. 

Another decision analysis with a 5-year time horizon assessed the effectiveness of extending 

anticoagulation from 3 months for FVL carriers and non-carriers following an initial lower-limb 

DVT to 1, 2, 3, 4, or 5 years in a hypothetical cohort. The authors stated that the risk of bleeding 

must be below 2.5 percent/year in order for prolonged anticoagulation to be the more effective 

strategy. 

Finally, one group used a decision-analytic model with a 12-month time horizon to assess 

the cost-effectiveness of universal or selective screening for FVL and resultant changes in 
9

management for carriers in four cohorts at high risk of VTE. In all four cohorts, selective 

screening was more cost-effective than universal screening. 

One group used the cost and outcomes data from a prospective cohort of 967 pregnant 

women in the United Kingdom to assess the cost-effectiveness of FVL testing and enoxaparin 

anticoagulant prophylaxis to prevent pregnancy-related vascular complications over the 8-month 

time horizon, from 12 weeks of gestation to 6 weeks postpartum. No women actually received 

anticoagulant prophylaxis, but the hypothetical impact of treating FVL carriers with an assumed 

efficacy of 50 percent was modeled. Testing only those women with a personal or family history 

of VTE was the most cost-effective approach. 

Discussion 

Based on our review of the evidence, we graded the strength of the evidence for the key 

questions as follows: 

Key Question 1 

There was no direct evidence that testing for FVL or the prothrombin G20210A mutations 

improves clinical outcome in adults with a personal history of VTE or that it improves clinical 

outcomes in adult family members of mutation-positive individuals. The evidence supporting 

KQ 2 through 4 can be considered indirect evidence to answer this overarching question. 

Key Question 2 

• There was high-grade evidence that tests to detect FVL have excellent analytic validity. 

• There was high-grade evidence that tests to detect prothrombin G20210A have excellent 

analytic validity. 

• There was high-grade evidence that most, but not all, clinical laboratories can test for FVL 

and prothrombin G20210A very accurately. There may be some laboratories that, for 

technical or administrative reasons, report inaccurate results. 

We note that the majority of the tested assays are not presently in widespread use. The majority 

of U.S. laboratories use PCR or invader chemistry technologies. 

Key Question 3 

• There was moderate-grade evidence that homozygosity for FVL in probands is predictive 

of recurrent VTE. 

• There was moderate-grade evidence that heterozygosity for FVL in probands is predictive 

of recurrent VTE. 

• The evidence is insufficient regarding the predictive value in probands of homozygosity 

for prothrombin G20210A, which is a rare genotype. 

• There was moderate-grade evidence that heterozygosity for prothrombin G20201A in 

probands is not predictive of VTE. 
10

• The evidence is insufficient regarding the predictive value in probands of double 

heterozygosity (FVL and prothrombin G20210A). 

We note that there may be little predictive value in knowing the mutation status in patients with 

idiopathic VTE as the index event, since the odds ratios for this subgroup were close to one. 

• There was high-grade evidence that homozygosity for FVL in family members is 

predictive of VTE. 

• There was moderate-grade evidence that heterozygosity for FVL in family members is 

predictive of VTE. 

• The evidence is insufficient regarding the predictive value in family members of 

homozygosity for prothrombin G20210A, which is a rare genotype. 

• The evidence is insufficient regarding the predictive value in probands of heterozygosity 

for prothrombin G20210A. 

• There was low-grade evidence that double heterozygosity (FVL and prothrombin 

G20210A) in family members is predictive of VTE. 

• There was low-grade evidence that homozygosity for FVL in pregnant family members is 

predictive of VTE. 

• The evidence was insufficient regarding the predictive value in pregnant family members 

of heterozygosity for FVL. 

• The evidence was insufficient regarding the predictive value in pregnant family members 

of homozygosity for prothrombin G20210A. 

• The evidence was insufficient regarding the predictive value in pregnant family members 

of heterozygosity for prothrombin G20210A. 

• The evidence was insufficient regarding the predictive value in pregnant family members 

of double heterozygosity (FVL and prothrombin G20210A.) 

For clinical context, we note that the annualized rate of venous thromboembolic events for 

family members without a mutation was approximately 0.1 percent per year. This translates to an 

event rate in heterozygous family members of 0.3 percent per year, or an absolute increase of 0.2 

percent per year (a change from an average of 1/1000 person-years to 3/1000 person-years). 

Key Question 4 

• There was no direct evidence that management guided by test results reduces VTE relatedoutcomes in individuals who have had VTE or in the probands’ family members who have 

been tested.

• There was low-grade evidence that physicians may alter patient management based on the 

results of testing for FVL or prothrombin G20210A. 

• There was high-grade evidence that anticoagulation can reduce recurrent events in patients 

with FVL or prothrombin G20210A; however, there was only low-grade evidence that the 

relative reduction in risk is comparable to that seen in individuals without these mutations. 
11

• There was moderate-grade evidence that neither harms nor benefits have been 

conclusively demonstrated in individuals with VTE or in their family members when 

tested for FVL or prothrombin G20210A. 

• There was low-grade evidence, derived from models, that testing for FVL alone, 

prothrombin G20210A alone, or the two tests in combination may be cost-effective when 

caring for selected patients with VTE (those with a high risk of recurrence and/or low risk 

of bleeding) or their family members. 

Limitations 

In addition to the reported deficits in the literature, there are limitations to this report. In our 

assessment of clinical validity, we chose to pool odds ratios rather than time-dependent measures 

of recurrence (such as hazard ratios or incident rate ratios). This approach necessarily excluded 

some studies from the pooled estimates. 

The odds ratios should approximate the relative rates of events in most studies, as these were 

relatively rare outcomes. We pooled the results using the DerSimonian and Laird random effects 

methods; this is a conservative method that often results in wide confidence intervals. 

Many of these studies were observational studies, and physicians may have altered their 

management based on their knowledge of mutation status, thereby changing the likelihood of a 

particular outcome. Most studies mitigated this potential difficulty by excluding patients who 

were chronically anticoagulated or by using a pre-defined anticoagulation approach. In those 

studies that reported the duration of anticoagulation after the index event in mutation-positive 

and -negative subgroups, there was no obvious discordance in anticoagulation duration. 

In these cohort studies, ascertainment bias is possible. In the studies of probands, the 

individuals were not blinded to their mutation status. Patients with mutations may be more likely 

to seek medical attention for symptoms consistent with VTE and might have been overdiagnosed with recurrence (as a result of false-positive tests), and those without mutations might 

have been under-diagnosed (because they did not seek medical attention for a thrombotic event 

that ultimately resolved without therapy). Ascertainment bias would tend to augment the 

association between the mutations and recurrent thrombosis. None of the studies we included had 

scheduled periodic radiographic testing to limit the potential for ascertainment bias. 

The majority of the observational studies about family members were retrospective, with 

some notable exceptions. Retrospective studies are prone to important biases, including recall 

bias. Although such bias can be mitigated by interviewing participants before they have 

knowledge of their mutation status, this approach was variably described in these studies. 

Implications for Future Research 

Studies to directly address the overarching question (KQ 1) would ideally be designed as 

randomized trials, in which participants with venous thrombosis and/or their family members 

would be randomized to a test arm or a no-test arm. Individuals would be managed by their 

physicians on the basis of the results of the testing (with evidence-based recommendations). 

Sufficient follow-up time would be included so that venous thromboembolic events could be 

witnessed and compared between the tested and untested groups. 
12

Analytic Validity 

Although the mutation detection methods have been shown to have high analytic validity, a 

small minority of laboratories account for a disproportionate percentage of errors in the 

performance of these tests. 

• There is a need for ongoing programs aimed at monitoring molecular diagnostic 

laboratories, through quality assurance programs, to ensure the consistent provision of 

high-quality genetic testing services. 

Clinical Validity 

• Future studies should report event rates over time (and relative rates of recurrence 

between specified groups), rather than just the number of events. 

• Studies should consistently differentiate between heterozygosity and homozygosity. 

• Studies should continue to use objectively measured thrombosis (radiographically 

proven) as a criterion for both the index and recurrent thromboses and should include 

more detail about both the index events and recurrences, such as the precipitants of these 

events. 

• Additional studies are needed to quantify the effect size more precisely with regard to 

the prothrombin G20210A mutation (alone and in conjunction with FVL). 

• Future research would be appropriate in Caucasian populations outside of Europe or in 

other populations with appreciable frequencies of mutations. 

• Future research could better explore the age-mutation interaction. 

Clinical Utility 

• Studies should measure how actual clinician practices change in response to results of 

both FVL and prothrombin G20210A testing. 

• Studies should be powered sufficiently to evaluate the risks associated with prolonged 

anticoagulation, as they relate to patients with specific thrombophilic mutations. 

• Future studies in both probands and family members might focus on whether 

management decisions (duration of therapy, use of thromboprophylaxis) affect the rates 

of VTE, particularly during times of heightened thromboembolic risk. 

• Studies based in the United States may give a clearer understanding of how patients here 

might respond to the testing process and results. 

• Larger sample sizes should also be used to increase the ability to detect rarer events, such 

as stigmatization. 

• Efforts should be made to recruit representative patient populations, and relevant 

comparison groups should be included (e.g., carriers and non-carriers) to increase the 

practical applications of the study findings. Quantitative studies may be preferable, using 

standardized, validated questionnaires to evaluate patients’ experiences. 

• Clinical trials could include an assessment of the costs associated with a testing strategy, 

as compared to care without testing. 
13

Our literature review included articles through December 2008. We do not anticipate any 

important secular changes in the event rate that would markedly change the event rates in 

upcoming years. We also do not expect major changes in the coming years in terms of the 

methods used to detect mutations. The most anticipated change would be an increasing in 

options to reduce risk as new drugs become available. Future research will need to include an 

evaluation of the risks and benefits associated with use of new anticoagulant drugs in probands 

and family members at high risk of thromboembolic events. 
15

Evidence Report 
16
17

Chapter 1. Introduction 

Venous Thromboembolism 

Venous thromboembolism (VTE) refers to pathologic thrombosis in the venous circulation. 

Although the most frequent venous thromboembolic event is deep venous thrombosis (DVT) in 

the veins of the legs, thromboses can also occur in the veins of the upper extremities, pelvis, 

abdomen, and cerebral venous sinuses. Pulmonary embolism is the main life-threatening 

complication of DVT, in which a portion of the venous thrombus is carried to the pulmonary 

arteries by blood flow, potentially obstructing the pulmonary vasculature. VTE and its 

complications are a common cause of morbidity and mortality in the United States. Data from 

the Rochester Epidemiology Project indicate that the average annual incidence of isolated DVT 

is 48 per 100,000 individuals, and that of pulmonary embolism (with or without DVT) is 69 per 

100,000.1

 Others estimate the incidence rate to be similar, at approximately 100 per 100,000, 

with approximately one-third manifesting as pulmonary embolism and two-thirds as DVT alone.2

Incident VTE is triggered by a confluence of modifiable and unmodifiable risk factors. Some 

of these risks are situational. Trauma or surgery may lead to direct endothelial injury, exposing 

circulating clotting factors to the thrombogenic material of the vessel wall (such as tissue factor). 

Stasis of blood is a precipitant of thrombosis, particularly when blood pools in the deep veins of 

the legs. Acquired alterations in coagulability are also precipitated by systemic illnesses, 

particularly malignancies, infection, nephrotic syndrome, and the antiphospholipid syndrome. 

These conditions are associated with varying degrees of inflammation, which activates 

hemostasis and/or changes in blood constituents (such as a loss of anti-clotting proteins in 

nephrotic syndrome, or the activation of the thrombotic cascade by tumor products). Primary 

hematological disorders, including myeloproliferative disorders such as polycythemia vera and 

essential thrombocythemia, increase the risk of thrombosis,3

 as do sickle cell anemia, 

thalassemia, and paroxysmal nocturnal hemoglobinuria.4 Heparin-induced thrombocytopenia is a 

unique condition associated with venous or arterial thrombosis in up to 50 percent of patients in 

whom it develops.5 Hyperhomocysteinemia is associated with increased risk of both venous and 

arterial thrombosis and can result from inherited enzymopathies or from acquired disorders of 

homocysteine metabolism, including renal failure and folate and vitamin B12 deficiency.6-8

Hormonal therapies, primarily estrogens, are prothrombotic, as is pregnancy and the post-partum 

state.3

 Advanced age, too, is a potent risk factor for VTE.9 Genetic risk factors for venous 

thrombosis include: deficiencies of endogenous anticoagulant proteins, antithrombin III, protein 

C, or protein S; and elevated levels of clotting factors VIII, IX, and XI; and possibly the 5,10-

methylenetetrahydrofolate reductase C677T polymorphism.3

 Although one would expect that 

disturbances of normal fibrinolytic function (e.g., excessive levels of plasminogen activator 

inhibitor-1 or α2-antiplasmin, or deficiencies in Factor XII or tissue plasminogen activator) 

would contribute to a hypercoaguable state, clinical evidence for such effects is lacking.10-12

Rarely, dysfibrinogenemia is associated with an increased tendency toward clot formation.13

This report focuses on two specific genetic factors associated with an increased risk of VTE, 

the Factor V Leiden (FVL) mutation and the prothrombin G20210A mutation. These mutations 

will be described in detail below. 

The risk factors for recurrent venous thrombosis differ from the risks for incident events; 

men are more likely to have recurrent events than are women.14 In patients who stop taking 
18

anticoagulant medications following treatment for an acute event, recurrent DVT occurs in about 

20 percent of patients within 5 years of the first event and in 30 percent within 10 years.15 16

However, continued anticoagulation treatment is highly effective in suppressing recurrent 

disease.17

Diagnosis and Treatment 

Many diagnostic tests and testing strategies have been evaluated with the goal of maximizing 

both the sensitivity and specificity of the diagnosis, so that an expeditious diagnosis can be made 

and treatment promptly initiated. Treatment of the incident event happens in two phases: 

Treatment begins with short-term use of a parenteral anticoagulant (and sometimes thrombolytic 

therapy) and then usually continues with a vitamin K antagonist, most commonly warfarin. In 

some instances, parenteral therapy is continued instead of warfarin.18 The duration of therapy 

depends on whether the patient is considered to have continuing risk factors for recurrence. 

Much effort has been devoted recently to quantifying the risk of recurrent thrombosis, with 

methods aimed at detecting whether a thrombophilic state persists. This effort has included 

measurement of D-dimer production19 and assessment of persistence of thrombosis by 

ultrasonography,20 as well as assessment of ongoing clinical risk factors. If the patient has a 

persistent risk factor for thrombosis, anticoagulant therapy is often continued, sometimes for the 

lifetime of the patient. The clinician and patient also try to reduce exposure to any modifiable 

prothrombotic risks as a secondary means of preventing additional events, and they may use 

mechanical or pharmacological means of preventing thrombosis at high-risk times, such as 

during hospitalization or pregnancy. 

The question of whether a patient has ongoing risk is directly associated with the subject of 

this report, an evaluation of the value of testing for FVL and prothrombin G20210A. A 

diagnostic test has value only if the process of testing or the results of testing lead to a change in 

outcome. In the case of venous thrombosis, a test that predicts recurrent events is appropriate if 

there is an intervention that can modify the risk of events. In this case, use of anticoagulants can 

modify the risk. Also, removal or avoidance of prothrombotic contributors (such as hormonal 

therapy) modifies the risk. It is then easily argued that identification of genetic risks may be 

important if it permits actions can be taken that alter outcomes. Even if the risk factor itself is not 

modifiable, management of the patient based on knowledge of the risk factor can change 

outcomes. 

Genetic Mutations Associated with VTE: FVL and 

Prothrombin G20210A 

FVL refers to a base change (from G to A at position 1691) in the gene coding for the Factor 

V protein, an amino acid substitution that eliminates one of three activated protein C cleavage 

sites in Factor V. As a result, Factor V is inactivated to a lesser extent and persists for a longer 

period in the circulation, leading to more thrombin generation.21 In the United States, a single 

FVL allele is present in about 5, 2.2, and 1.2 percent of the Caucasian, Hispanic, and African 

American populations, respectively; overall, about 1 in 5,000 individuals is homozygous for the 

mutation.22 23 The FVL mutation has been estimated to be present in 15 to 20 percent of patients 

with first venous thrombosis21 and is the most common heritable prothrombotic risk factor in the 
19

United States. In population-based studies, FVL increases the risk of a first venous thrombosis 4- 

to 7-fold in heterozygous individuals and 40- to 80-fold in homozygous individuals.21 22

The prothrombin (Factor II) mutation is the second most common inherited risk factor for 

thrombosis. In the United States, the prevalence of this allele is 1.1 percent in Caucasians and 

Hispanics and 0.3 percent in African Americans.24 The mutation in the gene, G20210A, is 

associated with an elevation of prothrombin levels to about 30 percent above normal in 

heterozygotes and to 70 percent above normal in homozygotes. This mutation is present in about 

2 percent of the population and in 6 percent or more of patients who present with a first VTE. Six 

to 12 percent of individuals who are heterozygous for FVL and have VTE also have the 

prothrombin G20210A mutation (double heterozygotes).21 22 25 Heterozygotes are at a two- to 

four-fold increased risk of an initial thrombosis.22 26 Individuals who are doubly heterozygous for 

FVL and prothrombin G20210A (about 1 in 1,000 Americans) have an estimated 20-fold 

increased risk when compared to individuals without either mutation, suggesting a multiplicative 

elevation in risk.21 22 25

Methods for Identifying Mutations

 Testing for these mutations is widely offered in the United States. In December 2003, the 

U.S. Food and Drug Administration (FDA) approved the first DNA-based laboratory tests 

specifically designed for FVL and prothrombin G20210A detection, manufactured by Roche 

Diagnostics Corporation (LightCycler Instrument). In 2007, Autogenomics Inc. received 

approval for their INFINITI System Assay for detection of the mutations. These tests are FDAapproved for use as an “aid to diagnosis in the evaluation of patients with suspected 

thrombophilia.”27 28 These commercially available tests have demonstrated, in their pre-approval 

testing, sufficient analytic validity to receive approval for use. They were not required to 

demonstrate clinical validity or clinical utility (to be defined below). Laboratories have also 

developed numerous other methods for detecting these mutations and do not use the 

commercially available systems; the analytic validity of these tests, however, is not well known. 

Objectives of this Evidence Report 

Our goal was to review the evidence regarding the value of testing for these mutations in two 

specific populations: (1) individuals who have had a VTE event (probands), and (2) the family 

members of probands with mutations. We did not aim to review the evidence regarding testing 

for these mutations as a screening strategy (for example, in a population with no known risks for 

disease, or in other clinical settings such as before use of oral contraceptives or hormonal 

therapy). Similarly, it was not our intention to review the use of these genetic tests in other 

clinical settings, such as in the investigation of fetal loss. 

To achieve our goal, we developed an analytic framework (described in Chapter 2). To 

understand this framework, one must be familiar with the definitions of analytic validity, clinical 

validity, and clinical utility.29 Analytic validity is a measure of an assay’s performance, which 

includes its analytic sensitivity and specificity as well as its robustness. Robustness refers to how 

resistant the test is to changes in pre-analytic and analytic variables, such as the source of the 

specimen or the temperature of the environment. Included in the framework of analytic validity 
20

is the assessment of laboratories’ quality control activities. These are procedures that ensure that 

the results fall within specified limits. 

Clinical validity is the extent to which the presence of the mutation predicts the clinical 

condition of interest. Clinical validity, therefore, includes the sensitivity and specificity of the 

test, as well as the predictive values of positive and negative tests, taking into account the 

disorder prevalence. Clinical validity may also be affected by penetrance, which is the 

relationship between the genotype and phenotype. In this report, the clinical condition of interest 

is recurrent venous thrombosis in the probands (because they have all had an incident event) and 

a first thrombotic event in the family members. Therefore, our assessment of clinical validity 

involves identifying the extent to which the presence of the mutations predicts venous 

thrombosis. 

Clinical utility refers to the impact of testing on outcomes. This evaluation involves knowing 

the availability and effectiveness of interventions employed in response to the test results, and an 

understanding of the risks and benefits when the test is used in routine practice. 

The report is structured as follows: In Chapter 2, we present our methods, which include a 

description of the Key Questions addressed in this evidence report, the analytic framework we 

constructed, and the methods used in our literature search, data abstraction, and synthesis of 

results. In Chapter 3, we present our results and assessment of the strength of the evidence for 

each Key Question, in terms of the analytic validity, clinical validity, and clinical utility of 

testing. Chapter 4 is our discussion and includes a review of the grading of the strength of the 

evidence and the implications of our findings for future research. 
21

Chapter 2. Methods 

The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) initiative was 

established by the Office of Public Health Genomics at the Centers for Disease Control and 

Prevention (CDC) to address the increasingly urgent need for timely and objective information 

that would allow health care providers and payers, policymakers, and consumers to identify 

genetic tests that are safe and useful. The CDC expects the EGAPP initiative to provide guidance 

on the appropriate use of genetic tests in practice, based on available evidence. The independent, 

multidisciplinary EGAPP Working Group, established in April, 2005, requested this review on 

the topic of testing for Factor V Leiden (FVL) and prothrombin G20210A. 

Establishing a Technical Expert Panel 

We began by recruiting experts to form a Technical Expert Panel. These experts were asked 

to provide input that would help us to refine the key questions to be answered with this review, 

to examine our list of articles identified for inclusion, and to suggest additional sources to search 

for evidence. These experts were also invited to be the peer reviewers of our finished report. The 

panel included four faculty members from the Johns Hopkins University. One of the four 

Hopkins panelists was a bioethicist and the former Assistant Director of the National Human 

Genome Research Institute, one was a clinical investigator with extensive experience with 

genetic testing for colon cancer, one was the director of our special coagulation laboratory, and 

one was the director of our anticoagulation management service and an expert on 

hypercoagulable states. Our external panelists included experts in thrombophilia and the 

management of venous thromboembolism (VTE); a genetic counselor; three members of the 

EGAPP Working Group with expertise in medical screening, genetics, and methodologies of 

genetic testing; a family physician; and a patient affected by these mutations who is a leader in 

the National Alliance for Thrombosis and Thrombophilia (see Appendix A). 

Analytic Framework 

We used an analytic framework to identify the evidence required to answer our Key 

Questions. The analytic framework (see Figure 1) was developed with the involvement of our 

Task Order Officer from the Agency for Healthcare Research and Quality (AHRQ) and the 

Technical Expert Panel. 
22

* harms include management-related outcomes and emotional/behavioral/social sequelae of testing 

** benefits include VTE-related outcomes, management-related outcomes, emotional/behavioral/social sequelae of testing 

† intervention (or change in management or recommendations) may or may not vary with test result 

Q = Question; VTE = venous thromboembolism 

Key Questions 

Guided by the analytic framework, we developed the following key questions (KQs). Our 

population was defined as adults with a personal history of VTE, or adult family members of 

individuals identified as having an FVL and/or prothrombin G20210A mutation(s). 

KQ1 [Overarching question] Does FVL testing, alone or in combination with 

prothrombin G20210A testing, lead to improved clinical outcome (e.g., avoidance of 

a recurrent VTE) in adults with a personal history of VTE or to improved clinical 

outcome (e.g., avoidance of an initial VTE) in adult family members of mutationpositive individuals? Are the testing results useful in medical, personal, or public 

health decisionmaking? 

KQ2 What is the evidence regarding the analytic validity of existing diagnostic 

tests for the FVL mutation and the prothrombin G20210A mutation, specifically their 

Q2

Factor V 

(±Prothrombin) 

mutation testing

Q1 

Benefits**

Reduced 

morbidity/mortality 

due to avoided

thromboembolic 

events & sequelae 

Risk of 

recurrence 

of VTE

Family members of 

mutation-positive 

individuals 

Q3 Q4

Uptake Rate 

 Factor V 

(±Prothrombin) 

Testing 

Factor V 

(±Prothrombin) 

mutation 

testing 

results

Patients with 

a personal 

history of VTE 

Harms*

Harms*

Benefits**

Reduced 

morbidity/mortality 

due to avoided

thromboembolic 

events & sequelae 

Factor V Intervention†

(±Prothrombin) 

mutation 

testing 

results

Risk of 

occurrence 

of VTE

Q3

Feasibility and 

costs of testing 

Feasibility and 

costs of testing 

Intervention†

Q4 Q4

Figure 1. FVL and prothrombin testing for the prevention of venous thromboembolic events: Analytic framework 
23

analytic sensitivity and specificity, reproducibility, and robustness (sources of 

variability)? 

KQ3a What is the evidence that the presence of FVL alone, prothrombin G20210A 

alone, or the two in combination predicts the risk of recurrent VTE in individuals 

(probands) who have had VTE and predicts the risk of VTE in the probands’ family 

members who have been tested? Does the testing add predictive information beyond 

clinical data? 

KQ3b What is the evidence that demographic or clinical factors modify the 

relationship between the presence of FVL or prothrombin G20210A and the risk of 

VTE? 

KQ4a What is the evidence that clinicians manage patients differently based on the 

results of testing for FVL or prothrombin G20210A? How do clinicians manage 

anticoagulation in individuals who have had testing, as compared to those who have 

not had testing? What other diagnostic tests do clinicians order or not order, based on 

testing results? What recommendations do clinicians make regarding other therapies 

and exposures, based on testing results? 

KQ4b What is the evidence that testing, and the resultant management, reduces VTE 

related-outcomes or has other benefits in individuals who have had VTE or in the 

probands’ family members who have been tested?

KQ4c What is the evidence of harms to individuals with VTE or to the probands’ 

family members who are tested for FVL or prothrombin G20210A as a result of 

testing or as a result of changed management based on the test results? 

KQ4d What is the evidence that testing for FVL alone, prothrombin G20210A alone, 

or the two tests in combination is a cost-effective strategy when caring for a patient 

with VTE or a family member of a proband? 

Search Strategy 

Searching the literature involved identifying reference sources, formulating a search strategy 

for each source, and executing and documenting each search. We also searched for medical 

subject heading (MeSH) terms that were relevant to the genetics of Factor V and prothrombin. 

We used a systematic approach for searching the literature, with specific eligibility criteria, to 

minimize the risk of bias in selecting articles for inclusion in the review. The systematic 

approach was intended to help identify gaps in the published literature. 

Our comprehensive search included electronic and hand searching. We searched five 

databases, MEDLINE® (1950 through December 2008), EMBASE® (1974 through December 

2008), The Cochrane Library (Issue 2, 2008), the Cumulative Index to Nursing & Allied Health 

Literature (CINAHL®; 1982 through December 2008) and PsycInfo©, to identify primary 

literature on the analytic validity, clinical validity, and clinical utility of testing for FVL and 

prothrombin G20210A. Hand searching for possibly relevant citations took two forms. First, 
24

from our electronic search, we identified the 11 journals (see Appendix B) that were most likely 

to publish articles on this topic (i.e., these journals had the highest number of abstracts and 

articles included in the review). We scanned the table of contents of each issue of these journals 

for relevant articles from March 2008 through September 2008. For the second form of hand 

searching, reviewers reviewed eligible articles and flagged references of interest for the team to 

compare to the existing database. 

Search strategies specific to each database were designed to enable the team to focus the 

available resources on articles that were the most likely to be relevant to the key questions. We 

initially developed a core strategy for MEDLINE®, accessed via PubMed®, based on an analysis 

of the MeSH terms and text words of key articles identified a priori. The PubMed® strategy 

formed the basis for the strategies developed for the other electronic databases (see Appendix C). 

The results of the searches were downloaded and imported into ProCite® version 5 (the 

Thompson Corporation, Stamford, CT). We used the duplication scan feature in ProCite® to 

delete citations already retrieved. From ProCite®, the articles were uploaded to SRS 4.0 

(TrialStat! Corporation, Ottawa, Ontario, Canada), a Web-based software package developed for 

systematic review data management. This database was also used to store citations in portable 

document format (PDF) and to track the search results at the title review, abstract review, article 

inclusion/exclusion, and data abstraction levels. A list of excluded articles is presented in 

Appendix D. 

Study Selection 

There were a total of six reviewers who were paired, so that each pair included an expert in 

the content of this review. Two reviewers independently conducted title scans in a parallel 

fashion. For a title to be eliminated at this level, both reviewers had to indicate that it was 

ineligible. If the two reviewers did not agree on the eligibility of an article, it was promoted to 

the next level (see Appendix F, Title Review Form). The title review phase was designed to 

capture as many studies as possible that reported on the analytic validity, clinical validity, and 

clinical utility of testing for FVL and prothrombin G20210A. All titles that were identified as 

potentially addressing these issues were promoted to the abstract review phase. 

Abstracts were reviewed independently by two investigators. Abstracts were excluded if both 

investigators agreed that the article met one or more of the following exclusion criteria: It (1) 

was not relevant to any of the key questions; (2) did not include any human data; (3) contained 

no original data; (4) was not conducted among adults; or (5) was not published in English. 

Differences of opinion regarding abstract eligibility were resolved through consensus 

adjudication. At this level of review, the reviewers were also asked to identify to which Key 

Question(s) the article might apply if it was eligible for review. 

Because of the broad array of potentially eligible articles obtained at the abstract review 

phase, full articles selected for review underwent another independent parallel review by two 

investigators to determine whether the articles should be included in the full data abstraction. In 

addition to the exclusion criteria used for the abstract review, there were additional exclusion 

criteria for each Key Question (see Table 1). 
25

Table 1. List of additional exclusion criteria by Key Question 

Question Criteria Explanation 

KQ2 • Published prior to 2000 

• Evaluated only APC resistance 

• Evaluated only DNA extraction methods 

• Did not report concordance, discordance, 

or reproducibility 

• Evaluated fewer than 10 samples 

KQ3 • Did not report results separately for FVL 

and/or prothrombin G20210A 

• Studied fewer than 10 probands or fewer 

than 10 family members 

• Did not objectively confirm VTE event 

• Was a case-control or retrospective cohort 

study of probands 

• Patients described in another publication 

We opted to exclude retrospective studies of 

probands, since this study design is more subject 

to biases than is a prospective design. We opted 

to include retrospective studies of family 

members because we expected to have few, if 

any, prospective studies. 

KQ4 • Did not study probands or family members 

• Fewer than 80% of the probands had the 

FVL or prothrombin G20210A mutations 

• Fewer than 80% of the probands had VTE 

We required that 80% of the study population 

had the mutations of interest if it was a study 

including family members with other 

hypercoaguable conditions and results were not 

reported separately by mutation. We required 

that 80% of the probands had VTE if it was a 

study of individuals with mutations, in keeping 

with our stated population of interest. 

APC = activated protein C; DNA = deoxyribonucleic acid; FVL = Factor V Leiden; KQ = Key Question; VTE = venous 

thromboembolism 

At this phase of the review, the investigators determined which of the Key Questions each 

article addressed (see Appendix E, Article Inclusion/Exclusion Form). If the articles were 

deemed to have applicable information, they were included in the full data abstraction. 

Differences of opinion regarding article eligibility were resolved through consensus adjudication. 

Data Abstraction 

We used a systematic approach to extracting data in order to minimize the risk of bias in this 

process. By creating standardized forms for data extraction, we sought to maximize consistency 

in identifying all pertinent data available for synthesis. 

Each article underwent double review by study investigators for full data abstraction and 

assessment of study quality. For all data abstracted from studies, we used a sequential review 

process. In this process, the primary reviewer completed all data abstraction forms. The second 

reviewer checked the first reviewer’s data abstraction forms for completeness and accuracy. 

Reviewer pairs were formed to include personnel with both clinical and methodological 

expertise. Reviewers were not masked to the articles’ authors, institutions, or journal.30 In most 

instances, data were directly abstracted from the article text. If possible, relevant data were also 

abstracted from figures. Differences of opinion were resolved through consensus adjudication. 

For assessments of study quality, each reviewer independently judged study quality and rated 

items on quality assessment forms (see Appendix F, Data Abstraction Review Forms). 
26

For articles that applied to Key Question 2, we abstracted information on the sample selection 

criteria, the test operators, the characteristics of the DNA extraction process, the experimental 

test, the reference standard, the commercial instruments used, the concordance rates, and the 

explanation of any false positives or false negatives, if applicable. For articles that applied to 

Key Question 3, we abstracted information on study characteristics (i.e., study design, 

enrollment dates, location, inclusion criteria, genetic test used, duration of follow-up, and 

radiographic and clinical surveillance), population characteristics (i.e., age, gender, idiopathic 

VTE, oral contraceptive or hormone use, pregnancy, other precipitating factors, and other 

thrombotic mutations), and the results. For articles that applied to Key Question 4, we abstracted 

information on the study characteristics (i.e., study design and location), population 

characteristics (i.e., patients or providers, age, race, gender, genotype, socioeconomic status, and 

specialty of the providers), the main objective of the study, and the results. 

All information from the article review process was entered into the SRS 4.0 database by the 

individual completing the review. Reviewers entered comments into the system whenever 

applicable. The SRS 4.0 database was used to maintain and clean the data, as well as to create 

detailed evidence tables and summary tables (see Appendix G and Summary Tables). 

Study Quality Assessment 

The study aspects considered in our quality assessment varied according to the question 

being addressed and according to the study design. As part of our dual, independent review of 

study quality, we judged articles on several aspects of each study type’s internal validity. Quality 

assessment of diagnostic studies for Key Question 2 was designed by selecting elements from the 

Standards of Reporting of Diagnostic Accuracy (STARD) Initiative31 and included: (1) adequate 

descriptions of the setting, the experimental test, and the reference standard; (2) a statement 

about testing being conducted without knowledge of the reference standard results; (3) a 

statement about all specimens being tested with both the experimental test and reference 

standard; (4) the reporting of a summary index and a measure of variability; and (5) a description 

of the funding source. Quality assessment for Key Question 3 was based on quality forms 

previously developed by our Evidence-based Practice Center (EPC).32 33 This assessment 

included items about the setting, inclusion/exclusion criteria, key characteristics of the enrolled 

subjects, losses to follow-up, and the funding source. Quality assessment of the qualitative 

studies for Key Question 4 included items from The Joanna Briggs Institute Qualitative 

Assessment and Review Instrument,34supplemented with quality items previously developed by 

our EPC. The qualitative study quality assessment form included: (1) a statement locating the 

researcher culturally or theoretically, (2) a statement of ethical approval, (3) a description of the 

theoretical basis for the study, (4) a description of why patients were selected, (5) adequate 

representation of the participants and their voices, (6) composition of the interview setting to 

maximize data gathering, (7) reporting of theme exhaustion, (8) a clear description of the data 

collection process, (9) addressing the influence of researchers on research and vice versa, (10) 

the use of triangulation, and (11) the development of a concept, model, or theory based on the 

data collected. Quality assessment of surveys for Key Question 4 was based on quality forms 

previously developed by our EPC and included: (1) a description of the theoretical basis for the 

study; (2) a statement of ethical approval; (3) a description of the setting, inclusion/exclusion 

criteria, and key characteristics of the study participants; (4) survey completion rates; and (5) a 

discussion of the validity, reliability, and interpretability of the survey instrument. Quality 
27

assessment of cost-effectiveness analyses for Key Question 4 was based on a review of 

guidelines for good practice in decision-analytic modeling in health technology assessment35 and 

included items regarding the structure, data, and consistency. 

A senior reviewer adjudicated any discrepancies in the quality assessment. 

Applicability 

Throughout the report, we describe the applicability of studies in terms of the extent to which 

the study population and testing procedure were relevant to the process of testing patients and 

their family members in the United States. We evaluated the applicability of studies in terms of: 

(1) the study population, (2) the genetic tests used to determine mutation status, (3) the eligibility 

criteria, (4) the outcome rates and measures, (5) the treatment regimen, (6) the interventions, (7) 

the follow-up time, and (8) the standards of care. 

Data Synthesis 

For each Key Question, we created a set of detailed evidence tables containing all the 

information extracted from the eligible studies. The investigators reviewed the tables and 

eliminated items that were rarely reported. 

In calculating the concordance rates for the studies that examined the analytic validity of the 

tests for detecting the mutations, we used the initial test results before any resolution by repeat 

testing. In the tables presenting these results (in Chapter 3), if the concordance was less than 100 

percent, we provide details about the discordant results. 

We conducted meta-analyses of the studies addressing the clinical validity of the tests, when 

there were sufficient data (three or more studies) and the studies were qualitatively homogeneous 

with respect to key variables (population characteristics, study duration, mutation status, and 

length of follow-up). When it was inappropriate to combine studies quantitatively, we 

qualitatively summarized the results. 

For the pooling, we used the number of events and count of the patients under observation in 

each group. We calculated a pooled estimate of the odds ratio for VTE in probands and 

separately in family members. We used a random effects model with the DerSimonian and Laird 

method for calculating between-study variance.36 The random effects model was chosen because 

we anticipated some heterogeneity among the studies. We repeated the pooling using MantelHaenszel (M-H) fixed effect methods and Peto fixed effects methods. As the outcome events 

were not always rare, the Peto methods yielded markedly different pooled estimates for some of 

the comparisons. The M-H fixed effects and random effects pooled estimates were quite similar. 

We assessed heterogeneity among the studies considered using a standard chi-squared test 

and a significance level of alpha ≤ 0.10. We also examined heterogeneity among studies with an 

I

2

 statistic, which describes the variability in effect estimates that is due to heterogeneity rather 

than random chance.37 A value greater than 50 percent may be considered to indicate substantial 

variability. 

We tested for publication bias in two ways. First, we used the Duval and Tweedie 

nonparametric "trim and fill" method of accounting for publication bias in meta-analysis.38 This 

methodology explores whether the inclusion of “missing” studies would alter the pooled odds 

ratio. We also used the Egger’s test to evaluate the likelihood of missing studies.39 Finally, we 

sequentially removed each study from the calculation of the pooled estimates and recalculated 
28

the pooled odds ratios. In this way, we were able to assess the impact on the odds ratios of one 

particularly influential study. 

All statistical analyses were conducted using STATA (Intercooled, version 9.0, StataCorp, 

College Station, TX). 

Data Entry and Quality Control 

Initial data were abstracted by the investigators and entered directly into Web-based data 

collection forms using SRS® 4.0 (TrialStat! Corporation, Ottawa, Ontario, Canada). After a 

second reviewer reviewed the data, the adjudicated data were re-entered into the Web-based data 

collection forms by the second reviewer. Second reviewers were generally more experienced 

members of the research team, and one of their main priorities was to check the quality and 

consistency of the first reviewers’ answers. If problems were recognized in a reviewer’s data 

abstraction, the problems were discussed at a meeting with the reviewers. 

Rating the Body of Evidence 

At the completion of our review, we graded the quantity, quality, and consistency of the best 

available evidence addressing the Key Questions by adapting an evidence-grading scheme 

recommended by the Grading of Recommendations Assessment, Development and Evaluation 

(GRADE) Working Group.40 We assessed the strength of the study designs for each question. To 

assess the quantity of evidence, we focused on the number of studies with the strongest design. 

We also assessed the quality and consistency of the best available evidence, including 

assessment of the limitations affecting individual study quality (using the individual study 

quality assessments), certainty regarding the directness of the observed effects in the studies, the 

precision and strength of the findings, and the availability (or lack) of data to answer the Key 

Question. We classified evidence bodies pertaining to the Key Questions into the following 

categories: (1) “high” grade, indicating confidence that further research is very unlikely to 

change our confidence in the estimated effect in the abstracted literature; (2) “moderate” grade, 

indicating that further research is likely to have an important impact on our confidence in the 

estimates of effects and may change the estimates in the abstracted literature; (3) “low” grade, 

indicating the further research is very likely to have an important impact on confidence in the 

estimates of effects and is likely to change the estimates in the abstracted literature. We also 

noted when there was no available evidence. This framework for grading the body of evidence is 

consistent with that suggested by the EGAPP working group.41
29 

Chapter 3. Results 

Results of the Search 

The literature search process identified 13,711 citations that were potentially relevant to the 

Key Questions. An additional 165 articles were found by hand searching; thus, the total number 

of citations retrieved was 13,876 (see Figure 2). We excluded 6,099 duplicate citations. Most 

duplicates came from concurrently searching MEDLINE® and EMBASE®. The search strategy 

we used in EMBASE®was modeled on that which we used in MEDLINE®, with similar search 

terms. (see Appendix C). Also, the EMBASE® search engine allows the user to search the 

MEDLINE® database as well as EMBASE®. Using this strategy often leads to many duplicates 

between the two search sites. However, this Evidence-based Practice Center (EPC) employs this 

strategy in order to improve the sensitivity of the search. 

In the title review process, we excluded 5,553 citations that were ineligible for inclusion. In 

the abstract review process, we excluded 1,827 citations that did not meet one or more of the 

eligibility criteria (see the list in the Methods chapter, and Figure 2). At article review, we then 

excluded an additional 273 articles that did not meet one or more of the eligibility criteria. That 

left 124 articles eligible for inclusion in the review of one or more of the Key Questions. 

Description of Types of Studies Retrieved 

No articles were identified that directly answered Key Question 1. One recent article nearly 

met our inclusion criteria and is described in Chapter 4.42 We identified 66 articles that were 

appropriate for Key Question 2. For Key Question 3, 23 articles were relevant to probands with 

the mutations, and 22 articles were relevant to family members with mutations. An additional 14 

articles were identified relevant to Key Question 4, including 7 decision analyses.

Key Question 1: 

Association of FVL testing, alone, or in combination with 

prothrombin G20210A testing, with improved clinical 

outcomes in adults with a personal history of VTE or in adult 

family members of mutation-positive individuals as a result 

of medical, personal, or public health decision making 

 We found no evidence that allowed us to directly answer this question. 
30 

Electronic Databases 

MEDLINE® (6096)

Cochrane (132)

EMBASE® (6744)

CINAHL (711)

PsycInfo (28)

Retrieved

13876

Title Review

7777

Duplicates

6099

Abstract Review

2224

Excluded

5553

Article Review

397

Excluded

1827

Included Articles

124**

Excluded

273

Reasons for Exclusion at Article Review Level*

Exclude from KQ2, KQ3 and KQ4:

No original data: 22

Non-English article: 32

Does not address study question: 136

Other: 81

Exclude from KQ2 because:

Published before 2000: 68

Evaluates only APC resistance: 6

Evaluates only DNA extraction methods: 5

Does not report concordance, discordance, or 

reproducibility: 23

Evaluates fewer than 10 samples: 13

Exclude from KQ3 because:

Does not report results separately for FV Leiden and/or 

PT 20210A: 15

Studies only children: 0

Studies fewer than 10 probands or 10 family members: 

10

Does not study venous thrombosis: 3

No objective confirmation of VTE event: 9

Case-control or retrospective cohort study of probands 

that does not have objective validation of both the 

index event and the recurrent event: 21

Case-control or retrospective cohort study of family 

members that does not have objective confirmation 

of VTE: 5

Exclude from KQ4 because:

Studies only children: 1

Does not study probands or family members: 5

Less than 80% of the study population has the FV 

Leiden or PT 20210A mutation: 7

Less than 80% of the study population has had VTE: 5

Reasons for Exclusion at Abstract Review Level*

Does not apply to a key question: 1461

Animals only: 2

No original data: 577

In vitro testing with no human specimen: 10

Studies only children: 25

Not in English: 116

Hand Searching Other: 130

165

Figure 2. Summary of literature search (number of articles)

* Total may exceed number in corresponding box, as articles could be excluded for more than one reason at this level. 

**3 articles addressed both KQ3 and KQ4. 

APC = activated protein C; CINAHL = Cumulative Index to Allied Health and Nursing Literature; DNA = deoxyribonucleic acid; 

FV Leiden = Factor V Leiden; KQ = Key Question; PT 20210A = prothrombin G20210A; VTE = venous thromboembolism 
31 

Key Question 2: 

Analytic validity of tests to identify FVL and prothrombin 

G20210A mutations 

As described early in this report, we employed the same definition of analytic validity that is 

used in the Analytic Validity, Clinical Validity, Clinical Utility and Associated Ethical, Legal 

and Social Implications (ACCE) framework.29 Accordingly, our review of analytic validity 

assessed the analytic sensitivity (the analytic detection rate), analytic specificity, laboratory 

quality control, and assay robustness. Calculations of sensitivity and specificity, however, are 

most applicable to tests with dichotomous results. Given that there are two alleles, genetic test 

results have three outcomes: no mutations, a single mutation (heterozygous), or two mutations 

(homozygous). The studies we reviewed uniformly and appropriately described analytic validity 

as concordance or discordance between the experimental test and the reference standard, rather 

than sensitivity and specificity. 

 Study quality was generally high (see Appendix G, Evidence Table 1). In our quality 

assessment, we focused mainly on biases that could result from commercial conflicts of interest. 

The majority of these studies were published before journals began requiring disclosure of 

funding sources and potential conflicts of interest. More studies than not, however, did report on 

the source of funding; we considered these sources of funding to be unlikely to bias the results, 

since most of the reported funding was from governmental grant support. 

 Studies varied greatly in the extent to which they described the population from which the 

participants came; 17 studies adequately reported the setting or population from which the blood 

samples were obtained.43-46 46-48 48-57 Most described the experimental test adequately; 

approximately 60 to 70 percent of the studies gave a detailed description of their reference 

methods. 

 An important quality-related consideration was whether the study indicated whether the 

experimental test was conducted without knowledge of the reference standard results. In only a 

minority of the studies, the staff members were unaware of the reference standard results at the 

time they performed the experimental tests.46 50 52 55 58-64

The conventional “gold standard” method for Factor V Leiden (FVL) and prothrombin 

G20210A detection is the bidirectional sequencing of the specific genetic region of the gene of 

interest. The Food and Drug Administration (FDA) requires that manufacturers of new assays 

compare the assay to an established method and considers polymerase chain reaction and 

restriction fragment length polymorphism (PCR-RFLP) to be an acceptable reference standard. 

Indeed, many of the studies used PCR-RFLP or allele-specific polymerase chain reaction (ASPCR) as their reference standards. In the absence of a substantial number of studies comparing 

the test under evaluation to the gold standard (bidirectional DNA sequencing), we included in 

our review studies that used traditionally accepted reference tests, typically PCR-RFLP or ASPCR. We acknowledge that a “methods comparison” may be a lower level of evidence regarding 

analytic validity than a gold standard comparison would be. 

All the studies included used blood as the source of DNA for analysis. In the table below, the 

studies are grouped according to the underlying chemical/biochemical experimental methods 

they employed (Tables 2-4; Appendix G, Evidence Tables 2-4). We observed a high level of 

concordance between the experimental methods and the reference standards, with many studies 

reporting 100 percent concordance (range of concordance, 98 to 100 percent). 
Table 2. Analytic validity of testing for the FVL mutation 

Experimental Test 

Flap Endonuclease + FRET (Invader Assay) 

FRET + Melting Curve Analysis (Light Cycler) 

Taqman Real-Time PCR Assay 

Electrochemical Genosensors 

Reference Standard AS-PCR or PCR-RFLP PCR or PCR-RFLP PCR-RFLP PCR-RFLP Studies Hessner, 200043 Ledford, 

200044 Patnaik, 200467 Schroell-Metzger, 200368 Mammo, 

200669 Cooper, 200370 Nauck, 200045

Parks, 200148

Louis, 200462Benson, 200147Behrens, 200471

Ozkan, 200265 Ozsoz, 

200372 Huang, 200273 

Total Population* 1369 + 371 + 368 132 + 2131 + 200 + 

110 + 155 

115 + 100 + 100 90 + 90 + NR 

Concordance 99.5% - 100% 100% 100% 93% - 100% Source of Zygosity Discordance**

5 samples were typed WT by reference standard but heterozygous by Invader assay. With retyping, there was concordance, with all genotypes unequivocally WT. In Ledford, 2 samples were discordant. Retesting yielded results concordant with the original PCR-RFLP results. In one case, a heterozygous sample was called homozygous by the Invader assay; the discrepancy was due to a failure to add genomic DNA to the well. In the second case, a heterozygous sample was called WT. 

NA NA In Ozkan et al., the source 

and nature of the 7% discordance were not explained. Ozsoz et al. reported neither reference standard nor quantitative results. Rather, it stated that there was "good agreement" and ongoing work to achieve 100% accuracy. Huang et al. did not report sample size or reference standard. 

Did Repeating Resolve the Discordance*? 

Yes (technical/operator error) NA NA Not reported 

32 
Table 2. Analytic validity of testing for the FVL mutation (continued) 

Experimental Test 

MALDI-TOF Mass Spectrometry 

PCR-Single Strand Conformation Polymorphism 

Simultaneous AlleleSpecific Amplification Followed by RFLP 

Fluorogenic Probe-Based Allelic Discrimination PCR Assay 

Melting Analysis of Labeled Probes System 

Reference Standard PCR-RFLP PCR-RFLP PCR-RFLP PCR-RFLP 

PCR-RFLP or Conventional DualHybridization Probe Genotyping 

Studies Hung, 200250

Humeny, 200155

Simundic, 200374 DelRio-LaFreniere, 200164 Sanders, 200075 El Housni, 200380

(dual-labeled probes); Vaughn, 200481 Crockett, 200182 (singlelabeled fluorophorebased systems) 

Total Population* 27 + 70 150 49 120 100 + 100 + 100 Concordance 100% 100% 100% 100% 100% Source of Zygosity Discordance** 

NA NA NA NA NA 

Did Repeating Resolve the Discordance**? 

NA NA NA NA NA 

33 
Table 2. Analytic validity of testing for the FVL mutation (continued) 

Experimental Test 

Rolling Circle Amplification Assay Using Open Circle Probes Ligation 

High-Resolution Melting Analysis of Small Amplicon 

Pyrosequencing 

READIT SYSTEM (Pyrophosphorolysis) ELISA 

Reverse AllSpecific Oligonucleo(ASO) HybridizatioAssay 

Reference Standard PCR-RFLP Light Cycler 

LightCycler or Direct Sequencing 

PCR-RFLP or Not Specified 

Direct Sequencing or PCR-RFLP PCR-RFLP 

Studies Alsmadi, 200383 Liew, 200476 Verri, 200577 Tsongalis, 200184

Rhodes, 200159 Lopez, 200766 Carmi, 200485 Kowalski, 20

Leyte, 20008

Total Population* 216 104 100 280 + 510 264 + 284 256 + 99 Concordance 99.4% 100% 100% 99-100% 95-100% 100% Source of Zygosity Discordance** 

Not reported NA NA Unclear (in 

Tsongalis et al., one discordant result was due to an equivocal relative light unit, while the other was indeterminate); NA59

No explanation was given as to the source of the discrepancies in Lopez et al.; NA85

NA 

Did Repeating Resolve the Discordance**? 

Yes (if tests were performed in triplicate). 

NA NA Yes84; NA59 Yes NA 

34 
Table 2. Analytic validity of testing for the FVL mutation (continued) 

Experimental Test 

Linked Linear Amplification - ASO Capture 

Nanochip Electronic Microarray 

Photo-Cross-Linking Oligonucleotide Hybridization Assay 

MGB-NFQ Probes (7700 SDS) 

Reference Standard 

PCR-RFLP & PCRASO 

PCR-RFLP, Light Cycler, Invader Monoplex Assay PCR-Based Assay 

FRET + Melting Curve Analysis (LightCycler) 

Studies Reyes, 200178 Schrijver, 200387 Erali, 

200388 Evans, 200289

French, 200490 Castley, 200579 

Total Population 111 197 for the comparison 

to RFLP and 195 + 224 + 758 for the comparison to LightCycler 

1054 151 

Concordance 100% 98-100% 98.6% 100% Source of Zygosity Discordance*

NA NA87 88; In Evans et al., 

the reference test did not correctly identify 17 samples (details as in footnote). 

2 false-negatives: For one, 

the reference indicated homozygous, but the experimental reported heterozygous. For the other, the reference indicated heterozygous, but the experimental test indicated WT. Repeat testing confirmed the results of the reference standard for both. False-positives: Reference 

indicatedWT, but the experimental test reported heterozygous initially. Repeat testing confirmed WT. 

NA 

Did Repeating Resolve the Discordance?* 

NA NA87 88; No89 Yes; 15 samples were 

discordant. All 15 samples were genotyped correctly (i.e., 100% concordance) upon retesting (reextraction of genomic DNA; resolution of the indeterminate, invalid, and discordant samples). 

NA 

* the order of the counts corresponds to the order of the studies in the row above; 

35 
**if <100% concordance ASO = allele-specific oligonucleotide; AS-PCR = allele-specific PCR; DNA = deoxyribonucleic acid; ELISA = enzyme-link

FRET = fluorescence resonance energy transfer; FVL = Factor V Leiden; MALDI-TOF = matrix-assisted laser desorption/ionization – time of flight; Mbinding non-fluorescent quencher; NA = not applicable; NR = not reported; PCR = polymerase chain reaction; READIT = reversed enzyme activity D= restriction fragment length polymorphism; SDS = sequence detection system; TGCE = temperature gradient capillary electrophoresis; WT = wild-tyTable 3. Details of studies on the analytic validity of testing for the FVL mutation 

Study(s), Year(s) Description 

Hessner, 200043 Seven samples (0.5%) were classified as equivocal; upon retesting, they were correctly typed as WT/WT (1

(1.2%) generated invalid results because of unacceptable signal strength. These 16 samples were repeated 

preparations, and all were successfully typed as WT/WT. 

Louis, 200462 The aim of the study was to assess the cost-effectiveness and specificity of a high-throughput real-time PC

specific fluorescent oligonucleotides that contain a 3' minor groove binding (MGB) probe. 

Benson, 200147 6295 adult subjects from a large managed-care population in Southern California were analyzed for FVL an

discrepancy rate of 0% was found on duplicate testing for all results different from WT among the first 1000 

heterozygous/homozygous samples were also analyzed by the traditional method involving restriction digesGeneScan analysis, with complete agreement between both analyses. 

Murphy, 200361 A low PCR product peak height on initial TGCE analysis was the source of the false homozygous result not

heterozygosity was confirmed by repeat testing using both methodologies. The false WT/Leiden involved a downstream of the Leiden site. Thus, this sample was WT/WT for Leiden allele but contained a new polymo

to Leiden position that caused the sample to be reported as heterozygous by TGCE. 

Huang, 200273

A method to identify SNP alterations by combining hairpin-forming DNA probes and electrochemical detectihybridization was reported. The results obtained using electrochemical detection of mismatches in nucleic acompletely with the results determined independently at the UCLA Diagnostic Molecular Pathology Laborato

Hung, 200250 A matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) method was used. Blood samples w

healthy controls and 16 patients with activated protein C resistance. The genotype of all 12 heterozygous aindividuals was not known to the MALDI-TOF group before genotyping of all samples was completed. 

Humeny, 200155 Samples: Women from the Obstetrics and Gynecology outpatient clinic who presented with various endocriPotter, 200149 Detection of FVL w/lanthanide-labeled probe: an assay using oligonucleotide probes for normal and mutant 

europium and samarium, respectively, and measured by time-resolved fluorescence. 

Verri, 200577 Failure to make a genotype call at the first attempt was infrequent (about 5%) and was mostly attributable to 

noise ratios caused by poor PCR amplification. 

Tsongalis, 200184 The Reversed Enzyme Activity DNA Interrogation Test (READIT) System was claimed to offer the following 

SNP analysis, (2) accuracy and precision, (3) cost effectiveness, (4) decreased turn-around times, (5) high 

excellent analysis software. 

Rhodes, 200159 Residual coded samples (n=510) from three independent laboratories were analyzed in a blinded format for 

gene using the READIT Assay. A blinded retrospective analysis of the FVL mutation generated by independto determine the concordance. 

36 
Table 3. Details of studies on the analytic validity of testing for the FVL mutation (continued) 

Study(s), Year(s) Description Reyes, 200178 A simple LLA model might not hold true for all situations (e.g., degradation of downstream primer extension 

complete at each cycle because of premature termination of primer extension or <100% efficiency of the po

exonuclease activity. Results of a simulated amplicon contamination experiment showed that LLA carryove

was substantially lower than that seen in PCR. In practical terms, this suggests that LLA is less susceptible 

results that are attributable to amplicon contamination. 

Schrijver, 200387 Some of the samples failed to amplify with the NanoChip system. In some of these cases, the authors were 

analysis because there was no sample remaining. The others failed re-amplification on the NanoChip Syste

Evans, 200289

Of the 635 samples classified by the Third Wave Assay (Invader Monoplex Assay) as FV WT, 10 were idenby the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were identified as WT bOf the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV WT by tresults that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctNanoChip technology. The Nanochip system was 100% accurate in characterizing WT/WT, heterozygous, aas compared to accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analyses. 

Castley, 200579 MGB-NFQ: minor groove–binding nonfluorescent quencher; 7700 SDS: ABI PrismTM 7700 sequence detecMammo, 200669

El Housni, 200380

Verri, 200577

Pecheniuk, 200046

Samples reported were FVL and prothrombin G20210A combined. 

DNA = deoxyribonucleic acid; EDEMNA = electrochemical detection of mismatches in nucleic acids; FVL = Factor V Leiden; LLA = linked linear a

matrix-assisted laser desorption/ionization – time of flight; MGB-NFQ = minor groove–binding non-fluorescent quencher; PCR = polymerase chain reEnzyme Activity DNA Interrogation Test; SDS = sequence detection system; SNP = single nucleotide polymorphism; SSCP = single strand conforma

temperature gradient capillary electrophoresis; UCLA = University of California at Los Angeles; WT = wild-type.

37 
38 

Outliers were the results reported by Ozkan et al.65 and by Lopez et al.,66 which detected FVL 

using electrochemical and enzyme-linked immunosorbent assay (ELISA) methods, respectively. 

In many studies, the discordances resolved after the tests were repeated. We describe the studies 

in detail below. 

Detection of FVL 

Forty-one studies compared at least two methods for FVL detection (Tables 2 and 3; 

Appendix G, Evidence Table 2). Three studies compared flap endonuclease combined with 

fluorescence resonance energy transfer (FRET/invader assay) to the reference standard.43 44 67

The concordance ranged from 99.5 percent to 100 percent. Five samples were typed as wild-type 

by the reference standard but were initially found to be heterozygous by the invader assay. 

Retyping of these samples by both methods was concordant, with all genotypes being 

unequivocally wild-type. In Ledford et al.,44 two samples were discordant with the PCR-RFLP 

method. Repeating both the invader and PCR-RFLP testing yielded results concordant with the 

original PCR-RFLP results. In one case, in which a heterozygous sample was called homozygous 

mutant by the invader assay, the discrepancy was due to a failure to add genomic DNA to the 

well. In the second case, a heterozygous sample was called wild-type, and there was no obvious 

cause for the discrepancy. The source of discordance was reported to be technical or operator 

errors. 

Five studies compared FRET combined with melting curve analysis (Light Cycler Assay) to 

the reference standard.45 48 68-70 The concordance was 100 percent for all four studies. 

Three studies compared Taqman real time polymerase chain reaction (PCR) to PCR-RFLP.47 

62 71 The concordance was again 100 percent in all these studies. 

In three studies, electrochemical genosensors were used to detect the FVL mutation in a 

research, rather than a clinical setting.65 72 73 The concordance ranged from 93 percent to 100 

percent. In the study by Ozkan et al., the source and nature of the 7 percent discordance were not 

explained.65 The study by Ozsos et al. reported neither the reference standard nor quantitative 

results.72 Rather, the authors stated that there was "good agreement" between the experimental 

and reference methods and that efforts to achieve 100 percent accuracy were underway. Huang et 

al. did not report the sample size or specify the reference standard.73

Murphy et al., in a study with more than 300 samples, used temperature gradient capillary 

electrophoresis (TGCE) technology to detect FVL.61 One heterozygous sample was falsely 

identified as being homozygous by TGCE but was corrected in repeat testing. A low PCR 

product peak height on the initial capillary electrophoresis analysis was identified as the source 

of the false homozygous result. One wild-type sample was falsely reported as being 

heterozygous by TGCE. Repeating the tests did not resolve this inconsistency. Further 

investigation revealed that the false heterozygous mutation involved a G ÆA mutation 95 base 

pairs downstream of the Leiden site. Thus, this sample was really wild-type for the Leiden allele 

but contained a new polymorphism in close enough proximity to the Leiden position that TGCE 

detected it as a heterozygous mutant. 

Two small studies used mass spectrometry to detect FVL and reported 100 percent 

concordance.50 55 There are several single studies, each of which compared a research-based 

experimental method to the reference standard and reported a concordance of 100 percent.46 64 74-

79 Three studies used a fluorophore probe to detect FVL,80-82 and all three reported 100 percent 

concordance. Potter et al. tested a lanthanide-labeled probe for FVL detection.49 There was a 1 
39 

percent discordance on the first test, and 0.26 percent remained discordant after repeating the 

test. Alsmadi et al. tested the rolling circle amplification assay using open circle probes ligation 

and reported a 99.4 percent concordance rate with PCR-RFLP.83

Taking advantage of pyrophosphorolysis chemistry, Tsongalis84 and Rhodes59 used the 

Reversed Enzyme Activity DNA Interrogation Test (READIT) System for FVL detection.59 84

The concordance rate was greater than 99 percent when compared to the reference standard. 

Tsongalis et al. reported one discordant result that was due to an equivocal relative light unit and 

another that was indeterminate.84

Two studies used ELISA to detect FVL:66 85 Carmi et al.85 reported 100 percent concordance 

with the reference method. The initial concordance in the other study was just 95 percent.66 No 

explanation was given regarding the source or nature of the discrepancies. Upon repeat testing, 

the discordance resolved. 

Three studies compared nanochip electronic microarray technology to various reference 

standards for the detection of FVL.87-89 Two reported 100 percent concordance rates.87 88

Interestingly, in the study by Evans et al., of the 635 samples classified by the Third Wave Assay 

(Invader Monoplex Assay) as wild-type, 10 were identified as heterozygous FVL by the 

NanoChip technique.89 Similarly, of the 114 putative heterozygous samples, 4 were identified as 

wild-type by the NanoChip technique. Of the 9 reported homozygous samples, 6 were read as 

homozygous, 2 as heterozygous, and 1 as wild-type by the NanoChip assay. All 17 results that 

were discordant were confirmed by DNA sequencing to have been correctly classified by the 

NanoChip technology. Compared to DNA sequencing, the NanoChip system was 100 percent 

accurate in characterizing wild-type, heterozygous, and homozygous samples, as compared to 

accuracies of 99.2, 90.2, and 100 percent for the comparable Third Wave analyses. 

Detection of Prothrombin G20210A 

The concordance rates between the experimental methods and the reference standards for the 

detection of prothrombin G20210A were 100 percent in nearly all of the 23 studies (Tables 4 and 

5, Appendix G, Evidence Table 3). Three studies had some initial discordance between ELISA 

and direct sequencing or PCR-RFLP (0 to 2 percent), which upon repetition completely 

resolved.54 66 85 Alsmadi et al. and Pecheniuk et al. reported concordance rates of 99.5 and 99 

percent, respectively.46 83

Simultaneous Detection of FVL and Prothrombin G20210A Mutations 

All 12 studies that employed multiplex technologies for the simultaneous detection of the 

two mutations reported 100 percent concordance between these technologies and the matched 

reference standard (Tables 6 and 7; Appendix G, Evidence Table 4).51 56 86 92-100

External Quality Assurance 

We identified three studies that addressed external quality assurance or laboratory 

performance relative to the gold standards.63 101 102 Studies assessing quality assurance that were 

published before the year 2000 were excluded by our search strategy, including that by Lutz et 

al.103
40 

Table 4. Analytic validity of testing for prothrombin G20210A 

Experimental Test PCR-RFLP 

Flap Endonuclease + FRET (Invader Assay) 

FRET + Melting Curve Analysis (Light Cycler) 

Taqman RealTime PCR Assay 

Electrochemical Detection 

Single StranConformatioPolymorphi(SSCP) and Denaturing Gel Electrop(DGGE) 

Reference Standard PCR-RFLP 

PCR-RFLP or ASPCR PCR or PCR- RFLP PCR-RFLP PCR-RFLP Direct DNA Sequencing 

Studies (ref. no.) Bravo-Osorio, 

2000104

Hessner, 2004105

Patnaik, 200467

SchroellMetzger, 200368

Cooper, 200370

Nauck, 200045

Parks, 200148

Louis, 200462Happich, 2000106

Behrens, 200471

Gellings, 200153 Meyer, 2000

Total Population* 98 522 + 377 132 + 200 + 110 

+ 157 

122 + 233 + 100 

12 527 

Concordance 100% 100% 100% 100% 100% 100% (based 

samples) 

Source of Zygosity Discordance** 

NA NA NA NA NA NA 

Did Repeating Resolve the Discordance**? 

NA NA NA NA NA NA 

40 
41 

Table 4. Analytic validity of testing for prothrombin G20210A (continued) 

Experimental Test 

SASA-PCR Followed by RFLP ELISA 

Rolling Circle Amplification Assay Using Open Circle Probes Ligation 

First Nucleotide Change Technology 

Nanochip Electronic Microarray 

High-ResMelting Analysis Small Am

Reference Standard PCR-RFLP 

Direct Sequencing or PCR-RFLP PCR-RFLP PCR-RFLP 

PCR-RFLP & Light Cycler Light Cyc

Studies DelRioLaFreniere, 

200164

Lopez, 200766Gilchrist, 200154

Carmi, 200485

Alsmadi, 200383 Pecheniuk, 200046 Schrijver, 

200387 Erali, 

200388

Liew, 2004

Total Population*

50 122 + 459 + 283 298 500 199 for the 

comparison to RFLP and 198 + 224 for the comparison to LightCycler 

22 

Concordance 100% 98-100% 99.5% 99% 100% 100% Source of Zygosity Discordance** 

NA No explanation of 

discrepancies in Lopez.54 Sensitivity 

was 100% with no false negatives, specificity was 99.2% with 2 false positives; 2 samples corrected on second attempt. 

Not reported No explanation was 

given on the source of discrepancies. 

NA 

Did Repeating Resolve the Discordance**? 

NA Yes66 85, NA54 Yes (If tests 

were performed in triplicate). 

Unclear: 1% misclassification was in accordance with 2.2% error noted when establishing the cut-off criteria. 

NA 

* the order of the counts corresponds to the order of studies in the row above; **if <100% concordance AS-PCR = allele-specific PCR; DGGE = denaturing gradient gel electrophoresis; DNA = deoxyribonucleic acid; ELISA = enzyme-linked immunosor

fluorescence resonance energy transfer; MALDI-TOF = matrix-assisted laser desorption/ionization – time of flight; MGB-NFQ = minor groove–bindiNA = not applicable; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; SASA-PCR = simultaneous allele-specific 

sequence detection system; SSCP = single strand conformation polymorphism 

41 
42 

Table 5. Details of studies on analytic validity of testing for prothrombin G20210A 

Study(s), Year(s) Description 

Bravo-Osorio, 2000104 Since control of digestion is of importance to avoid false results in RFLP, this study described a method for det

G20210A and methylenetetrahydrofolate reductase (MTHFR) mutations, in which a second restriction site was 

control the enzyme digestion of the respective PCR products. There were 49 individuals with either a prothrommutation, and 49 individuals with neither mutation as negative controls. Those performing the tests were aware 

Patnaik, 200467 AS-PCR reference standard mistyped 14 samples that the invader experimental assay typed correctly.Louis, 200462 The aim of the study was to assess the cost-effectiveness and specificity of a high-throughput real-time PCR a

specific fluorescent oligonucleotides that contain a 3' minor groove binding (MGB) probe.

Happich, 2000106 Samples of 233 individuals previously genotyped for PT-G20210A and MTHFR by restriction fragment analysis 

5’nuclease fluorescence assay (Taqman).

Gellings, 200153 Genotype was determined based on the electrochemiluminescence signal generated by the ruthenium compleMeyer, 200052

A series of 383 patients with thrombosis and 144 controls were tested with SSCP and DGGE. Eight of the normheterozygous samples, and the homozygous samples were tested with direct DNA sequencing, and the results prevalence of prothrombin mutation in thrombotic and healthy patients was reported.

Humeny, 200155 Sample: women from the Obstetrics and Gynecology outpatient clinic who presented with various endocrine pGilchrist, 200154 Only 100 patients were tested with RFLP, although 459 patients were tested with microparticle enzyme immunPecheniuk, 200046 Samples reported FVL and prothrombin G20210A combined.Erali, 200388 Two prothrombin samples were identified as containing mutations at sites other than position 20210. Both wer

was heterozygous at A20218G and the other at C20209T. Both were correctly identified as WT G20210 in the fluorescence signal for the WT probe was approximately one-half of the signal for the WT control sample. The the lower signal is that the WT probe was binding only to the exact WT strand and did not bind to the strand wThe mutant probe did not bind to either strand.

Castley, 200579 The goal of the study was to show that whole-blood PCR after formamide addition is as good as DNA-extracte

EXTRACTION). There were two comparisons: (1) whole blood vs. DNA-extracted real-time detection (RTD) PC(concordance results in Tables 2 and 3 of online Data Supplement [not available in this program]). (2) Whole-b

RTD-PCR and SDS. MGB-NFQ: minor groove–binding nonfluorescent quencher, 7700 SDS: ABI PrismTM 770system.

AS-PCR = allele-specific PCR; DGGE = denaturing gradient gel electrophoresis; DNA = deoxyribonucleic acid; FVL = Factor V Leiden; MEIA = mi

immunoassay; MGB-NFQ = minor groove–binding non-fluorescent quencher; MTHFR = methylenetetrahydrofolate reductase; PCR = polymerase ch

prothrombin G20210A mutation; RFLP = restriction fragment length polymorphism; RTD = real-time detection; ; SDS = sequence detection system; S

conformation polymorphism; WT = wild-type. 

42 
43 

Table 6. Analytic validity of simultaneous detection (multiplex) of FVL and prothrombin G20210A mutations 

Experimental Test 

FRET + melting Curve Analysis (Light Cycler) 

Mutagenically Separated PCR Followed by Gel Electrophoresis 

Multiplex PCRRFLP 

Multiplex PCR – Reverse Hybridization Line Probe Assay 

Multiplex AS-PCR - Ion-Pair Reversed Phase HPLC 

Reference Standard PCR-RFLP PCR-RFLP 

PCR-RFLP or Light Cycler PCR-RFLP PCR-RFLP 

Studies (ref. no.) van den Bergh, 

200092

HobsonPeters, 200593

Ameziane, 200394

Endler, 200156 Baris, 200495

Angeline, 200551

Huber, 200096

Lucotte, 200397

Koksal, 200757

Leyte, 200086 Nietzel, 200399 

Total Population* 47 + 43 + 200 153 408 + 72 + 205 + 

175 + 104 

99 > 100 

Concordance 100% 100% 100% 100% 100% Source of Zygosity Discordance** 

NA NA NA NA NA 

Did Repeating Resolve the Discordance**?

NA NA NA NA NA 

* The order of the counts corresponds to the order of studies in the row above; **if <100% concordance ASO = allele-specific oligonucleotide; AS-PCR = allele-specific PCR; FRET = fluorescence resonance energy transfer; HPLC = high performance (prchromatography; NA = not applicable; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism 

43 
44 

Table 7. Details of studies on the analytic validity of simultaneous detection (multiplex) of FVL and prothrombin G20210A mutations 

Study(s), Year(s) Description 

Hobson-Peters, 200593 Used FRET with a dual-probe quenching system that is similar to the Light Cycler assay.Endler, 200156 70 known patients with recurrent venous thrombosis and 83 selected healthy controls from the Austrian Study 

Recurrent VTE for whom previous determination of the genotypes for the FV:R506Q (G1691A), the FII:G20210(C677T) variants had been established by PCR followed by RFLP

Baris, 200495 No reference method was reported. For concordance, known patients with recurrent venous thrombosis with "p

genotypes for FVL and prothrombin G20210A were used. FVL was amplified significantly less than prothrombinprimer concentration of 2:1. The effects of various factors, including magnesium cation concentration, primer co

conditions, on PCR specificity and efficiency were determined and optimized in this study. 

Huber, 200096 Concordance was only tested on FVL. For prothrombin, the multiplex PCR-RFLP identified 9 heterozygous and 

independently verify prothrombin results, DNA sequencing confirmed 9 heterozygous mutant and 11 wild-type The study also genotyped 123 samples with multiplex PCR-RFLP and the invader assay for Factor II, with 100% 

Lucotte, 200397 Duplex PCR-RFLP for simultaneous detection of FVL and prothrombin G20210A was used.Koksal, 200757 Significantly less amplification was noted for FV relative to prothrombin; the FV primer was therefore 2x concen

DNA = deoxyribonucleic acid; FII = Factor II; FV = Factor V; FRET = fluorescence resonance energy transfer; FVL = Factor V Leiden; MTHFR = mreductase; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; VTE = venous thromboembolism 

44 
45 

Jennings et al have described the results of the United Kingdom National External Quality 

Assessment Scheme (UK NEQAS) Thrombophilia Screening Program.101 Two hundred eighty 

centers participated in the thrombophilia screening exercises. This group reported the details of 

their first three evaluation exercises in an earlier publication by Preston et al.107 They described 

an important error rate in 6 years of UK NEQAS experience.107 Among centers performing 

genetic analysis for FVL and the prothrombin mutation, an error rate of 3 to 6 percent was 

identified, with both transcription and analytical errors being observed. For example, a staff 

member transcribing the wrong genotype in preparing a report of the results was considered a 

transcription error; misinterpretation of the results of the testing procedure was an analytic error. 

Hertzberg et.al. have reported the results of The Royal College of Pathologists of 

Australasia’s external quality assurance program.63 This program sent 133 DNA samples with 

known mutations to laboratories in 10 separate surveys. For the 3,799 responses received, the 

overall successful identification rate was 98.63 percent; the poorest individual sample result was 

a 15 percent incorrect identification of a homozygous FVL sample. Success rates in identifying 

specific mutations were 98.13 percent for FVL and 98.84 percent for prothrombin G20210A. Of 

the 39 responding laboratories, 20 (51 percent) made at least one error. Importantly, 3 of the 39 

laboratories were responsible for 46 percent of all errors. 

Finally, Tripodi et al.102 confirmed the findings of Preston et al.107 described above. Their 

survey was organized by the Subcommittee on Hemostasis of the Italian Committee for 

Standardization of Laboratory Tests (CISMEL). They sent four samples with known genotypes 

to 52 participating laboratories and received 41 responses. One laboratory misidentified a 

heterozygous FVL as a wild-type specimen, one misidentified a homozygote as a heterozygote, 

and one misidentified a heterozygote as a homozygote. Eight samples were given no 

interpretation. Similarly, one wild-type prothrombin G21210A was called a heterozygote, four 

heterozygotes were misclassified, and one homozygote was called heterozygote, with 10 being 

uninterpreted. The authors concluded that regular quality control programs aimed at identifying 

causes of failure are warranted. 

Summary of the Evidence for Key Question 2 

• There was high-grade evidence that tests for the detection of FVL have excellent analytic 

validity. 

• There was high-grade evidence that tests for the detection of prothrombin G20210A have 

excellent analytic validity. 

• There was high-grade evidence that most, but not all, clinical laboratories can test for FVL 

and prothrombin G20210A very accurately. There may be some laboratories that, for 

technical or administrative reasons, report inaccurate results. 
46 

Key Question 3: 

Clinical Validity of Testing for FVL 

and/or Prothrombin G202010A 

The next section of evidence describes the clinical validity of testing for these mutations. 

While Key Question 2 asked whether the mutations could be detected, Key Question 3 asked 

whether the detection of the mutations could predict patient-relevant outcomes (i.e., thrombosis). 

FVL and Recurrent Venous Thrombosis in Probands 

We identified 22 articles that examined the rates of recurrent VTE in individuals with a history 

of thrombosis (the probands) and the FVL mutation (Table 8 and Appendix G, Evidence Tables 

5-12). Eighteen were conducted in Europe14 19 108-123 and four in North America.124-127 As we 

required, all studies followed patients prospectively for the occurrence of VTE and objectively 

diagnosed venous thrombosis with radiographic testing. Most were prospective cohort studies, 

but six were cohorts nested within randomized controlled trials. Of these, five tested 

anticoagulation management strategies116 119 123-125 and one tested the effect of hormone 

replacement on acquired activated protein C resistance.114 Two articles were derived from the 

same cohort (the Physicians’ Health Study).126 127 Two other studies came from a cohort of 

Austrian patients.14 112Total sample sizes for the studies ranged from 30 to 953 genotyped 

probands. Ages were variable; one study targeted patients under 40 years old.117 Follow-up 

ranged from a median of 0.5 years to over 8 years. 

Given our selective criteria for article inclusion, the study quality was generally high (Appendix 

G, Evidence Table 5). Some studies did not have a primary objective of quantifying the rates of 

thrombosis recurrence according to mutation status. Specifically, nine articles had different 

objectives, including evaluation of the prognostic value of other variables (such as D-dimer 

levels, positive family history of thrombosis, or sex),14 19 115 118 assessing anticoagulation 

strategies,123 124 examining the natural history of treated upper-extremity thrombosis,109

evaluating the relationship between mutation status and non-clinical hematologic variables,121

 or 

assessing the impact of post-menopausal hormone replacement on resistance to activated protein 

C.114 Most studies described the outcomes separately for individuals who were homozygous and 

those who were heterozygous for FVL, but some combined homozygotes with heterozygotes 

when quantifying the number of recurrent events.14 109-111 115 117 119 121 122 Two studies did not 

include an FVL mutation-free comparison group.108 122 

The FVL mutation-free comparison groups varied in terms of whether other mutations or 

thrombophilic defects were included or excluded, as shown in Appendix G, Evidence Table 5. 

However, those studies that included individuals with other thrombotic defects in the control 

group generally also included them in the group of FVL-positive individuals. Many studies did 

not present descriptive data separately for individuals with and without FVL mutations. It was 

therefore not possible to confirm that those with and without the mutation were broadly similar. 

No study described interactions between FVL and other mutations (besides prothrombin 

G20210A). We saw no evidence of funding support that would have been likely to bias the 

results. 
47 

Table 8 Pooled results describing the risk of recurrent VTE for probands with VTE compared to probands without 

mutations

Mutation Studies, N 

Studies With 

Data Appropriate 

for the Pooled 

Odds Ratio, n 

Individuals 

Contributing to the 

Pooled Odds Ratio*, 

n 

Pooled Odds Ratio†

(95% CI) 

Heterozygous FVL 14 13‡ 4730 1.56 (1.14-2.12) 

Homozygous FVL 9‡ 8‡ 2382 2.65 (1.18-5.97) 

Unspecified FVL§ 8 3 362 1.56 (0.75-3.25) 

Double Heterozygous FVL 

and PT 20210A 

4 3 843 4.81 (0.50-46) 

Heterozygous PT 20210A 11‡ 10‡ 3636 1.45 (0.96-2.21) 

Mixed PT 20210A 7 4 1143 0.73 (0.37-1.44) 

Homozygous PT 20210A 2 NA NA NA 

*studies without comparison groups or having zero events in both arms are not included 

†

unadjusted odds ratios, relative to probands without mutations ‡

the count includes both arms of a study that reported results separately for drug- versus placebo-treated patients 

§

unspecified means that heterozygous and homozygous individuals were not distinguished in the study 

CI = confidence interval; FVL = Factor V Leiden; NA = not applicable; PT 20210A = prothrombin G20210A; VTE = venous 

thromboembolism

 Heterozygous FVL in probands. Thirteen studies described event rates in probands who 

were heterozygous for FVL as well as probands without mutations (Table 9; Appendix G, 

Evidence Table 6).19 111-114 116 118 120 123-127 Since two studies were derived from the same 

cohort,126 127 we included only the more recent and larger of the two in our pooled analysis, so as 

to not count patients twice.126 One of the studies presented data separately for two treatment arms 

(ximelagatran extended prophylaxis versus placebo) and presented adequate data to allow us to 

analyze these two arms separately; therefore, we counted these arms as two different studies.123

In one article, the number of mutation-free patients was not given but could be calculated from 

percentages presented in the article; this calculation may have been subject to a small degree of 

rounding error.118 The number of probands heterozygous for FVL in these studies ranged from 

19 to 161 (median = 83). The number of probands without this mutation ranged from 55 to 724 

(median = 204). There were a total of 979 heterozygous individuals who suffered a total of 161 

recurrent thrombotic events, and a total of 3751 mutation-free individuals who suffered a total of 

473 thrombotic events. The pooled odds ratio for recurrent thrombosis was 1.56 (95 percent CI, 

1.14-2.12) for FVL heterozygous individuals when compared to patients without the mutation 

(Figure 3). There was a fair amount of heterogeneity (I2

 = 48 percent), but no significant 

evidence of publication bias. In our sensitivity analysis, in which we removed each study 

sequentially, the odds ratios were little changed. Annualized event rates in heterozygous 

individuals were reported in three studies and ranged from 3.1 to 7.5 percent.108 113 127
48 

Odds Ratios Venous Thromboembolism for

Probands Heterozygous for FV Leiden

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Lindmarker, 1999 1.35 (0.75,2.43)

 Kearon, 1999 0.38 (0.08,1.87)

 Simioni, 2000 3.62 (1.74,7.53)

 Hoibraaten, 2001 1.61 (0.30,8.62)

 Miles, 2001 1.99 (0.67,5.90)

 Eichinger, 2002 0.94 (0.50,1.76)

 Palereti, 2003 3.12 (1.61,6.04)

 Christensen, 2005 1.30 (0.73,2.31)

 Wahlander(a), 2006 1.36 (0.73,2.51)

 Wahlander(b), 2006 1.26 (0.26,6.14)

 Gonzalez-Porras, 2006 1.43 (0.48,4.29)

 Prandoni, 2007 2.33 (1.51,3.61)

 Kearon, 2008 0.38 (0.10,1.50)

 Overall (95% CI) 1.56 (1.14,2.12)

 Homozygous FVL in probands. Seven studies described event rates in probands 

homozygous for FVL and also in those without mutations (Table 5; Appendix G, Evidence Table 

7).19 111 114 116 123-125 One of the studies presented data separately for two treatment arms within 

one study (ximelagatran extended prophylaxis versus placebo), and we analyzed these arms 

separately as two different studies.123 The number of FVL-homozygous probands in these 

studies ranged from 1 to 11 (median = 6). The number of FVL-negative probands ranged from 

55 to 469 (median = 360). There were a total of 49 homozygotes who suffered a total of 7 

recurrent thrombotic events, and a total of 2333 mutation-free controls who suffered a total of 

225 thrombotic events. The pooled odds ratio was 2.65 (95 percent CI, 1.18-5.97) (Figure 4). 

There was no evidence of significant heterogeneity (I2

 = 0 percent). In our sensitivity analysis, in 

which we removed each study sequentially, the odds ratios changed little. One study specifically 

sought to determine whether FVL homozygosity carried a higher rate of recurrence than did FVL 

heterozygosity.108 This study did not include a control group without FVL. In this study, 5 of 32 

individuals homozygous for the mutation (16 percent) developed recurrent thrombosis, and 12 of 

108 individuals with heterozygosity developed recurrence (11 percent). This difference was not

Figure 3. Odds ratios for VTE for probands with heterozygous FVL mutation, as compared 

to probands without the mutation* 

 Study Odds Ratio (95% CI) 

Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence intervals (CI) 

for the pooled results are derived from a random effects model 
49 

statistically significant, but the relative risk for recurrence was marginally significant after 

excluding from both groups those patients who also carried the prothrombin G20210A mutation 

(relative risk, 1.8; 95 percent CI, 1.0-6.2). The annualized event rate was reported for 

homozygotes in only one study, as 5.8 percent (95 percent CI, 4.9-7.4).108

Unspecified FVL in probands. The authors of eight articles combined the individuals who were 

heterozygous and those who were homozygous for FVL in their analyses,14 109 110 115 117 119 121 122

without providing adequate detail in the text or tables to allow us to report the results separately 

for these individuals (Table 8; Appendix G, Evidence Table 8). Only three of these reported the 

number of events in the group with mutations as well as a group of probands without mutations, 

allowing for pooling.109 119 121 The number of probands with FVL in these studies ranged from 3 

to 50, and the number of mutation-free individuals ranged from 24 to 138. There were 68 

individuals with FVL who suffered a total of 12 events. Among 294 control patients, there were 

46 events. The pooled odds ratio was very similar to that for heterozygotes, at 1.56 (95 percent 

CI, 0.75-3.25) (Figure 5). There was no evidence of heterogeneity (I2

 = 0 percent). Among the 

studies that did not report the actual number of events, and therefore could not be included in our 

pooled analysis, four did test whether the FVL mutation was associated with recurrent 

thrombosis.14 110 115 117 None of these individual analyses demonstrated that the mutation was a 

significant predictor of recurrent events. The reported hazard ratio (HR) or relative risk values 

ranged from 1.2-1.35. One study simply reported the results as non-significant without further 

quantification.115

Odds Ratios of Venous Thromboembolism for

Probands Homozygous for FV Leiden

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Lindmarker,1999 4.03 (1.13,14.34)

 Kearon, 1999 9.44 (0.36,245.69)

 Hoibraaten, 2001 15.33 (0.85,276.51)

 Palerti, 2003 2.18 (0.10,46.20)

 Christiansen,2005 0.64 (0.08,5.26)

 Wahlander(b), 2006 2.50 (0.13,46.47)

 Wahlander(a),2006 0.98 (0.05,18.50)

 Kearon, 2008 0.89 (0.05,16.72)

 Overall (95% CI) 2.65 (1.18,5.97)

Figure 4. Odds ratios for VTE for probands with homozygous FVL mutation, as compared 

to probands without the mutation* 

Study Odds Ratio (95% CI) 

Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence intervals (CI) for 

the pooled results were derived from a random effects model
50 

We pooled these studies of unspecified zygosity with the studies of heterogeneous FVL, 

since we presumed that most of the unspecified individuals were indeed heterogeneous for the 

mutation. The pooled estimate from combining these 17 studies was an odds ratio of 1.61 (95 

percent CI, 1.24 – 2.10), which is just slightly above the estimate for heterogenous FVL alone. 

Odds Ratios Venous Thromboembolism for

Probands with Mixed FV Leiden

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Baarsla, 2004 2.50 (0.18,34.67)

 Santamaria, 2005 1.88 (0.60,5.93)

 Strandberg, 2007 1.25 (0.45,3.49)

 Overall (95% CI) 1.56 (0.75,3.25)

Individuals with an idiopathic index event. Patients with idiopathic events (unprovoked VTE) 

are often advised to take life-long anticoagulation. There is a great deal of interest in whether this 

patient population can be better stratified according to risk of recurrence. Among the studies that 

included only patients with idiopathic index thromboses112 124 125 127 and the studies in which 

results were reported separately for individuals with idiopathic index events,119 120 the pooled 

odds ratio for recurrence was 1.17 (95 percent CI, 0.63 - 2.18.) for individuals with FVL, as 

compared to individuals without. Only one of these studies was not exclusively composed of 

probands with heterogeneous FVL (the zygosity was unspecified).119 A single study described 

recurrence rates for individuals with FVL who had a clearly provoked VTE (non-idiopathic 

VTE), with an odds ratio of 6.5 (95 percent CI, 2.5 –18).120

Double (or compound) heterozygous (FVL plus the prothrombin G20210A mutation) 

probands. Three articles presented event rates in individuals having a FVL mutation as well as a 

prothrombin G20210A mutation and in mutation-free control patients (Table 8; Appendix G, 

Evidence Table 9).19 120 126 There were 10 doubly heterozygous individuals in all, 4 of whom 

suffered recurrent events. Among the mutation-free controls, there were 95 recurrent thromboses 

in 833 patients. The pooled odds ratio was 4.81 (95 percent CI, 0.50-46.3) (Figure 6). In one 

study, all three double heterozygotes developed recurrent thrombosis,126 requiring a continuity 

Figure 5. Odds ratios for VTE for probands with unspecified FVL mutations, as compared to 

probands without the mutation* 

Study Odds Ratio (95% CI) 

Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence intervals (CI) 

for the pooled results were derived from a random effects model
51 

correction for calculating pooled odds ratios. The odds ratio was lower when this study was 

excluded during sensitivity testing analysis (odds ratio, 1.64; 95 percent CI, 0.25-11). Annual 

incidence rates were not reported in these studies. 

Prothrombin G20210A and recurrent venous thrombosis in probands. We identified 18 

articles that examined the rates of recurrent VTE in probands with the prothrombin G20210A 

mutation (Table 8; Appendix G, Evidence Table 10).14 19 109-112 115-120 122-126 128 All of these 

studies were included in the 22 studies examining FVL (above), and therefore they generally 

shared common control groups with the FVL analyses. They also suffered from similar 

limitations, as discussed above in the section FVL and recurrent venous thrombosis in probands. 

These limitations included: a limited description of covariates, such as age and sex, in those with 

and without the mutation; combining individuals with 

homozygous and heterozygous mutations when reporting results; and, in some studies, not 

specifying the exact number of thrombotic events in each arm in the case of those with and 

without the mutation. 

Heterozyous prothrombin G20210A in probands. Nine articles quantified event rates in the 

probands who were heterozygous for prothrombin G20210A and also in mutation-free probands 

(Table 8; Appendix G, Evidence Table 11).19 113 116 118 120 123-126 One of the studies presented data 

separately for two treatment arms (ximelagatran extended prophylaxis versus placebo), and we 

analyzed these two arms separately.123 The number of probands with a heterozygous 

prothrombin G20210A mutation in these studies ranged from 3 to 58 (median = 26.5). The 

number of comparison individuals without the mutation ranged from 72 to 724 (median = 307). 

There were a total of 281 heterozygous individuals who suffered 38 recurrent thrombotic events, 

Odds Ratios Venous Thromboembolism for

Probands who are Double Heterozygous

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Simioni, 2000 2.01 (0.18,22.81)

 Miles, 2001 57.26 (2.85,1150.32)

 Palareti, 2003 1.21 (0.06,22.90)

 Overall (95% CI) 4.81 (0.50,46.31)

Figure 6. Odds ratios for VTE for probands who were doubly heterozygous for the FVL and 

prothrombin G20210A mutations, as compared to probands without mutations* 

Study Odds Ratio (95% CI) 

 Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence 

intervals (CI) for the pooled results are derived from a random effects model 

 
52 

and a total of 3355 mutation-free individuals who suffered 385 thrombotic events. The pooled 

odds ratio was 1.45 (95 percent CI, 0.96-2.21) (Figure 7). There was little evidence of 

heterogeneity (I2

 =8 percent), and no evidence of publication bias. In our sensitivity analysis, one 

study did have a major impact on the pooled odds ratio: This was the study that had an odds ratio 

of 4.0.120 Removal of this study from the meta-analysis decreased the pooled odds ratio to 1.30. 

Annual recurrence rates were reported in two studies and were 0 and 2.9 percent.113 125

Odds Ratios Venous Thromboembolism for

Probands Heterozygous for PT 20210A

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Kearon, 1999 1.90 (0.16,22.39)

 Lindmarker, 1999 1.08 (0.36,3.24)

 Simioni, 2000 4.03 (1.67,9.68)

 Miles, 2001 1.86 (0.37,9.25)

 Palareti, 2003 0.95 (0.28,3.21)

 Gonzalez-Porras, 2006 1.15 (0.30,4.33)

 Wahlander(b), 2006 1.02 (0.06,18.03)

 Wahlander(a), 2006 0.66 (0.15,2.88)

 Prandoni, 2007 1.45 (0.72,2.94)

 Kearon, 2008 0.16 (0.01,2.85)

 Overall (95% CI) 1.45 (0.96,2.21)

Unspecified prothrombin G20210A in probands. The authors of seven articles combined 

individuals who were heterozygous and those who were homozygous for prothrombin 

G20210A14 109-111 115 119 128 (Table 8; Appendix G, Evidence Table X). Four of these articles 

described event rates in this combined prothrombin G20210A group as well as in a group of 

mutation-free probands.109 111 119 128 In our pooled analysis, there were a total of 86 individuals 

with the prothrombin G20210A mutation who had 10 recurrent events, and 1057 patients without 

the mutation who suffered 158 recurrent events. The pooled odds ratio was 0.73 (95 percent CI, 

0.37-1.44) (Figure 8). There was no evidence of significant heterogeneity (I2

 = 0 percent). 

Relative risk or HR values were reported in five of the studies14 110 111 115 128and ranged from 0.7 

to 2.1. Of note, three of the studies that presented relative risk or HR were not included in the 

pooled analysis, since the specific number of events was not reported.14 110 115 Two of these 

studies reported adjusted relative risks that were significantly different from 1.0, with adjusted 

Figure 7. Odds ratios for VTE for probands who were heterozygous for prothrombin 

G20210A, as compared to probands without the mutation* 

Study Odds Ratio (95% CI) 

 Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence intervals (CI) 

for the pooled results are derived from a random effects model 

 
53 

Odds Ratios Venous Thromboembolism for

Probands with Mixed PT 20210A

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Eichinger, 1999 0.56 (0.17,1.89)

 Baarslag, 2004 1.52 (0.05,43.69)

 Santamaria, 2005 1.03 (0.27,3.92)

 Christiansen, 2005 0.67 (0.23,1.97)

 Overall (95% CI) 0.73 (0.37,1.44)

relative risk estimates of 2.1 in both cases.14 115 The annual incident rate of recurrent thrombosis 

was reported in only one study, at 1.9 percent.111

 Since probands with unspecified prothrombin G20210A mutations are almost certainly 

heterozygous for the mutation, given the rarity of homozygous prothrombin G202010A, we 

pooled these studies with the studies of probands known to be heterozygous. The combined odds 

ratio was 1.23 (95 percent CI, 0.87-1.7). 

Homozygous prothrombin G20210A in probands. Only two articles quantified event rates in 

individuals homozygous for prothrombin G20210A and in mutation-free probands.123 125 There 

were a total of only three homozygous individuals in these studies, and none of them developed 

recurrent thrombosis. 

Summary of the Evidence for Key Question 3 (Probands) 

Based upon our review of the published literature, we can summarize the evidence regarding 

the clinical validity of testing for FVL and prothrombin G20210A as follows: 

• There was moderate-grade evidence that homozygosity for FVL in probands is predictive 

of recurrent VTE. 

• There was moderate-grade evidence that heterozygosity for FVL in probands is predictive 

of recurrent VTE. 

Figure 8. Odds ratios for VTE for probands with unspecified prothrombin G20210A 

mutation, as compared to probands without the mutation* 

Study Odds Ratio (95% CI) 

 Odds Ratio 

*The size of the box is proportional to the study sample size; the confidence intervals 

(CI) for the pooled results are derived from a random effects model 

 
54 

• Homozygosity for prothrombin G20210A is a rare genotype, and its association with 

recurrent venous thrombosis in probands is currently unknown, because of a lack of 

sufficient evidence. 

• There was only low-grade evidence that heterozygosity for prothrombin G20201A in 

probands is not predictive of recurrent VTE. 

• There is insufficient evidence to allow us to draw any conclusions about double 

heterozygosity (FVL and prothrombin G20210A) in probands. 

FVL and Prothrombin G20210A and Venous Thrombosis in Family 

Members 

We identified 18 articles that evaluated venous thrombosis in family members of probands. 

All were conducted in Europe, with the majority in the Netherlands or France (Table 9, 

Appendix G, Evidence Tables 13-17). Five studies were prospective or included a prospective 

component,129-133 and the rest were retrospective cohort studies. One was a letter to the editor.133

The smallest study enrolled 13 people from a single family;134 the largest enrolled 1,093 family 

members.26 Inclusion criteria were not always stated, except for requiring that subjects be family 

members of probands with VTE. Three of the five prospective studies had scheduled clinical 

surveillance for VTE events, either annually or biannually.129 130 132 The individuals in the 

mutation-free comparison groups described below were generally the same in each of the 

comparisons within a given study. Only two studies explicitly excluded family members with 

prothrombin G20210A in their calculation of the odds ratio associated with FVL positivity.26 130

Table 9. Pooled results describing the risk of VTE for family members of probands with VTE, comparing those with a 

mutation to those without a mutation 

Mutation Studies, N 

Studies With Data 

Appropriate for the 

Pooled Odds Ratio, 

n 

Individuals 

Contributing to the 

Pooled Odds Ratio*, 

n 

Pooled Odds 

Ratio†

[95% CI] 

Heterozygous FVL 9 6 2,091 3.5 [2.5 to 5.0] 

Homozygous FVL 6 5 896 18 [7.8 to 40] 

Mixed FVL 6 6 3831 2.9 [1.8 to 4.8] 

Double Heterozygous FVL 

and PT 20210A 

4 3 727 6.7 [2.9 to 15] 

Mixed PT 20210A 1 NA NA NA 

Homozygous PT 20210A 1 NA NA NA 

Heterozygous PT 20210A 3 3 291 1.9 [0.35 to 10] 

*Studies without comparison groups or having zero events in both arms not included †

Unadjusted odds ratios, relative to family members without mutations 

CI = confidence interval; FVL = Factor V Leiden; NA = not applicable; PT 20210A = prothrombin G20210A; VTE = venous 

thromboembolism
55 

Study quality was mixed (see Appendix G, Evidence Table 13). To some extent, it was 

challenging to assess the quality of these studies, since they were not necessarily designed to 

answer our question (e.g., the clinical validity question was secondary to their primary aims). In 

our quality assessment, we focused mostly on biases that might result from the sampling scheme. 

For the prospective studies, we also were attentive to the description of losses to follow-up and 

the percentage of participants lost; this was only relevant to five of these studies. Three described 

the number lost, and it was low;129-131 the others provided no description.132 133 Studies varied 

greatly in terms of the degree to which they described the population from which the participants 

came. Since these were family members of probands, studies with adequate description generally 

described the source of the probands, and then provided a description of the recruitment process 

used to enroll family members. Several of the studies had very little description of the inclusion 

and exclusion criteria for the family members.133 135-137 Even among those that had explicit 

inclusion criteria, not all uniformly provided a detailed description of the enrolled participants. 

Even age and sex were inconsistently described. The majority of these studies were published 

before journals began requiring disclosure of funding sources and potential conflicts of interest. 

More studies than not, however, did report on the source of funding; we considered these sources 

of funding to be unlikely to bias the results, since most of the reported funding was from 

governmental grant support. 

 

Heterozygous FVL in family members. Nine studies described results for people heterozygous 

for FVL (Table 9, Appendix G, Evidence Tables 14 and 15).129 130 132 134 137-141, although one of 

these included a single person homozygous for the mutation among the 91 studied.138 Two of the 

studies did not report on a comparison group without mutation.129 141 The seven studies with 

comparison groups enrolled between 5 and 299 affected individuals, for a total of 1,066 

heterozygous family members. We show the rates of events among the affected family members, 

as compared to the rates of events among 940 unaffected family members (Appendix G, 

Evidence Table 15). The odds ratios across these studies were quite comparable except for the 

one small study by Simioni, in 2000, in which there were no events among the eight 

heterozygous individuals.137

 
56 

The pooled odds ratio was 3.49 (95 percent CI, 2.46 to 4.96), with very little heterogeneity 

between studies (I2

= 4 percent) (Figure 9). The small study by Rintelen contributed little to the 

pooled estimate. 

Three of these studies reported person-years of observation to allow calculation of the annual 

rate of VTE events.129 130 132 Our sensitivity analysis, in which we removed each study 

sequentially, demonstrated that removal of the study by Couturaud et al.132 raised the pooled 

odds ratio to close to 4.0, although the CI values overlapped the pooled odds ratio when that 

study was included. The annualized event rate among heterozygous individuals in this study was 

0.36 percent (95 percent CI, 0.24 to 0.49), which is lower than those in the other two studies in 

which rates were reported.129 130 It is not apparent from either the inclusion criteria or the patient 

characteristics why the rates were lower in this study, which was of adequate quality. The event 

rates in the mutation-free groups were fairly comparable across the three studies. There was no 

evidence of important publication bias. 

Homozygous FVL in family members. Six studies described results for 48 people homozygous 

for FVL130 132 134 137 139 140. The annualized rates were given in only three studies and are shown in 

Table 9, Appendix G, Evidence Tables 14 and 15.129 130 132 The odds of events were compared to 

the odds for 850 family members without mutations (Figure 9). The pooled odds ratio was 18 (95 

percent CI, 7.8 to 40), with very little heterogeneity between studies (I2

=0 percent) (Figure 10). 

One small study was excluded from pooling because it had no events in either arm and only a 

single individual in each arm.134 One study, a French study by Couturaud et al.,139 was 

Odds Ratios Venous Thromboembolism for

Family Members Heterozygous for FV Leiden

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Middeldorp, 1998 4.09 (1.66,10.07)

 Le Cam-Duchez, 1999 3.91 (2.28,6.70)

 Lensen, 2000 4.79 (1.71,13.41)

 Rintelen, 2001 0.45 (0.02,12.49)

 Simioni, 2002 6.79 (0.84,54.67)

 Coutraud, 2006 2.36 (1.20,4.66)

 Simioni, 2000 (Excluded)

 Overall (95% CI) 3.49 (2.46,4.96)

Figure 9. Odds ratios for VTE for family members with a heterozygous FVL mutation, as compared to 

family members without the mutation* 

Study Odds Ratio (95% C.I.) 

 Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence intervals 

(CI) for the pooled results are derived from a random effects model 

 
57 

particularly influential in raising the odds ratio. Removal of that study in the sensitivity analysis 

resulted in a pooled odds ratio of roughly 16, with an upper limit of the CI that did not reach 18. 

It is not apparent why the event rate was so high in this French study (five of the six individuals 

with homozygosity had VTE events). The annualized rate was not described for their mutationfree comparison group. There was no evidence of important publication bias for this comparison.

Mixed or unspecified FVL in family members. Nine studies reported the results from an 

unspecified group of homozygous and heterozygous individuals (Table 9, Appendix G, Evidence 

Tables 14 and 15).26 131-133 135 136 140 142 143 Three of these studies also described the results 

separately for these groups, so we will not discuss these composite groups here.26 132 140 The 

odds ratios for each study as well as the pooled odds ratio are given in Figure 11. The pooled 

odds ratio was 2.9 (95 percent CI, 1.8 to 4.8), with little heterogeneity between studies (I2

=4 

percent). One large study was particularly influential.142 The odds ratio of this study was 5.2; 

when it was removed from pooling, the aggregated odds ratio dropped to close to 2.0, with the 

upper limit of the CI extending only to 2.8. This influential study involved 1,437 family 

members from nine European countries and was part of the European Prospective Cohort on 

Thrombophilia (EPCOT) study of familial thrombophilia. Thirteen of the 225 family members 

with FVL were homozygous for the mutation. The event rate in members without mutations was 

quite low, at 0.03 percent (95 percent CI, 0.02 to 0.05). The prospective portion of the EPCOT 

study described very few VTE events among the family members with FVL, with an odds ratio 

of only 0.92 and wide confidence intervals (95 percent CI, 0.11 to 7.5).131

Odds Ratio 

Odds Ratios of Venous Thromboembolism for

Family Members Homozygous for FV Leiden

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Middeldorp, 1998 18.76 (4.30,81.82)

 Le Cam-Duchez, 1999 33.33 (3.71,299.48)

 Rintelen, 2001 11.00 (0.35,345.06)

 Simioni,2002 5.69 (0.22,145.88)

 Coutraud, 2006 17.75 (5.36,58.83)

 Simioni, 2000 (Excluded)

 Overall (95% CI) 17.84 (7.98,39.89)

Couturaud, 2006 

Figure 10. Odds ratios for VTE for family members with homozygous FVL mutation, as compared to 

family members without the mutation* 

Study Odds Ratio (95% CI) 

 Odds Ratio 

*the size of the box is proportional to the study sample size; the confidence 

intervals (CI) for the pooled results are derived from a random effects model 
58 

Family members doubly heterozygous for FVL and prothrombin G20201A. Four studies 

described VTE events in a total of 59 doubly heterozygous individuals (Table 9, Appendix G, 

Evidence Tables 14 and 15).26 132 134 137 These individuals were compared to a total of 674 

individuals without mutations. The very small study134 observed no events in either arm. The 

odds ratios for each study as well as the pooled odds ratio are given in Figure 12. The pooled 

odds ratio was 6.7 (95 percent CI, 2.9 to 16), with very little heterogeneity between studies (I2

=0 

percent). The odds ratio associated with double heterozygosity in the large study by Martinelli et 

al. was higher than in the other two studies (odds ratio, 8.0; 95 percent CI, 2.8 to 23). This 

difference may be partially explained by the low rate of events in their study among the family 

members without mutations. The rate of events in the mutation-free group was 0.06 percent per 

year, whereas in most studies it was closer to 0.1 percent per year. In the case of this comparison, 

there may have been some degree of publication bias, given the paucity of small studies 

reporting large effect sizes (high odds ratios).

Odds Ratios Venous Thromboembolism for

Family Members with Mixed FVL

s ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Samama, 1996 1.91 (0.50,7.26)

 Simioni, 1999 3.08 (1.02,9.34)

 Simioni, 1999 1.40 (0.23,8.47)

 Lensen, 2001 1.91 (0.57,6.43)

 Vossen, 2004 5.29 (2.52,11.13)

 Vossen, 2005 0.92 (0.11,7.55)

 Overall (95% CI) 2.93 (1.79,4.81)

 Odds Ratio 

*The size of the box is proportional to the study sample size; the confidence 

intervals (CI) for the pooled results are derived from a random effects model 

 

Figure 11. Odds ratios for VTE for family members with an unspecified FVL mutation, as 

compared to family members without the mutation*

Study Odds Ratio (95% CI)
59 

Prothrombin G20210A and venous thrombosis in family members. Five studies evaluated 

prothrombin G20210A and venous thrombosis in family members of probands.26 132 134 137 144 As 

mentioned above, all five studies were conducted in Europe. Only one of these studies was not 

described above in the discussion of FVL.144 This was a small study of adequate quality 

conducted in Italy that exclusively investigated outcomes from heterozygosity for prothrombin 

G20210A. This earliest study, by Castaman, had rigorous inclusion criteria and excluded women 

on oral contraceptive or hormonal therapies.144 Similarly, Martinelli et al. excluded people with 

malignancies or known autoimmune disease.26 

Homozygous prothrombin G20210A in family members. Only a single study described a 

single patient who was homozygous for this mutation (Table 9, Appendix G, Evidence Tables 16 

and 17).137 This patient was identified among 44 family members from four families and had no 

reported VTE events.

Heterozygous prothrombin G20210A in family members. Family members who were 

heterozygous for this mutation were compared to family members without the mutation in three 

studies (Table 9, Appendix G, Evidence Tables 16 and 17).132 137 144 In one study, an age-adjusted 

relative rate of events of 3.4 (95 percent CI, 0.2-56) was reported, when compared to family 

members without mutation.144 The other studies were very small, with one event in eight patients 

in one study137 and no events in six people in the other.132 Pooling these small studies yielded an 

odds ratio for VTE events of 1.89 (95 percent CI, 0.35-10), with an I2

 of 0 percent, suggesting 

that family members who are heterozygous for this mutation do not have an elevated risk of 

thrombosis (Figure 13).

Odds Ratios Venous Thromboembolism for

Family Members Compound Heterozygous

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Martinelli, 2000 7.99 (2.76,23.15)

 Rintelen, 2001 4.71 (0.41,54.83)

 Coutraud, 2006 5.07 (0.95,27.09)

 Simioni, 2000 (Excluded)

 Overall (95% CI) 6.69 (2.88,15.55)

Figure 12. Odds ratios for VTE for family members with double heterozygous mutations, 

as compared to family members without mutations* 

Study Odds Ratio (95% CI)

 Odds Ratio 

*The size of the box is proportional to the study sample size; the confidence 

intervals (CI) for the pooled results are derived from a random effects model 

Couturaud, 2006 
60 

Odds Ratios Venous Thromboembolism for

Family Members with Mixed PT 20210A

Odds ratio

.1 1 10

Study

 Odds ratio

 (95% CI)

 Castman, 2000 3.22 (0.18,56.88)

 Rintelen, 2001 1.57 (0.08,29.41)

 Coutraud, 2006 1.31 (0.07,24.67)

 Overall (95% CI) 1.89 (0.35,10.17)

The doubly heterozygous individuals were described above in the discussion of FVL. The 

risk of VTE events was notably high for doubly heterozygous individuals. 

Association of FVL and Prothrombin G20210A With Venous 

Thrombosis During Pregnancy and the Postpartum Period 

Testing asymptomatic family members for the FVL and prothrombin G20210A mutations 

would be important if the mutations raise the risk of venous thrombosis and if other risk factors 

can be modified or prophylactic interventions can be employed to lower the risk. A particularly 

important time with regard to risk is pregnancy and the postpartum period. These are high-risk 

times for VTE because hormonal and mechanical changes shift the balance in favor of 

hypercoaguability.145 

Several of our included articles described the experience of family members of probands 

during their pregnancies. Here we report the results of studies that quantified the risk of VTE 

associated with FVL and prothrombin G20210A specifically during pregnancy and the 

postpartum period for these family members of patients with VTE. Occasionally these studies 

included probands and evaluation of their subsequent pregnancies; this situation was rare, and we 

excluded these probands from our analyses. 

We identified four articles that specifically addressed the risk of venous thrombosis 

attributable to FVL and prothrombin G20210A during the pregnancies of family members,146-149

and one additional study that included useable pregnancy data.132 All were retrospective cohort 

Figure 13. Odds ratios for VTE for family members with heterozygous prothrombin 

G20210A, as compared to family members without the mutation* 

Study Odds Ratio (95% CI)

 Odds Ratio 

*The size of the box is proportional to the study sample size; the confidence 

intervals (CI) for the pooled results are derived from a random effects model 

Couturaud, 2006 
61 

studies conducted in Europe; one included a prospective component132 (Appendix G, Evidence 

Tables 18 and 19). Two explicitly excluded women who had had a venous thrombosis prior to 

pregnancy.147 149 

Although not explicitly stated, we believe that the two studies by Martinelli et al. include 

some of the same women, so we did not extract data about the women who were homozygous for 

FVL from the more recent of the two studies.146 149 The mutation-free groups described in these 

two studies may also include many of the same women. We show the results in Appendix G, 

Evidence Tables 18 and 19. The odds ratios compare the odds of a VTE event per pregnancy 

across two groups, with the reference group being women with neither FVL nor the prothrombin 

G20210A mutation. 

Two studies evaluated the risk associated with homozygous FVL.146,147 The number of 

pregnant women with homozygous FVL was small, with only nine in one study146 and six in the 

other,147 and a total of 33 pregnancies among them. The odds ratios for venous thrombosis were 

very high, although with wide CIs, suggesting that homozygosity for FVL is associated with 

venous thrombosis in the pregnancy or post-partum periods. 

The odds ratios associated with heterozygous FVL were close to 1.0 and quite similar in the 

two studies reporting this outcome. The rates of events were low, at 2.5 percent per pregnancy 

(95 percent CI, 0.9-5.4)147 and 1.5 percent per pregnancy (95 percent CI, 0.5-4.3).149 Two 

studies evaluated the risk associated with heterozygosity for the prothrombin G20210A mutation. 

A total of 247 women with this mutation were studied. The rates of VTE were low, at 0.3 

percent per pregnancy (95 percent CI, 0.1 to 1.6)148 and 1 percent per pregnancy (95 percent CI, 

0.2-3.6).149 These odds ratios were also close to 1.0. 

Individuals who were doubly heterozygous were evaluated in two studies.147,149 The larger of 

the two studies reported an odds ratio that was quite similar to the odds ratios associated with 

heterozygosity of either of the two mutations.149 The small study (five doubly heterozygous 

women with pregnancies) reported an odds ratio comparable to that seen in the women with 

homozygosity for FVL.147 The study with a prospective component only reported the results for 

a group of women with unspecified FVL that may have included some women who were 

homozygous for the mutation. The number of pregnancies was not described, only the number of 

women with pregnancies. The risk ratio for venous thrombosis in women with FVL as compared 

to those without was 2.9 (95 percent CI, 0.96-8.4). 

Age and VTE Among Family Members With Mutations 

We sought to examine whether the risk attributable to the mutations varied with age. Few 

studies reported results stratified by age. None of the studies of prothrombin G20210A described 

the results according to age. Three studies examining the risk from the FVL mutation described 

age-specific rates of events.138,140,143 One additional study described the age of the individuals at 

the time of their first thromboses in the family members with and without mutations.139 The 

mean age at thrombosis was 40 years in the family members without a mutation, 34 years in 

people who were heterozygous for FVL, and 21 years in homozygous individuals. The difference 

was not statistically significant when heterozygous and mutation-free individuals were compared 

(p=0.07), but it was significant when homozygous and mutation-free individuals were compared 

(p=0.009). 

Middledorp et al. described age-specific relative rates of events for a group (homozygous, 

heterozygous, double heterozygous) with FVL mutation, as compared to family members 
62 

without mutations (Appendix G, Evidence Tables 14 and 15). The relative risk posed by the 

mutation was highest in the youngest patients, aged 15 to 30 (relative rate of approximately 15) 

and was lower and more comparable across the older age strata.140 Similarly, Simioni et al. 

described age-specific rates in a group of family members with unspecified FVL mutations.143 

The relative rate in the youngest age group was much lower than that reported in the Middeldorp 

study. Similarly, Lensen et al. described age-specific rates among heterozygous individuals that 

were not importantly different across age strata.138 

The study by Lensen et al. presented a relative hazard value from a Cox model across the 

lifetime of these heterozygous relatives, as compared to the mutation-free relatives. The relative 

hazard was 3.4 (95 percent CI, 1.3-9.2), which is remarkably similar to the odds ratio we 

calculated for the pooled studies of heterozygous individuals, 3.42 (95 percent CI, 2.40 to 4.89). 

Another study reported an HR (adjusted for age, sex and other factor deficiencies).26 The relative 

hazard for the FVL heterozygous individuals was 2.7 (95 percent CI, 1.4-18) and 6.5 (95 percent 

CI, 2.4-18) for the doubly heterozygous family members. This study also reported an HR for 

individuals with an isolated prothrombin G20210A mutation (Appendix G, Evidence Tables 16 

and 17). 

Other High-risk Periods 

In addition to analyzing the pregnancy and postpartum periods, we sought information on 

whether FVL or prothrombin G20210A additively or multiplicatively raised the risk of VTE 

during other high-risk periods, such as following surgery or during the use of hormonal therapy. 

Very few studies reported event rates stratified by high-risk periods,135 143 and we were unable to 

draw any conclusions regarding such periods. 

Summary of the Evidence for Key Question 3 (Family Members) 

 The literature supported the following conclusions about the clinical validity of testing for 

FVL and prothrombin G20210A: 

• There was high-grade evidence that homozygosity for FVL in family members is 

predictive of VTE. 

• There was moderate-grade evidence that heterozygosity for FVL in family members is 

predictive of VTE. 

• The evidence was insufficient to allow us to draw any conclusions about the predictive 

value of heterozygosity for prothrombin G20201A in family members. 

• There was low-grade evidence that double heterozygosity (for FVL and prothrombin 

G20210A) in family members is predictive of VTE. 

• There was low-grade evidence that homozygosity for FVL in pregnant family members is 

predictive of VTE. 

• The evidence was insufficient to allow us to draw any conclusions about the predictive 

value of heterozygosity for FVL in pregnant family members. 

• Homozygosity for prothrombin G20210A is a rare genotype, and its association with 

recurrent VTE in family members is unknown, because of the lack of sufficient evidence. 
63 

• The evidence was insufficient to allow us to draw any conclusions about the predictive 

value of heterozygosity for prothrombin G20210A in pregnant family members. 

• The evidence was insufficient to allow us to draw any conclusions about the predictive 

value of double heterozygosity for FLV and prothrombin G20210A in pregnant family 

members. 

Key Question 4 

Clinical Utility of Testing for FVL 

and/or Prothrombin G202010A 

We identified 10 studies addressing the clinical utility of testing for these mutations. The 

studies varied substantially in their methodology and design, sources of the participants, 

geographical locations, and outcomes. We summarize our findings, including our assessment of 

the quality of these studies, according to the four main sub-questions of KQ4 (Tables 10-12). 

Effect of Testing on Clinicians’ Management (KQ 4a) 

We found a single study addressing how physicians’ management decisions are affected by 

FVL testing (Appendix G, Evidence Table 21).150 Rodger et al surveyed 662 Canadian 

obstetrical care providers (65 percent of whom were obstetricians, 28 percent family 

practitioners, and 5 percent perinatologists), about their management recommendations in 

response to four clinical scenarios involving pregnant women with a history of FVL. The survey 

asked whether the practitioners would recommend peripartum thromboprophylaxis and whether 

they would refer the patient to a hematologist or to another specialist. The survey response rate 

was 46 percent (1,448 members of the Society of Obstetricians and Gynecologists of Canada had 

been invited to participate); 57 percent of the respondents were male. For scenarios involving 

asymptomatic women, the recommendation for thromboprophylaxis almost doubled, from 26-34 

percent to 58 percent, if the patient was described as having a family history of VTE. For 

scenarios involving symptomatic women, 60-68 percent of clinicians recommended prophylaxis 

for women with recurrent fetal loss, and 83-84 percent of clinicians recommended it for women 

with a personal history of VTE. Uncertainty was prevalent regarding the use of prophylaxis: 14 

percent of clinicians were unsure as to whether or not to recommend prophylaxis when the 

patient had a history of prior VTE, and 29 percent were unsure in cases in which the patient had 

a family history of VTE or recurrent fetal loss. More than 80 percent of clinicians would refer a 

pregnant woman with a history of FVL to a specialist. This study suggests a high degree of 

uncertainty regarding the management of pregnant patients with a history of FVL, with clinicians 

reporting a preference to recommend prophylaxis and referral, particularly in cases of 

symptomatic women. 

Effect of Testing and Resultant Mnagement on VTE-related Otcomes 

(KQ 4b) 

 There were four studies yielding VTE recurrence rates among probands with FVL or 

prothrombin G20210A during anticoagulation therapy (Table 10, Appendix G, Evidence Tables 
64 

22-24).122,123,125,151 Each was a subgroup analysis; three involved individuals participating in 

randomized controlled trials,123,125,151 while the fourth was nested within a prospective cohort 

study.122 Three of the four studies included participants with FVL or prothrombin G20210A,123, 

125,151 while the fourth addressed only FVL.122 Two studies investigated the effect of warfarin on 

recurrence rates,125,151 one the effect of ximelagatran,123 and one did not specify the treatment 

that patients received.122 None of these studies was designed to examine how testing effects 

changes in management or whether choosing treatment based on test results alters outcomes. 

As required for inclusion in this report, all index events and recurrences were objectively 

documented. There were quality flaws across the studies, including inadequate description of 

how patients were recruited, raising the possibility of selection bias (Appendix G, Evidence 

Table 20). Only one of the studies described the number of patients lost to follow-up.122

Although some of the studies had stringent inclusion and exclusion criteria, for the most part 

these were the criteria for safely using these drugs. Ridker et al assessed thromboembolism 

recurrence rates among individuals with FVL or prothrombin G20210A who received either a 

low-intensity warfarin regimen (international normalized ratio [INR] target between 1.5 and 2.0) 

or placebo151 (Table 10). Each participant had experienced a prior unprovoked VTE and had 

completed at least 3 months of uninterrupted treatment with warfarin. The patients randomized to 

the placebo and treatment groups had similar demographic and clinical characteristics. Of 77 

patients with FVL or prothrombin G20210A in the placebo group, 14 had recurrences (8.6 events 

per 100 person-years), as compared to 3 of 66 such patients assigned to the low-intensity 

warfarin group (2.2 events per 100 person-years). Low-intensity warfarin reduced the rate of 

recurrence among thrombophilic patients by 75 percent (HR, 0.25; 95 percent CI, 0.07 to 0.85). 

This risk reduction, however, was not significantly different than the 58 percent reduction seen in 

patients without either mutation (HR, 0.42; 95 percent CI, 0.20 to 0.86; p-value for the 

interaction, 0.51). These results suggest that low-intensity warfarin significantly decreases the 

rate of recurrence of VTE in patients with FVL or prothrombin G20210A; however, this 

protective effect was not different than that observed for individuals without the mutations. 

Kearon et al asked whether VTE recurrence rates differed among patients with and without 

FVL or prothrombin G20210A who were receiving either low-intensity (INR goal, 1.5-2.0) or 

conventional (INR goal, 2.0-3.0) warfarin therapy.125 All patients were participants in the 

Extended Low-intensity Anticoagulation for Unprovoked Thromboembolism (ELATE) trial and 

were separately invited to participate in this study. Among 171 patients with FVL, three had 

recurrences. No recurrences were seen in the 60 patients with prothrombin G20210A. The 

recurrence rate for patients with FVL while receiving low- or conventional-intensity warfarin 

therapy was 0.8 percent per year (95 percent CI, 0.2 to 2.2). This rate was not statistically 

different than the rate among those participants without FVL (HR, 0.7; 95 percent CI, 0.2 to 2.6). 

The HR for recurrent VTE in the low-intensity warfarin group as compared to the conventionalintensity group was 2.7 among those with thrombophilia (not just FVL or prothrombin 

G20210A; 95 percent CI, 0.5 to 14), and 5.6 in those without thrombophilia (95 percent CI, 0.7 

to 47; p-value for interaction, 0.6). These results were not reported separately according to 

mutation. 
65 

Table 10. Four studies examining the effect on VTE-related outcomes of changes in therapy based on testing for FVL or prothrombin G20210A 

Author, year Type of Study Population Sample Size Outcome of Interest Key ConcluRidker, 2003151 Randomized 

controlled trial 

• FVL or PT 20210A 

(pooled) 

• Prior unprovoked 

VTE 

• Had 3 months of 

uninterrupted warfarin 

• United States-based 

study 

• Age range 46-65 • 53% males • Mean follow-up 2.1 

years (maximum 4.3) 

508 participants: 253 assigned to placebo (26.6% FVL carriers, 4.8% PT G20210A carriers), 255 assigned to low-intensity warfarin (22.0% FVL carriers, 4.7% PT G20210A carriers) 

• VTE recurrence while 

treated with low-intensity warfarin 

• VTE recurrence rate among 

carriers 

Low-intensitrecurrence i(HR, 0.42; 9reduces rec(HR, 0.25; 9

Kearon, 2008125

Cohort analysis nested within randomized controlled trial 

• FVL or PT 20210A • Prior VTE • Had 3 months of 

uninterrupted warfarin 

• Canada-based 

study 

• Mean age, 57 (SD, 

15) 

• 54-58% males • Mean follow-up 2.3 

years 

661 participants (pooled): 337 assigned to low-intensity warfarin (91 FVL, 32 PT G20210A), 324 assigned to conventional-intensity warfarin (80 FVL, 28 PT 20210A) 

• VTE recurrence while 

treated with either lowintensity or conventionalintensity warfarin 

• VTE recurrence rate among 

carriers, as compared to non-carriers 

Conventionareduces recto low-intenrecurrence wamong the pintensity thepatient-year allocated to therapy (HR13.2). The afor FVL carrwas 0.8% pand was lesdefect (HR 0

65 
66 

Table 10. Four studies examining the effect on VTE-related outcomes of changes in therapy based on testing for FVL or prothrombin G20210A (c

Author, year Type of Study Population Sample Size Outcome of Interest Key ConcluWahlander, 2006123 Cohort 

analysis nested within randomized controlled trial 

• FVL or PT 20210A • Prior VTE • Had completed 6 

months of uninterrupted warfarin 

• European-based 

study 

• 18-month study 

period 

1223 participants: 612 oral ximelagatran (111 FVL, 27 PT 20210A), 611 placebo (125 FVL, 27 PT G20210A) 

• VTE recurrence while 

treated with ximelagatran 

• VTE recurrence rate among 

carriers 

Oral ximelagin non-carrie(Figure 2, H

Vossen, 2005122

Prospective cohort 

• FVL or combined 

FVL-PT 20210A 

• Prior VTE • European-based 

study 

• Age range 14-78 • 35-48% males • Followed for 1710 

person-years (mean, 5.6; range, 1-7) 

304 participants: 124 on long-term anticoagulation (13 FVL, 1 combined FVLPT 20210A), 180 not on long-term anticoagulation (79 FVL, 7 combined FVLPT G2021A) 

• VTE recurrence while 

treated with long-term anticoagulation 

• VTE recurrence rate among 

carriers 

Long-term arecurrence irecurrence wyear among anticoagulatamong the panticoagulatThe rate of carriers not anticoagulatno events in anticoagulat

CI = confidence interval; FVL = Factor V Leiden; HR = hazard ratio; PT G20210A = prothrombin G20210A; SD = standard deviation; VTE = venous thr

66 
67 

Wahlander et al. assessed the risk of VTE recurrence in relation to thrombophilic factors in 

probands receiving ximelagatran, an oral direct thrombin inhibitor (not presently available), or 

placebo.123 This study involved a group of participants recruited from the Thrombin Inhibitor in 

VTE (THRIVE III) trial.152 Among 111 FVL carriers in the treatment group, two had recurrences 

of VTE, as compared to 16 in the 125 with mutations assigned to placebo; this difference in 

recurrence rates was described as statistically significant (HR under 0.25; p-value not reported). 

The recurrence rate with treatment was very similar for individuals without FVL, suggesting that 

there was no interaction between FVL and the effect of ximelagatran in preventing recurrent 

VTE (p-value for the interaction, 0.92). Among 27 prothrombin G20210A carriers in the 

treatment group, none experienced a recurrence, while 2 of 27 carriers in the placebo group had a 

recurrence (HR not calculable). Recurrence rates with treatment were very similar for individuals 

without FVL or prothrombin G20210A, suggesting that there was no interaction between FVL or 

prothrombin G20210A and the effect of ximelagatran in preventing recurrent VTE (p-value for 

the interaction, 0.92 for FVL and 0.98 for prothrombin G20210A). There were no significant 

differences in the risk of bleeding in carriers of FVL or prothrombin G20210A when compared 

to non-carriers (p-value for the interaction, 0.60 for FVL and 0.16 for prothrombin G20210A). 

Vossen et al studied the rate of recurrence of VTE in a cohort of patients belonging to 

thrombophilic families, classified according to whether they were receiving long-term 

anticoagulation or not.122 They focused on probands with a family history of VTE because of the 

potentially higher rates of recurrence among these individuals. All patients were recruited as part 

of the EPCOT trial. The mean follow-up was 5.6 years (range 1 to 7 years). Of 304 patients, 124 

were on long-term anticoagulation, and 180 were not. The details of the anticoagulation 

regimens were not reported. The proportion of women was higher in the anticoagulation group 

(48 versus 35 percent). There were fewer FVL carriers in the long-term anticoagulation group 

than in the comparison group (13 and 44 percent, respectively). Of 79 patients with FVL who did 

not receive long-term anticoagulation, 13 had recurrences during 366 person-years (incidence 

rate, 3.5 percent per year; 95 percent CI, 1.9 to 6.1). Among the 13 FVL carriers who received 

anticoagulation, none had any recurrence during 43 person-years. This study showed that longterm anticoagulation decreased the rate of VTE recurrence among probands belonging to 

thrombophilic families. However a quantitative estimate was not calculable because there were 

no events in the group of patients receiving treatment. Confidence in the study’s results is low 

because this study was observational, and there were important differences in the baseline 

characteristics and duration of follow-up in the two groups. Also, there was a paucity of detail 

about the anticoagulation regimens. 

Effect of testing and results on other outcomes (KQ 4c) 

Four studies addressed how probands’ and family members’ knowledge, behaviors, and 

healthcare experiences were affected by their being tested for FVL or prothrombin G20210A;153-

157 only one study evaluated individuals with prothrombin G20210A153 (Table 11 and Appendix 

G, Evidence Table 21). Two publications by Saukko et al. investigated the same population of 

patients but provided complementary information and are considered as one study here.155,156

Three studies were conducted in Europe154-157 and one in Canada.153 

Two studies employed cross-sectional surveys of convenience samples of probands and 

family members to assess risk perception and behavioral effects following genetic testing.153,154

The other two studies used qualitative, structured interviews of probands and relatives to 
68 

Table 11. Studies examining the effect of FVL and prothrombin G20210A testing on non-VTE-related outcomes AT = antithrombin deficiency; EPCOT = European Prospective Cohort on Thrombophilia; FVL = Factor V Leiden; PC = protein C deficiency; PS = p20210A = prothrombin G20210A; VTE = venous thromboembolism 

Author, year Type of Study Population Sample Size Outcome of Interest Key ConclBank, 2004157 Qualitative, 

structured interview 

• Asymptomatic first-degree 

relatives of probands 

• Tested for FVL • In the Netherlands • Age, 26-56 years • 53% males 

17 participants (65% response rate) 

Overall experience with testing; effects of testing on daily life 

• For most

received FVL testi

• More con

health tha

• No chang

majority o

Teutsch, 2008156 and Saukko, 2007155

Qualitative, structured interview 

• Probands or relatives 

referred for FVL testing 

• In the United Kingdom • 12% males 

42 participants (43% response rate) 

Understanding of testing process and implications of results on daily life 

• Most did 

to be diffeordered b

• Less und

of lower s

• Most did 

as a resu

Kaptien, 2008154

Cross-sectional survey 

• Probands and relatives 

recruited from EPCOT study 

• Tested for FVL, AT, PC, or 

PS deficiency 

• In Europe (multiple 

countries) 

• Age, 26-87 years • 46% males 

174 participants (78% response rate) 

VTE risk perception and disease-related worry 

• Increased 

worry am

• Level of w

FVL loweand simila

Heshka, 2008153

Cross-sectional survey 

• Asymptomatic first-degree 

relatives of probands, not on long-term anticoagulation 

• Tested for FVL or PT 

20210A 

• In Canada • Age, 21-78 years • 38% males 

70 participants: 44 carriers (86% response rate) 26 non-carriers (55% response rate) 

Perception of VTE risk and changes in behavior following FVL or PT 20210A testing 

• No signifi

recognitiobetween despite c

• Behavior 

testing wegroups 

68 
69 

describe their experience during the process of testing as well as their interpretation of the 

results.155-157 In all studies, except that of Saukko et al,155,156 subjects were recruited from 

cohorts already enrolled in larger trials investigating venous thromboembolic outcomes in 

probands or relatives. 

The quality of these studies was generally high (Appendix G, Evidence Table 20). Although 

all four studies used a cross-sectional design and looked at convenience samples of probands or 

relatives, the methods employed were appropriate to answer the research questions. The surveys 

used instruments that had been previously tested in patients with genetic mutations or that had 

been validated to measure specific constructs such as worry or risk perception.153,154 Both of the 

qualitative studies used appropriate data collection methods, including the exploration and 

exhaustion of predetermined themes, the use of trained interviewers, and the use of quotes to 

represent participants’ voices.155-157

Heshka et al surveyed the perception of VTE risk and changes in behavior following testing 

for FVL or prothrombin G20210A among first-degree relatives of probands.153 The survey 

response rate was 86 percent for relatives who carried the FVL mutation and 55 percent for noncarriers; otherwise, the 44 carriers and 26 non-carriers were similar in age, education, and 

income. More mutation carriers recognized trauma as a risk factor for VTE than did non-carriers, 

but otherwise there were no statistically significant differences between the two groups regarding 

their recognition of risk factors for VTE, despite the carriers having received counseling after 

testing. Behavior changes following testing were uncommon in both groups, but carriers were 

slightly more likely to have tried to avoid long trips since testing, to have stopped using oral 

contraceptive pills or hormone replacement therapy, to have discussed the results of the test with 

their surgeons or gynecologists, or to have been placed on anticoagulants during surgery or 

during or after childbirth. The results of this study suggest that testing for FVL and prothrombin 

G20210A and sharing the results with patients do not lead to a substantial change in risk 

perception or an important increase in preventive health behaviors. 

Kaptein et al investigated whether the type of thrombophilic mutation and history of VTE 

affected the perception of risk and level of worry among probands or their relatives with FVL, 

when compared to other thrombophilic mutations.154 The core of the survey tool was a version of 

the Illness Perception Questionnaire adapted for patients with thrombophilia. Of the 251 

individuals invited, 196 (78 percent) responded. Of these, 62 individuals had only FVL (no 

protein C, protein S, or antithrombin deficiency). This group included both relatives and 

probands, with and without a history of VTE. Overall, patients with a history of VTE had an 

increased perception of risk and worried more about VTE than did individuals without prior 

VTE. After controlling for age and prior VTE, patients with FVL had a significantly lower 

perception of risk of VTE than did individuals with antithrombin deficiency but a similar risk 

perception when compared to patients with protein C or S deficiency. Level of worry about 

thrombosis was similar across all thrombophilia groups. This study suggests that, among carriers 

of FVL, the level of worry is similar to that of carriers of other thrombophilic mutations, 

although individuals with antithrombin deficiency (which has the highest VTE risk of the four 

measured thrombophilias) had a higher perception of risk than did patients with the FVL 

mutation. However, worry and risk perception were not measured in non-carriers; therefore, 

although it appears that having the FVL mutation does not increase risk perception and worry 

beyond the levels seen with other relatively common thrombophilic mutations, this study did not 

specifically address how the testing process and knowledge of results affects individuals tested 

for FVL. 
70 

Bank et al conducted a qualitative study of asymptomatic relatives of probands with FVL to 

assess their overall experience with the testing process and how the results affected their daily 

lives.157 Of 26 relatives invited, 17 (65 percent) agreed to be interviewed. The topics covered 

during the open-ended interviews included stigmatization, perception of risk, and preventive 

behaviors. Among the participants, most found that the testing process itself was not stressful; all 

had received written information about the test prior to testing. Most exhibited knowledge of the 

most common risk factors for VTE (e.g., pregnancy, surgery), citing as the source of this 

knowledge the information provided during testing and the information obtained from providers 

after receiving the test results. Some participants related an increased level of worry about 

developing VTE, while some felt stigmatized by friends, family, or even their providers, who 

saw them as having a health condition. One person in the Netherlands who disclosed his test 

results to his employer was discriminated against in his disability insurance policy. Although the 

majority of participants indicated that testing had not altered their daily lives, many wanted to 

screen their children to decrease their risk of VTE from pregnancy or oral contraceptive use. This 

study suggests that although, overall, no drastic changes in mood or behavior were observed 

among individuals tested for FVL, testing generated concern about the individuals’ own health 

and the health of their children. The authors argued that the information provided on the day of 

testing and after the results were given played a crucial role in lessening patients’ concerns and 

improved their testing experience. 

Saukko et al. assessed the level of understanding of the testing process and the implications 

of the results among probands and relatives referred for FVL testing by their primary care doctor 

or specialist.155,156 The study, which was carried out in the United Kingdom, involved structured 

qualitative interviews of 42 individuals (43 percent of the 97 invited to participate). The themes 

explored during the interviews included reasons for testing, how the results were relayed to the 

participants, how they reacted to the results, what they did with the information, and what they 

thought of the testing process as a whole. Twenty of the 42 participants had been referred 

because of a family history of VTE or thrombophilia; 10 had a prior history of VTE; and 5 were 

referred because of prior miscarriage. Seven participants were unaware they had been tested. 

Sixteen participants tested positive for FVL, although the results were for the most part not 

reported according to carriership status. Most participants did not consider thrombophilia testing 

to be different from the other tests ordered by their providers, even if they understood the 

increased risk of VTE. 

The overall understanding of the FVL test and its implications fell along a spectrum: Most 

participants who understood the test well belonged predominantly to higher socio-economic 

strata, as compared to those with a poorer understanding of the test. Participants who received or 

obtained information about FVL prior to talking to their physicians (e.g., from relatives) were 

more likely to feel that the information given by their providers was sufficient or useful. Many 

did not receive written, detailed information about VTE risk factors or preventive behaviors from 

their providers when they were given their results. Participants also learned about the results of 

the tests in different ways, and this variability affected their behavior after receiving results. 

Those who were more informed about FVL sought information on their own, from relatives or 

the Internet, whereas the less-informed participants tended to assume that a lack of information 

from their providers meant that FVL was not an important condition. Most participants did not 

incorporate behaviors to reduce their risk for VTE into their daily routines as a result of the 

testing, although most participants who were aware of their positive status stated they had 
71 

undergone testing to inform their decision to take hormonal therapy or to advise relatives on the 

matter. 

These studies by Saukko et al. once again suggest that information given to patients at the 

time of testing affects their understanding of the process and the implications of the results.155,156

Furthermore, socio-economic factors such as income or literacy may potentially influence 

patients’ understanding of thrombophilia testing by limiting their access to supplementary 

information about the various tests. It is worth noting that although all patients were referred for 

FVL testing, many were actually referred for a thrombophilia panel, and so the attitudes or 

behaviors reported may not necessarily have been in response to FVL testing. 

Cost-effectiveness of FVL and Prothrombin G20210A Testing in the 

Case of Probands and Their Relatives (KQ 4d) 

We identified six studies that assessed the cost-effectiveness of genetic testing and resultant 

changes in management158-163 and one study that assessed only effectiveness.164 Six of the 

studies employed decision-analytic modeling, with a single decision giving rise to a series of 

multiple possible outcomes, each of which was weighted according to the probability of its 

occurrence.158,160-164 Of these six studies, only one did not incorporate a Markov state-transition 

model, a technique that permits the consideration of iterative events over time.158 The seventh 

study used a prospective cohort to derive cost and outcomes data, with which the authors 

retrospectively modeled the costs and effectiveness of hypothetical changes in management.160 

Four of the studies considered testing for FVL alone,160,161,163,164 while the remaining three 

evaluated testing for FVL along with prothrombin G20210A.158,159,162 Five of the studies 

modeled probands with a personal history of VTE,159,161-164 and the other two studies considered 

individuals with either a personal or family history of VTE.158,160

We first briefly describe the design, interventions, population, outcomes, results, 

conclusions, and quality deficits of each study (Table 12; Appendix G, Evidence Tables 25-27). 

In the tables, we present the results in 2007 U.S. dollars, although here in the text, we describe 

the results as reported in each paper. 

The seven studies were of variable quality. They all had a clear statement of the objective 

and perspective, although the primary decision-maker for whom the study was conducted was 

specified only in the case of the United Kingdom National Health Service.158,160 A societal 

perspective was stated twice, without the incorporation of any indirect costs.159,161 The structure 

of the models was generally stated clearly and justified, together with the assumptions 

underlying the structure, the interventions examined, the time horizon, and the disease states 

included. When used, the health state quality weights were clearly stated and referenced, but no 

study critically discussed the health valuation methodology or options available. Only two of the 

five Markov models reported the cycle length.162,163 Data were well identified, modeled, and 

incorporated, although only three studies undertook a systematic review with a meta-analysis to 

define at least one model parameter.158,162,163 All studies assessed parameter uncertainty with 

sensitivity or scenario analysis, but none of the studies addressed structural uncertainty, and only 

one explicitly conducted subgroup analysis.158 Finally, internal consistency (the methodology of 

the model development) was not discussed in any study, although all but two compared their 

results to previous models’ results, providing a measure of external consistency.160,163 
72 

Table 12. Summary of studies evaluating the cost-effectiveness of testing for FVL and/or prothrombin G20210A mutationsStudy Design 

Interventions Compared Population Outcomes Results* Quality D

Sarasin, 1998164

Decision analysis with a Markov model 

Extending oral anticoagulation up to 5 years vs. 3 

months oral anticoagulation for carriers and noncarriers of FVL 

Probands: Individuals having had a DVT and 3 months of anticoagulation 

DVT/PE prevented, major bleeding induced 

For those with FVL, for all durations of anticoagulation, the number of major iatrogenic bleeding episodes exceeded the number of PEs prevented, although the number of bleeding episodes was far less than the number of DVTs averted, by a ratio of 1:3 after 1 year, and decreased with longer duration. If the bleeding risk was <2.5% per year, continued anticoagulation was preferred. 

Structure model mafull treatmof quality made comoutcomes cycle lenData – thaddress mstructura

– internaconsisten

Marchetti, 2000163

CEA, decision analysis with a Markov model 

Screening for FVL with 2 years of warfarin vs. no 

screening with 6 months of warfarin 

Probands: 60-year-old males with a personal history of VTE 

Costs, QALYs, DVT/PE averted, major bleeding induced, life years 

Testing for FVL, followed by extended anticoagulation, was cost-effective ($15,451/QALY). ICER > $50,000/QALY for individuals with high bleeding risk (>0.34% per year), low VTE recurrence rate (<9% in first 2 years), low anticoagulation efficacy (<74%), or low anticoagulation compliance (<94%). 

Structure duration discussiojustificatioData – hadiscussenot addreinternal cdiscusse

Marchetti, 2001162

CEA, decision analysis with a Markov model 

Screening for double heterozygosity with 2 years of warfarin vs. no 

screening and 6 months of warfarin 

Probands: 60-year-old males with personal history of VTE 

Costs, QALYs, DVT/PE averted, major bleeding induced, life years 

Testing for both mutations, with subsequent extended anticoagulation for double heterozygous individuals, was cost-effective (ICER $15,959/QALY). ICER > $50,000/QALY for individuals with high bleeding risk (>1.6% in 6 months), low double-heterozygote prevalence (<1.4%), low PE mortality (<10%), or low anticoagulation efficacy (<65%). 

Structure duration discussiojustificatioData – hadiscussenot addreinternal cdiscusse

72 
73 

Table 12. Summary of studies evaluating the cost-effectiveness of testing for FVL and/or prothrombin G20210A mutations (continued)Study Design Interventions 

Compared 

Population Outcomes Results Quality 

Clark, 2002160

CEA, prospective cohort 

Universal FVL screening or selective FVL screening based on personal or family history of VTE (prophylactic enoxaparin for FVL carriers in both cases) vs. no screening 

Pregnant women attending antenatal care 

Costs, pregnancyrelated vascular complications 

$15,173/vascular complication prevented for selective screening with prophylactic anticoagulation relative to no screening; $26,744/vascular complication prevented for universal screening with prophylactic anticoagulation relative to no screening (not considered cost-effective). ICER for selective screening with 100% effective anticoagulant prophylaxis was $5758/vascular complication averted relative to no screening. 

Structurscope, ohorizon 

assumpefficacy justifiedtreatmenof life weof uncerparametefficacyassessmexternal 

Eckman, 2002161

CEA, decision analysis with a Markov model 

Screening for FVL with warfarin for 3 years or lifelong vs. no screening and 6 months of warfarin 

Probands: 35-year-old females with a personal history of VTE 

Costs, QALYs Testing and treating with 3 years of 

anticoagulation dominated (in base case); $20,937/QALY for testing and treating with lifelong anticoagulation relative to no screening with 6 months anticoagulation, with a constant VTE rate at 7.3%/year. Model very sensitive to rate of recurrent VTE. 

StructurclaimedhealthcaMarkov stated; Dcorrectioquality oonly cosstructuraaddressinternal address

73 
74 

Table 12. Summary of studies evaluating the cost-effectiveness of testing for FVL and/or prothrombin G20210A mutations (continued)Study Design Interventions 

Compared 

Population Outcomes Results Qua

Auerbach, 2004159 CEA, 

decision analysis with a Markov model 

Hypercoagulability testing with anticoagulation for 6 months to indefinite depending on results vs. no testing with anticoagulation for 6 months to indefinite 

Probands: 40-year-old following a DVT and 6 months of anticoagulation 

Costs, QALYs 

$13,365/QALY for testing followed by 24 months anticoagulation for positive individuals relative to no testing followed by 24 months anticoagulation for all individuals (base case). Cost-effective to test for disorders conferring a RR of recurrent VTE > 1.25 with a prevalence > 5%, followed by 2 years of anticoagulation for 2 years (or indefinitely if the relative risk exceeds 6). This would include testing for FVL and for double heterozygosity. 

StrupersdirecMarkunspcorreutility strucaddrinterdiscu

Wu, 2006158

CEA, decision analysis model 

Universal thrombophilia screening vs.

selective screening based on personal and family VTE history vs. no screening 

(1) women prior to OC use, (2) women prior to HRT, (3) pregnant women at 6 weeks of gestation, (4) patients prior to major orthopedic surgery 

Costs, adverse clinical events prevented 

Selective screening relative to no screening: $136,604/complication prevented in cohort 1, $4,226/complication prevented in cohort 2, $140,344/complication prevented in cohort 3 (not costeffective), $15,780/complication prevented in cohort 4. 

Struhorizhave treatabsemakoutcpregaggrdiffic

strucaddrinterdiscu

* Monetary values converted into U.S. dollars (2007) 

CEA = cost-effectiveness analysis; DVT = deep venous thrombosis; FVL = Factor V Leiden; ICER = incremental cost-effectiveness ratio; HRT = hor

OC = oral contraceptive; PE = pulmonary embolism; QALYs = quality-adjusted life years; RR = relative risk; VTE = venous thromboembolism 

74 
75 

Sarasin and Bounameaux used a decision analysis with a Markov model and a 5-year time 

horizon to assess the effectiveness of extending anticoagulation from 3 months for FVL carriers 

and non-carriers following an initial lower-limb DVT to 1, 2, 3, 4, or 5 years in a hypothetical 

cohort.164 Quality weighting was explicitly not employed, since the authors expressed doubt that 

sufficient evidence existed to make the adjustments “reliable.” Instead, the results were provided 

in terms of the health outcomes: major bleeding events induced and recurrent DVT and 

pulmonary embolisms (PE) prevented. The best-case scenario, in which the point estimates most 

favorable for extended anticoagulation were modeled from the range of values identified in the 

literature (a high rate of PE, maximum anticoagulant efficacy, and low bleeding risk), revealed 

that the number of bleeding events induced outnumbered the number of pulmonary emboli 

prevented across all extended anticoagulant durations among carriers and non-carriers of FVL. 

However, the number of DVTs prevented among FVL carriers greatly exceeded the number of 

bleeding events induced across all extended anticoagulant durations, and the incremental ratio of 

DVT prevented to bleeding events induced was highest for 2 years of anticoagulation. Assuming 

that PE are of equal importance with bleeding events, the authors stated that the risk of bleeding 

must be below 2.5 percent/year in order for prolonged anticoagulation to be the more effective 

strategy. A major limitation of this analysis is that it did not take into consideration differences in 

how patients might view these different types of complications. The time horizon of this model 

may not have captured the full treatment effects, and the Markov cycle length was unspecified. 

The authors did not address methodological or structural uncertainty or internal or external 

consistency. 

Marchetti et al used decision analysis with a Markov model and lifetime time horizon to 

assess the cost-effectiveness of testing for FVL with 2 years of warfarin anticoagulation for 

carriers following VTE in a hypothetical cohort of 60-year-old men.163 FVL testing with 2 years 

of anticoagulation for carriers was a cost-effective strategy (ICER = $12,833/QALY) when 

compared to no testing and 6 months of anticoagulation. However, this intervention was not costeffective (ICER > $50,000/QALY) for individuals with a high risk of fatal bleeding on warfarin 

(>0.34 percent/year), low VTE recurrence rate (<9 percent in first 2 years), low anticoagulation 

efficacy (<74 percent), or low anticoagulation compliance (<94 percent). Cost figures were 

derived from the Italian health system. Quality deficits are discussed with the next study. 

Building on their previous study, Marchetti et al. used the same model to assess the costeffectiveness of testing for prothrombin G20210A and FVL, followed by 2 years of warfarin 

anticoagulation, in doubly heterozygous individuals.162 Testing for both mutations was costeffective (ICER = $13,624/QALY) when compared to no testing and 6 months of 

anticoagulation, and if the testing costs were <$43, the testing strategy was cost-saving. Testing 

was not cost-effective (ICER > $50,000/QALY) for patient populations with a high bleeding risk 

(>1.6 percent in 6 months), low double-heterozygote prevalence (<1.4 percent), low pulmonary 

embolism mortality (<10 percent), or low anticoagulation efficacy (<65 percent). The quality 

deficits of this study apply to the previous study as well.163 The model structure examined a 

single anticoagulant duration, and the choice of this strategy was not discussed or justified. A 

half-cycle correction was not discussed, and structural uncertainty was not addressed. Internal 

consistency was not discussed. 

Eckman et al. used decision analysis with a Markov model and a lifetime time horizon to 

assess the cost-effectiveness of testing for FVL and extending warfarin anticoagulation for 3 

years or for life for carriers following a first VTE in a hypothetical cohort of 35-year-old 

women.161 The cost-effectiveness of extending the duration of anticoagulation was highly 
76 

dependent on the rate of VTE recurrence used in the model. Testing followed by 3 years of 

anticoagulation was the dominant strategy (more effective and less costly) in scenarios that 

assumed a high rate of recurrence (16.3 percent/year) for the first 3 years and no or low (2.3 

percent/year) recurrence beyond. If the rate of recurrence remained constant (7.3 percent/year), 

lifelong anticoagulation was the most cost-effective strategy (ICER = $16,823/QALY) when 

compared to no testing with 6 months of anticoagulation. Lifelong anticoagulation would be less 

cost-effective in patient populations with a low FVL prevalence, low risk of recurrent VTE, or 

risk factors for bleeding on anticoagulant therapy. Structurally, the authors claimed a societal 

perspective, although only direct healthcare costs were modeled, and the cycle length of the 

Markov model was not explicitly stated. A half-cycle correction was not mentioned, the quality 

of life weights were not justified, and only the costs were discounted. Structural uncertainty and 

internal consistency were not addressed. 

Auerbach et al. used decision analysis with a Markov model and lifetime time horizon to 

assess the cost-effectiveness of a hypercoagulability testing panel and warfarin anticoagulation 

for 6, 12, 18, 24, or 36 months, or for life, following an apparently idiopathic deep vein 

thrombosis in a hypothetical cohort of 40-year-old individuals.159 In the base case analysis, 

extending warfarin anticoagulation for 24 months following a positive test result was the most 

cost-effective option (ICER $11,100/QALY) when compared to the least costly option of not 

testing and treating for 24 months. Each test was evaluated separately in the hypercoagulability 

panel, and the authors concluded that tests detecting disorders present in at least 5 percent of the 

population that confer a relative risk exceeding 1.25, including FVL and prothrombin G20210A, 

should be included. The authors claimed a societal perspective but incorporated only direct costs, 

and the cycle length of the Markov model was unspecified. A half-cycle correction was not 

mentioned, the utility weights were not justified, and structural uncertainty was not addressed. 

Internal consistency was not discussed, although the results were discussed in relation to similar 

studies for individual hypercoagulable disorders. 

Clark et al. used the cost and outcomes data from a prospective cohort of 967 pregnant 

women in the United Kingdom to assess the cost-effectiveness of FVL testing and enoxaparin 

anticoagulant prophylaxis in preventing pregnancy-related vascular complications over the 8-

month time horizon from 12 weeks gestation to 6 weeks postpartum.160 All women were 

screened for FVL, but the results of the test were not disclosed to the women until the conclusion 

of the study. No women actually received anticoagulant prophylaxis, but the hypothetical impact 

of treating FVL carriers with an assumed efficacy of 50 percent was modeled ex post facto. 

While testing all women and treating the FVL carriers identified was the most effective 

approach, testing only those women with a personal or family history of VTE was a more costeffective approach (ICER = ₤7,535/vascular complication prevented) when compared to no 

screening or prophylaxis. The structure of the model was not described, and the scope, outcomes, 

and time horizon were not justified. Data were largely derived from the cohort, but the 

assumption of prophylactic efficacy was stated without discussion or justification. There was no 

discussion of treatment alternatives, and quality of life weighting was not used or discussed. 

There was also no assessment of internal or external consistency or uncertainty beyond the 

parameter of anticoagulant efficacy. 

Wu et al. used a decision analysis model with a 12-month time horizon to assess the costeffectiveness of universal or selective screening for FVL and the resultant changes in 

management for carriers in four cohorts with high VTE risk: women prior to combined oral 

contraceptive prescription, women prior to hormone replacement therapy, pregnant women at 6 
77 

weeks of gestation, and patients prior to major orthopedic surgery.158 FVL carriers in the first 

two cohorts did not receive hormonal therapy, and carriers in the second two cohorts received 3 

months of thromboprophylaxis in an effort to prevent adverse events (VTE or pregnancy-related 

vascular complications). Selective screening was based on a personal or family history of VTE. 

While universal screening was more effective in all cohorts, selective screening was more costeffective in all cohorts. Selective screening was most cost-effective when compared to no 

screening in the case of the hormonal therapy cohort (ICER = ₤2,447/VTE prevented), followed 

by the surgery cohort (₤9,136/VTE prevented), and it was less cost-effective in the case of the 

oral contraceptive cohort (₤79,085/VTE prevented) and the pregnancy cohort 

(₤81,250/pregnancy-related vascular complication prevented). The short time horizon of the 

model may not have captured the full treatment effects, and the absence of quality weights made 

comparing the health outcomes difficult. Structural uncertainty was not addressed, and internal 

consistency was not discussed. 

Summary of the evidence for Key Question 4 

We summarize the evidence regarding the clinical utility of testing for FVL and prothrombin 

G20210A as follows: 

• Low-grade evidence indirectly supported the hypothesis that patient management by 

physicians may change on the basis of the results of testing for FVL or prothrombin 

G20210A. 

• There was no direct evidence that testing for these mutations, and the resultant 

management, can reduce VTE related-outcomes in individuals who have had VTE or in 

the probands’ family members who have been tested.

• There was moderate-grade evidence that treatment can reduce recurrent events in patients 

with FVL or prothrombin G20210A; however, the magnitude of this relative reduction was 

comparable to that seen in individuals without mutations. 

• There was moderate-grade evidence that neither harms nor benefits have been 

conclusively demonstrated in individuals with VTE or their family members when tested 

for FVL or prothrombin G20210A. 

• There was only low-grade evidence, coming from models, to suggest that testing for FVL 

alone, prothrombin G20210A alone, or the two tests in combination is cost-effective when 

caring for selected patients with VTE (those with a high risk of recurrence and/or low risk 

of bleeding) or their family members. 
79

Chapter 4. Discussion 

Our literature review was designed to identify evidence to inform our Key Questions. We 

identified literature that was directly applicable to answering the questions about analytic and 

clinical validity (Key Questions 2 and 3), but these studies were only indirectly relevant to the 

overarching question (Key Question 1). The literature was also less directly applicable to 

addressing the question about the clinical utility of testing for these mutations (Key Question 4). 

Key Question 1: 

Association of FVL Testing, Alone or in Combination With 

Prothrombin G20210A Testing, With Improved Clinical 

Outcomes in Adults With a Personal History of VTE or in 

Adult Family Members of Mutation-positive Individuals. 

We found no evidence that directly informed our overarching question (Key Question 1): 

Does FVL testing, alone or in combination with prothrombin G20210A testing, lead to improved 

clinical outcomes (e.g., avoidance of a recurrent VTE) in adults with a personal history of VTE 

or to improved clinical outcomes (e.g., avoidance of an initial VTE) in adult family members of 

mutation-positive individuals? In our discussion of Key Question 4, below, we address the 

implications of the absence of direct evidence, the value of indirect evidence, and the 

implications for future research. 

 One recent study that did not meet our inclusion criteria nevertheless provides some 

information to answer this overarching question.165 We could not include this study by Coppens 

et al. in our review because the patients had a variety of different thrombophilic conditions for 

which they were tested. Using a case-control design, the authors investigated whether 

thrombophilia testing reduced the rates of recurrent VTE among persons with a history of VTE. 

From a cohort of patients enrolled in the Dutch-based Multiple Environmental and Genetic 

Assessment (MEGA) trial, 197 patients with VTE recurrence and 324 without recurrence were 

studied. Physicians had ordered thrombophilia testing in 35 percent of cases and 30 percent of 

controls. The odds ratio for recurrence in the tested versus the non-tested patients was 1.2 (95 

percent CI, 0.8-1.9) and changed little after adjusting for age, sex, and VTE risk factors. These 

results suggest that thrombophilia testing did not affect the rate of VTE recurrence. 

Key Question 2: 

Analytic Validity of Tests to Identify FVL and 

Prothrombin G20210A Mutations 

An assessment of the analytic validity of a genetic test refers not only to the test’s accurate 

identification of genotypes but also its reliability and robustness. Many of the studies we 

reviewed were preclinical (and pre-commercial) evaluations of assays. The majority of 

laboratories in the United States still use PCR-RFLP or invader chemistry technology, and less 

frequently the assays described in this report. The studies we reviewed demonstrated the high 

analytic validity of both the commercially available and pre-commercial tests. Most of the 
80

discordant results resolved with repetition of the respective test, suggesting that operator or 

administrative errors were responsible for the discordant results. 

The majority of the studies reviewed used the well established and commonly used PCRRFLP or AS-PCR as the reference (gold) standards. There is a theoretical concern that these nonsequence-based methods may not distinguish between the single nucleotide mutations of interest 

(e.g., FVL) and other nearby benign polymorphisms and could potentially yield false-positive 

results. For example, a rare silent A1692C Factor V polymorphism would be mistakenly 

genotyped as a FVL allele in an RFLP assay. We saw false-positives attributable to this 

mechanism only very infrequently in the studies we reviewed. 

In the quality assurance studies, we found evidence that most laboratories are highly 

accurate, even perfectly accurate, when asked to classify a sample with a known mutation. The 

majority of errors came from a limited numbers of laboratories. As we described, among the 

responding laboratories in one study, 51 percent made at least 1 error; however, three of the 39 

laboratories were responsible for 46 percent of all errors.63 We suspect that this situation 

illustrates a systematic quality assurance defect in these isolated laboratories. We did not review 

the evidence about how to identify laboratories that may perform poorly. We suggest that our 

findings underscore the need for ongoing internal and external quality control programs. 

There are abundant technologies that can be used to detect the FVL and prothrombin 

G20210A mutations. Given the almost equal analytic validity of these methods, considerations 

such as shorter turn-around time, cost-effectiveness, high throughput, and availability of userfriendly software become important factors in selecting one method over another. The choice of 

methodology will likely be driven by considerations other than analytic validity.

Key Question 3: 

Clinical Validity of Testing for FVL and Prothrombin G20210A 

Mutations 

Testing Probands 

We found moderate-grade evidence that individuals with at least one prior thrombotic event 

who are homozygous or heterozygous for FVL have a higher risk of recurrent VTE than do those 

without the mutation. For heterozygous individuals, the odds ratio was 1.56 (95 percent CI, 1.14-

2.12); for homozygous individuals, it was 2.65 (95 percent CI, 1.2-6). 

There was moderate-grade evidence that prothrombin G20210A is not predictive of recurrent 

thrombosis. The odds ratio was 1.45 (95 percent CI, 0.96-2.2), similar to that for heterozygous 

FVL, but the confidence interval overlapped 1.0. There were too few pieces of data to allow us to 

refute or support the contention that homozygosity for prothrombin G20210A, a rare condition, 

is associated with recurrent thrombosis. 

There was insufficient evidence that double heterozygosity (FVL plus prothrombin 

G20210A) is predictive of recurrent thrombosis. The pooled odds ratio was 4.8, but the 

confidence interval was wide, with very few studied individuals having double heterozygosity. 

When we separately evaluated patients with idiopathic VTE as the index event, we found that 

the odds ratio associated with heterozygous FVL was close to one (1.17; 95 percent CI, 0.63-

2.18), suggesting that that there may be little predictive value in knowing the mutation status in 

patients with idiopathic events. 
81

There were limitations in this body of evidence, although the quality of the studies overall 

was fairly high. There was often insufficient description of potential confounders of the 

relationship between the mutation and the recurrent event. However, in those studies employing 

multivariate models that included other potential predictors of recurrent thrombosis, statistical 

adjustment did not result in any attenuation of the mutation-specific effect size,14, 111,116,120,127

suggesting that these other clinical variables were not driving the relationship between mutation 

status and recurrent thrombosis. Although not all of the studies were primarily designed to 

answer our question about risk among probands, the prospective design that we required for 

inclusion in this review is the optimal design for answering this question. 

Testing Family Members

We found high-grade evidence that family members of probands who are homozygous for 

the FVL mutation have a substantially increased risk of VTE when compared to family members 

who do not have this mutation. Homozygous family members may have a thrombosis rate nearly 

20-fold that of individuals without homoyzgosity, or an event rate of 2 percent per year. There 

was moderate evidence that the risk of an event for heterozygous individuals is increased roughly 

three-fold (odds ratio, 3.4; 95 percent CI, 2.4-4.9). The absolute increase in the rate of events, 

however, was low. 

We saw that the annualized rate of venous thromboembolic events for family members 

without a mutation was approximately 0.1 percent per year. This value translates to an event rate 

in heterozygous family members of 0.3 percent per year, or an absolute increase of 0.2 percent 

per year (a change from an average of 1/1000 person-years to 3/1000 person-years). 

There was insufficient evidence regarding family members who are heterozygous for 

prothrombin G20210A, with an odds ratio of 1.9 (95 percent CI, 0.35-10). Doubly heterozygous 

individuals, with one FVL and one prothrombin G20210A mutation, can be expected to have 

event rates that are higher than those for singly heterozygous individuals but lower than those of 

family members who are homozygous for FVL (odds ratio, 6.7; 95 percent CI, 2.9-15) This 

conclusion was supported by only low-grade evidence. There was little information about the 

rare condition of homozygosity for the prothrombin G20210A mutation. 

There was insufficient evidence to allow us to draw any conclusions about the risks 

associated with heterozygosity in FVL among pregnant family members. The point estimates 

were above one in both studies, but with very wide CIs. Because pregnancy and the postpartum 

period are high-risk times for VTE, the absolute increase in risk with pregnancy is likely to be 

much greater than the absolute risk increase from FVL. We suspect that the risk attributable to 

FVL during pregnancy is very small. There was only low-grade evidence that homozygosity for 

this mutation may increase the risk of venous thrombosis in pregnancy beyond that which is 

usually seen. The evidence concerning women who are doubly heterozygous for FVL and 

prothrombin G20210A was insufficient, and we could not draw any conclusions about the risk. 

We reviewed one additional informative study that could not be included because the pregnant 

women were either those with a personal history of thrombosis or a family history of thrombosis 

or a family history of thrombophilia. This study suggested that double heterozygosity does 

modestly raise the risk to women during their pregnancies.166 
82

Key Question 4 

Clinical Utility of Testing for FVL and 

Prothrombin G20210A Mutations 

Effect of Testing on Clinicians’ Management 

There was low-grade evidence that patient management by clinicians is altered on the basis 

of the results of FVL testing, although there was no evidence to indicate whether this action 

improves patient outcomes or not. No information was available on management practices 

resulting from testing for prothrombin G20210A.We identified a single study addressing this 

question, which assessed practitioners’ responses when presented with clinical scenarios.150 The 

evidence to address this question can be considered to be only indirect, as this study asked 

clinicians to respond to hypothetical cases; there was no observation of practice patterns. This 

study was performed in Canada, where clinicians’ management decisions may be influenced by 

the availability of resources and the health policy environment specific to that country. Also, all 

of the scenarios described pregnant women, and therefore the results may not be applicable to 

other patient populations. 

 We identified one additional study published after the end of our search period.167 In this 

study by Hindorff et al., 112 primary care physicians in Washington state (60 frequent and 52 

infrequent prescribers of FVL testing) responded to a survey about their motivations for testing 

for FVL (response rate of 67 percent). Approximately 82 percent of the providers indicated they 

would order FVL in order to advise patients about VTE recurrence, while 67 percent would order 

the test to make decisions about VTE treatment or prevention. Fewer than 40 percent of the 

clinicians reported a high level of confidence in interpreting or communicating the results of 

FVL testing, or a high degree of confidence in determining when it is appropriate to order the 

test. As in the study by Rodger et al,150 this study did not provide direct evidence that clinicians 

manage patients differently based on FVL test results. However, the results suggest that 

physicians ordering this test may use results to inform management decisions. This study also 

highlighted a significant level of uncertainty regarding when to order FVL testing as well as how 

to interpret the results. 

Effect of Testing and Resultant Management on VTE-related 

Outcomes 

We conclude that there is no direct evidence that testing for FVL and prothrombin G20210A, 

and the resultant management, reduce VTE related-outcomes. In our search for indirect evidence 

to support or refute this hypothesis, we found high-grade evidence that anticoagulation reduces 

recurrent events in patients with FVL or prothrombin G20210A. However, there was only lowgrade evidence that the magnitude of this relative reduction in outcomes is comparable to that 

seen in individuals without these mutations. Thus, the mutation status of the patient apparently 

does not, of necessity, play a role in the decision to extend anticoagulation in a patient with a 

history of VTE. This conclusion is based on four studies, none of which was specifically 

designed to directly answer the question about testing or treatment changes based on testing. The 

studies did not describe bleeding associated with anticoagulation that was stratified according to 

mutation. 
83

The four studies we identified were heterogeneous in their designs and treatments. This body 

of evidence is limited by the relative lack of data on patients with prothrombin G20210A. 

Although Ridker et al. included individuals with prothrombin G20210A, these authors pooled 

them with the individuals having FVL for the analysis, so it was not possible to assess the effect 

of each mutation on the relationship between treatment and outcome.151 Also, some important 

safety outcomes were not analyzed according to the type of mutation of the participants. Of the 

three studies that addressed the risks associated with anticoagulation,122,123,151 only one reported 

these results as a function of mutation status.123

Effect of Testing and Results on Other Outcomes 

We found moderate-grade evidence that little change in knowledge and behavior results from 

testing for FVL or prothrombin G20210A. Four studies directly answered this question; each 

study was designed to explore how FVL or prothrombin G20210A testing affected nonthromboembolic outcomes. The main themes from these studies were: (1) an individual’s 

understanding of the risk factors for VTE or the significance of the test results was not improved 

after testing, unless structured counseling as well as access to information before and after 

testing was provided; (2) daily life changes were uncommon, although some patients used the 

test results to make important medical decisions; (3) most individuals did not regard carrier 

status as a serious condition but tended to worry about the implications for their children and 

relatives. We conclude that there is moderate evidence that the process of testing for these 

mutations does not have serious adverse consequences but may possibly improve understanding 

of VTE risk factors. 

The main limitation of these studies was that they were all performed outside the United 

States. The political and cultural factors in these countries, as well as the healthcare environment, 

may have affected how the patients experienced genetic testing, interpreted the results, or 

behaved in response to those results. Another limitation of this body of evidence is that all of the 

studies involved self-selected participants who were interested in the research question, a 

situation that could have skewed the study results. Only one study reported how outcomes 

differed based on the test result. It may be useful for clinicians to know how patients are affected 

by the results of the test, and not just by the process of testing. 

Two other important limitations were the paucity of studies addressing outcomes for patients 

undergoing prothrombin G20210A testing and the fact that all of the results were exclusively 

based on patient perceptions and behaviors. No study (except for the one by Wahlander et al.123) 

addressed clinical outcomes arising from changes in management as a consequence of testing 

(such as bleeding, mortality, or hospitalization rates) or quantifiable nonclinical outcomes, such 

as cost to patients or utilization of healthcare services. 

Cost-effectiveness of FVL and prothrombin G20210A testing in the 

care of probands and their relatives 

The cost-effectiveness studies all used decision analytic models. These models can point to 

the need for further testing of the utility of an intervention if the assumptions in the models are 

compatible with actual practice. The data ranges explored in the sensitivity analyses demonstrate 

the variables to which the cost-effectiveness of the interventions are most sensitive. 
84

The five studies that modeled the experience of probands following a VTE all suggested that 

testing for FVL alone or in combination with testing for prothrombin G20210A could be costeffective in certain patients.159,161-164 The strategy of testing for FVL alone161,163 or in 

combination with prothrombin G20210A,159,162 with extended anticoagulation for 2 or 3 years for 

identified carriers, was either the dominant or the most cost-effective option in patient 

populations having a high prevalence of the mutations (e.g., 5 percent or more), a high risk of 

recurrence (e.g., 10 percent or more within 2 years), and a low risk of bleeding. The models 

were not robust. They were extremely sensitive to the parameters chosen for the input, and we 

challenge some of the assumptions that were used for the base case analyses. 

The models’ results were most sensitive to the rate of venous thrombosis recurrence, the 

prevalence of FVL and prothrombin G20210A mutations, the risk of adverse bleeding events, 

and anticoagulant efficacy. We compared the model input to the summary results from our metaanalyses. In our analyses, the odds ratio for recurrence for individuals heterozygous for FVL 

relative to non-carriers was 1.56. This odds ratio is above the threshold suggested by Auerbach et 

al. at which a test for the mutation should be included in a hypercoagulability panel.159 Also, our 

pooled odds ratio was slightly higher than the value used in the model by Marchetti et al.; using 

our data for input would have more strongly favored FVL testing and extended 

anticoagulation.163

However, there are examples in these models of the use of parameters that seem flawed. One 

model made the assumption that there is an average risk of recurrence of 16 percent per year for 

the first 3 years, followed by 0 percent in subsequent years.161 These percentages were based on 

a single study with a rate of recurrence that we found to be an outlier.120 Similarly, one model 

assumed that prophylaxis against recurrent VTE with enoxaparin would be 50 percent 

effective.160 This assumption was based on a study of women with antiphospholipid syndrome 

and may not be applicable to women with FVL during pregnancy. 

Two studies considered the use of universal or selective testing for FVL160 with or without 

prothrombin G20210A158 in cohorts at high risk for thrombosis recurrence. The absence of 

quality-of-life weighting makes interpretation challenging. Without a way to standardize 

outcomes, the models’ results can only be meaningfully compared within a study, as in Wu et 

al,158 or across similar studies, as in the pregnancy cohorts of Wu et al 158 and Clark et al.160

Although these models shared many assumptions, the incremental cost-effectiveness ratios 

differed, presumably because of the different costs included in the models. However, selective

screening based on personal or family history of VTE was preferred in both studies, although the 

authors of both studies acknowledged that it may be challenging to obtain an accurate family 

history. 

While an incremental cost-effectiveness ratio must be interpreted carefully in the context of 

the study from which it is derived, the values obtained in these four studies, which ranged from 

$11,100 to $13,624/ QALY, were well within the range considered to be cost-effective in the 

economic evaluation literature. Lifelong anticoagulation was modeled in two studies159,161 but 

was the preferred option only in scenarios of high recurrent VTE risk and low risk of 

anticoagulant-induced bleeding. 

Testing for FVL or for FVL and prothrombin G20210A, followed by extended 

anticoagulation for 2 to 3 years in a carrier following a VTE could prove to be a cost-effective 

strategy, although these models have demonstrated that the level of cost-effectiveness depends 

heavily on the prevalence of the mutations, the risk of recurrent VTE, the risk of bleeding, and 

the effectiveness of anticoagulation. Lifelong anticoagulation after testing may prove to be cost-
85

effective for individuals with a very high risk of recurrent VTE and low risk of anticoagulantinduced bleeding events. The studies offer a robust model structure to assess the potential 

effectiveness and cost-effectiveness of various interventions. 

The models are a useful starting point in the evaluation of cost-effectiveness, but since the 

literature did not strongly support the effectiveness of testing, the results of cost-effectiveness 

models are challenging to interpret and apply to patient care. 

Summary of the Evidence 

In Tables 13-15 (see also Appendix G, Evidence Table 28), we summarize the evidence to 

answer our Key Questions. In brief, we found no direct evidence to indicate whether testing for 

FVL and prothrombin G20210A improves outcomes for probands or family members. There was 

high-grade evidence to support the conclusion that existing laboratory assays accurately detect 

these mutations, and most laboratories do an adequate job of detecting these mutations in a 

clinical setting. 

There was moderate-grade evidence that homozygosity or heterozygosity for FVL is 

predictive of recurrent thromboembolism among probands, and high-grade evidence that 

homozygosity is predictive of VTE events in family members of probands. There was moderategrade evidence that heterozygosity for FVL is predictive of thromboembolic events in family 

members and that heterozygosity for prothrombin G20210A is not predictive of VTE in 

probands; there was insufficient evidence as to whether double heterozygosity is predictive. 

There was low-grade evidence that double heterozygosity is predictive of VTE in family 

members and insufficient information to allow us to draw any conclusions about the predictive 

value of homozygosity for prothrombin G20210A. 

There was low-grade evidence that clinicians might change their practice based on testing 

results. There was high-grade evidence that anticoagulation can reduce VTE in individuals with 

these mutations, but only low-grade evidence that the relative risk reduction is comparable to 

that in individuals without mutations. There was moderate-grade evidence that there are neither 

harms nor benefits associated with these genetic tests. The modeling studies suggest that testing 

followed by treatment (for 2 to 3 years) for carriers of a mutation could be cost-effective, 

although the level of this evidence was only low-grade. 

Table 13. Strength of the evidence regarding Key Questions 1 and 2: overarching question and analytic validity 

KQ Evidence statement Strength of evidence 

1 Overarching question: Testing for FVL testing alone, or in 

combination with prothrombin G20210A testing leads to improved 

clinical outcomes (e.g., avoidance of a recurrent VTE) in adults 

with a personal history of VTE, or to improved clinical outcomes 

(e.g., avoidance of an initial VTE) in adult family members of 

mutation-positive individuals. 

No direct evidence 

supports this statement; 

the indirect evidence 

described below 

contributes to answering 

this question 

2 Tests for detection of FVL have excellent analytic validity. High-grade evidence 

2 Tests for detection of prothrombin G20210A have excellent 

analytic validity. 

High-grade evidence 

2 Most, but not all, clinical laboratories can test for FVL and 

prothrombin G20210A very accurately. 

High-grade evidence 
86

Table 14. Strength of the evidence regarding Key Question 3: clinical validity

Factor V Leiden Prothrombin G20210A 

Probands Pooled Odds 

Ratio [95% CI] 

Evidence 

Grade 

Probands Pooled Odds 

Ratio [95% 

CI] 

Evidence Grade

Heterozygous 1.56 [1.14-2.12] Moderate grade, 

predictive 

Heterozygous 1.45 [0.96-2.2] Moderate grade, 

not predictive 

Homozygous 2.65 [1.2-6] Moderate grade, 

predictive 

Homozygous --- --- 

Doubly 

heterozygous 

4.8 [0.50-46] Insufficient 

Family members Pooled Odds 

Ratio [95% 

CI] 

Evidence 

Grade 

Family members Pooled Odds 

Ratio [95% 

CI] 

Evidence Grade 

Heterozygous 3.4 [2.4-4.9] Moderate grade, 

predictive 

Heterozygous 1.9 [0.35-10] Insufficient 

Homozygous 18 [7.8-40] High grade, 

predictive 

Homozygous --- 

Doubly 

heterozygous 

6.7 [2.9-15] Low grade, 

predictive 

 

Pregnant family 

members 

Odds Ratios, 

Not Pooled 

[95% CI]

Evidence 

Grade

Pregnant Family 

Members 

Odds Ratios, 

Not Pooled 

[95% CI]

Evidence Grade

Heterozygous 5.4[0.65-46]; 

3.4[0.35-33] 

Insufficient Heterozygous 3.0 [0.12-74]; 

2.3 [0.21-25] 

Insufficient 

Homozygous 16 [0.9 - 278]; 

41[5.5-419] 

Low grade, 

predictive 

Homozygous --- 

Doubly 

heterozygous 

4.1 [0.37-44]; 

15.3 [1-232] 

Insufficient 
87

Table 15. Strength of the evidence regarding Key Question 4: clinical utility 

CI = confidence interval; FVL = Factor V Leiden; VTE = venous thromboembolism 

Limitations of This Report 

In addition to the reported deficits in the literature, there are limitations to this report. In our 

assessment of clinical validity, we used pooled odds ratios rather than time-dependent measures 

of recurrence (such as hazard ratios or incident rate ratios). This approach necessarily excluded 

some studies from the pooled estimates. We recognize that odds ratios may be biased if the 

follow-up duration varied systematically between individuals with and without the mutations. 

However, there is little reason to suspect that follow-up duration varied according to mutation 

status in these prospective studies. In the studies that reported time-dependent analyses, the 

results of these analyses were generally similar in direction and magnitude to the unadjusted 

odds ratios that we calculated from the raw event data.113 Odds ratios are often misinterpreted as 

being highly clinically significant when the absolute difference in the rates of events is very low. 

We did not calculate pooled rates of events, since we expected the rates of events in the probands 

to be very dependent on the timing of the study relative to the index event and change over 

time.26,110,111,125,138

The odds ratios should approximate the relative rates of events in most studies, since these 

were relatively rare outcomes. We pooled the results using the DerSimonian and Laird random 

effects methods, a conservative method that often results in wide confidence intervals. In our 

sensitivity analyses, we also repeated the pooling using several fixed effect methods. Given the 

near-absence of heterogeneity among the studies in our comparisons, the results were very 

similar. We opted to report the results from our pooling incorporating random effects because we 

think this more accurately represents the truth (the odds ratios for the individual studies coming 

from a distribution of the odds ratios). 

We opted not to pool time-dependent outcomes, including the rates of VTE. The reporting of 

time-dependent outcomes has inherent limitations in a study of thrombosis recurrence, since 

recurrence rates are highest in the months following anticoagulation cessation. This situation 

KQ Evidence statement Strength of evidence 

4 Patient management by physicians changes based on the results 

of testing for FVL or prothrombin G20210A. 

Low-grade evidence 

4 Knowledge of test results reduces VTE related-outcomes in 

individuals who have had VTE or in the probands’ family 

members who have been tested. 

No evidence supports this 

statement 

4 Anticoagulation can reduce recurrent events in patients with FVL 

or prothrombin G20210A 

High-grade evidence 

4 The magnitude of this relative reduction with anticoagulation is 

comparable to that seen in individuals without mutations. 

Low-grade evidence 

4 Neither harms nor benefits have been demonstrated conclusively 

in individuals with VTE or their family members when tested for 

FVL or prothrombin G20210A. 

Moderate-grade evidence 

4 Testing for FVL alone, prothrombin G20210A alone, or the two 

tests in combination may be cost-effective when caring for 

selected patients with VTE (those with a high risk of recurrence 

and/or low risk of bleeding) or their family members. 

Low-grade evidence (from 

models) 
88

renders absolute event rates (such as the number of events per 100-patient years) challenging to 

interpret, because a longer duration of follow-up after termination of anticoagulation will tend to 

bias the results in the direction of lower annualized incidence rates. For this reason, we did not 

primarily compare incidence rates across studies when the follow-up or anticoagulation duration 

varied or was unstated. 

Another potential source of bias was that anticoagulation practices are not independent of 

mutation status (e.g., the longer-duration anticoagulation or more aggressive preventive 

strategies in those with mutations). Most studies mitigated this potential difficulty by excluding 

patients who were chronically anticoagulated14,110,113,114,118,119,121,123-125 or by using a pre-defined 

anticoagulation approach.116,119,123-125

In those studies that reported the duration of anticoagulation after the index event in 

mutation-positive and -negative subgroups, there was no obvious discordance in the 

anticoagulation duration between the two groups.111,112,120,121,127 If any bias was introduced by 

changes in clinical management based on knowledge of mutation status, it would tend to reduce 

the association between the mutation(s) and recurrent events, presuming that mutation positivity 

led to more intensive anticoagulation. 

There was substantial heterogeneity in the composition of the control groups across studies, a 

matter of concern in that the rates of events in the control groups could have differed 

substantially. However, all studies were internally consistent, in that those that included other 

prothrombotic defects in the control group included those same defects in the group with 

mutations. When data were presented on the prevalence of other defects in groups with and 

without mutations, there was no evidence that the prevalence of other thrombophilic defects 

differed across groups.108,112,113 We cannot exclude, however, an interaction between other 

thrombophilic defects and our mutations of interest and thrombosis recurrence. In these cohort 

studies, ascertainment bias is possible. In the studies of probands, the individuals were not 

blinded to their mutation status. It is possible that patients with mutations were more likely to 

seek medical attention for symptoms consistent with deep vein thrombosis or pulmonary 

embolism and might have been over-diagnosed with recurrence (due to false-positive testing), or 

that those without mutations were under-diagnosed (because they did not seek medical attention 

for a thrombotic event that ultimately resolved without therapy). Ascertainment bias would tend 

to augment the association between the mutations and recurrent thrombosis. None of the studies 

we included had scheduled periodic radiographic testing to limit the potential for ascertainment 

bias. 

The majority of the observational studies concerning family members were retrospective, 

with some notable exceptions.129-133 Retrospective studies are prone to important biases, 

including recall bias. Although this potential source of bias can be mitigated by interviewing 

participants before they have knowledge of their mutation status, this process was variably 

described in these studies. 

The limitations that are specific to Key Question 4 – the clinical utility question – are 

described above in the discussion of that question. 

Implications for Future Research 

Studies to directly address our overarching question (Key Question 1) would ideally be 

designed as trials in which participants with venous thrombosis, and/or their family members, 

would be randomized to a test arm or a no-test arm. Individuals would be managed by their 
89

physicians on the basis of the test results (with evidence-based recommendations). Sufficient 

follow-up time would be included in the study design so that VTE events could be witnessed and 

compared between the tested and untested groups. An alternative approach would be a welldesigned prospective cohort study. The population of interest would be defined as individuals 

with recent VTE. The subgroup of these individuals who had testing for the mutation would be 

considered the “exposed” group. A comparison group of closely comparable individuals who 

were not “exposed” to testing would also be defined, and the outcomes in the groups would be 

compared, with careful attention to complete follow-up in both groups. 

Analytic Validity 

Although the mutation detection methods were found to have high analytic validity, a small 

minority of laboratories accounted for a disproportionate percentage of the errors in the 

performance of these tests. This result suggests an ongoing need for participation of molecular 

diagnostic laboratories in external quality assurance programs to assure consistent provision of 

high-quality genetic testing services. In the United States, laboratories doing molecular testing on 

human samples are required to follow the guidelines set by the Clinical Laboratory Improvement 

Amendments (CLIA) 1988 that include requirements for proficiency testing. In addition, a large 

majority of the laboratories performing these tests have additional accreditation from bodies such 

as from the College of American Pathologists, which also requires semi-annual proficiency 

testing to ensure accurate and precise testing. 

Clinical Validity 

Future studies should report event rates over time (and relative rates of recurrence between 

specified groups), rather than just the number of events. Studies should consistently differentiate 

between heterozygous and homozygous individuals, since there is a different rate of recurrence 

in these two groups. Future studies should continue to use objectively measured thrombosis 

(radiographically proven) as a criterion for the index and recurrent thromboses but should 

provide more detail about both the index events and the recurrence, including whether the events 

had other precipitants (e.g., peri-procedural, idiopathic, associated with hospitalization, or 

cancer-associated). Such data were presented in an inconsistent fashion in the studies we 

reviewed, and when reported, they were generally given for the entire study cohort, rather than 

separately for the groups of interest. By examining specific subsets of patients, it may be 

possible to clarify whether there are any interactions between mutation status and clinical 

variables in terms of predicting recurrence. With regard to the prothrombin G20210A mutation 

(alone or in conjunction with FVL), additional studies are needed to more precisely quantify the 

effect size. 

There remains uncertainty about the estimates of risk for family members, given the very 

wide confidence intervals surrounding the odds ratios and the rarity with which the studies 

reported actual rates of events (rather than counts). Also, the studies that we included were 

exclusively studies of European populations. It is well known that the mutation frequency varies 

markedly across populations (and is particularly low in African-derived populations), but it is 

still unclear whether the risk attributable to the mutation differs in other populations having 

different genetic or environmental contributors to VTE risk. Future research would be 
90

appropriate in Caucasian populations outside of Europe or in other populations with appreciable 

frequencies of mutations. Also, future research could better explore the age-mutation interaction. 

Clinical Utility 

Future studies should directly address whether clinicians change their recommendations in 

response to the results of FVL and prothrombin G20210A testing. Rather than surveys based on 

hypothetical situations, we suggest that chart reviews or analyses of utilization data (such as 

tracking prescriptions or the number of referrals) based on actual patients referred for testing 

would more directly and cogently answer this question. 

To assist clinicians in the management of patients with VTE, future studies should be 

powered sufficiently to evaluate the risks associated with prolonged anticoagulation, as they 

relate to patients with specific thrombophilic mutations. Future studies addressing this question 

might move away from primarily focusing on the effect of treatment on absolute recurrence rate 

toward whether management decisions based on testing results affect the rates of recurrence in 

carriers of each of these mutations. Even though the evidence suggested that neither FVL nor 

prothrombin G20210A attenuates the prevention of recurrence during ongoing anticoagulation in 

probands, future studies in both probands and family members might focus on whether 

management decisions (duration of therapy, use of thromboprophylaxis) affect rates of VTE, 

particularly during times of heightened thromboembolic risk. 

Future studies should ensure an adequate representation of patients with FVL and 

prothrombin G20210A. Studies based in the United States may give a clear understanding of 

how patients here might respond to the testing process and results. Larger sample sizes should 

also be used to increase the ability to detect rarer events, such as stigmatization and 

discrimination by insurers. Efforts should be made to recruit representative patient populations, 

and relevant comparison groups should be included (e.g., carriers and non-carriers) to increase 

the practical applicability of the study findings. Quantitative studies may be preferable, involving 

the use of standardized, validated questionnaires to evaluate patients’ experiences. 

The cost-effectiveness analyses should be updated when there are additional data to support 

the assumptions of the models and the factors on which the results most depend, including the 

magnitude and duration of VTE recurrence risk, anticoagulant efficacy in preventing recurrent 

VTE, and anticoagulant-induced bleeding risk. To more definitively determine the costeffectiveness of testing for these mutations, clinical trials could include an assessment of the 

costs associated with a testing strategy, as compared to care without testing. 

Our literature review included articles through December 2008. We do not anticipate any 

important secular changes in the event rate that would markedly change the event rates in the 

upcoming years. We also do not expect major changes in the coming years in terms of the 

methods used to detect mutations. The most anticipated change would be an increase in options 

to reduce risk as new drugs become available. Future research will need to include an evaluation 

of the risks and benefits associated with use of new anticoagulant drugs in probands and family 

members at high risk of events. 
91

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84(5):752-7. 

 164. Sarasin FP, Bounameaux H. Decision analysis 

model of prolonged oral anticoagulant treatment in 

factor V Leiden carriers with first episode of deep 

vein thrombosis. BMJ 1998; 316(7125):95-9. 

 165. Coppens M, Reijnders JH, Middeldorp S, Doggen 

CJ, Rosendaal FR. Testing for inherited 

thrombophilia does not reduce the recurrence of 

venous thrombosis. J Thromb Haemost 2008; 

6(9):1474-7. 
98

166. Samama MM, Rached RA, Horellou MH et 

al.Pregnancy-associated venous thromboembolism 

(VTE) in combined heterozygous factor V Leiden 

(FVL) and prothrombin (FII) 20210 A mutation and 

in heterozygous FII single gene mutation alone. Br 

J Haematol 2003; 123(2):327-34. 

167. Hindorff LA, Burke W, Laberge AM et al. Motivating 

factors for physician ordering of factor V Leiden genetic 

tests. Arch Intern Med 2009; 169(1):68-74. 
99

Acronyms 

Acronym Definition 

ACCE Analytic validity, Clinical validity, Clinical utility and Ethical, legal and social 

implications 

AHRQ Agency for Healthcare Research and Quality 

APC Activated protein C

AS-PCR Allele-specific polymerase chain reaction 

CDC Center for Disease Control and Prevention 

CI Confidence interval 

CISMEL Italian Committee for Standardization of Laboratory Tests 

DNA Deoxyribonucleic acid 

DVT Deep venous thrombosis 

EGAPP Evaluation of Genomic Applications in Practice and Prevention 

ELATE Extended Low-Intensity Anticoagulation for Unprovoked Thromboembolism 

ELISA Enzyme-linked immunosorbent assay 

EPC Evidence-based Practice Center 

EPCOT European Prospective Cohort on Thrombophilia 

FDA Food and Drug Administration 

FRET Fluorescent resonance energy transfer 

FVL Factor V Leiden 

GRADE The Grading of Recommendations Assessment, Development and Evaluation 

HR Hazard ratio 

ICER Incremental cost-effectiveness ratio 

INR International normalized ratio 

MeSH Medical Subject Heading 

M-H Mantel-Haenszel 

PCR Polymerase chain reaction 

PCR-RFLP Polymerase chain reaction and restriction fragment length polymorphism 

PE Pulmonary embolism 

PT 20210A Prothrombin G20210A 

QA Quality assurance 

QALY Quality-adjusted life year 

READIT Reversed Enzyme Activity DNA Interrogation Test 

STARD Standards of Reporting of Diagnostic Accuracy 

TEP Technical Expert Panel 

TGCE Temperature gradient capillary electrophoresis 

THRIVE Thrombin Inhibitor in Venous Thromboembolism 

UK NEQAS United Kingdom National External Quality Assessment Scheme

VTE Venous thromboembolism 
Appendix A: Technical Experts and Peer Reviewers 

A-1 

Frederick Chen, M.D., M.P.H., F.A.A.F.P. 

Department of Family Medicine 

University of Washington 

Seattle, WA 

William Gregory Feero, M.D., Ph.D. 

National Human Genome Research Institute 

National Institutes of Health 

Bethesda, MD 

Andrea Ferreira-Gonzalez, Ph.D. 

Virginia Commonwealth University 

Richmond, VA 

Steven Gutman, M.D. 

Office of In Vitro Diagnostic Device 

Evaluation and Safety 

Center for Devices and Radiological Health 

Food and Drug Administration 

U.S. Department of Health and Human 

Services 

Washington, DC 

James Haddow, M.D. 

Director, Division of Medical Screening 

Women and Infants' Hospital 

Providence, RI 

Denise L. Heaney, Ph.D. 

Roche Diagnostics Corporation 

Dept. of Medical and Scientific Affairs 

Indianapolis, IN 

John Heit, M.D. 

Division of Cardiovascular Diseases 

Department of Internal Medicine 

Mayo Clinic 

Rochester, MN 

Clive Kearon, M.D., Ph.D. 

Professor of Medicine, McMaster University 

Head, Clinical Thrombosis Service 

Henderson General Hospital 

Hamilton, CA 

Lori Preston, M.B.A. 

Vice President of National Alliance for 

Thrombosis and Thrombophila 

Towson, MD 

Sue Richards, Ph.D., F.A.C.M.G. 

Professor, Molecular & Medical Genetics 

Scientific Director, Molecular Diagnostic 

Center 

Oregon Health & Science University 

Portland, OR 

Steven Teutsch, M.D., M.P.H. 

Executive Director of Outcomes Research 

Merck & Co. 

West Point, PA 

Elizabeth Varga, M.S., C.G.C. 

Molecular and Human Genetics 

Columbus Children's Research Institute 

Columbus, OH 

Marc S. Williams, M.D., F.A.A.P., 

F.AC.M.G. 

Intermountain Healthcare Clinical Genetics 

Institute 

Salt Lake City, UT 
Appendix B: Hand Searched Journals 

B-1 

Clinical Chemistry 

British Journal of Haematology 

Thrombosis and Heamostasis 

Blood Coagulation and Fibrinolysis 

Journal of Thrombosis and Haemostasis

Thrombosis Research 

Molecular and Cellular Probes 

Haematologica 

NEJM 

Blood 

Annals of Internal Medicine 
Appendix C: Detailed Search Strategies 

C-1 

Database Terms Date Returns 

PubMed (((Inherit*[tiab] OR herit*[tiab] OR genetic*[tiab] OR genotype[tiab] OR 

familial[tiab]) AND (thrombophil*[tiab] OR coagula*[tiab] OR (venous[tiab] 

AND (thromboemboli*[tiab] OR thrombophili*[tiab])) OR "deep vein 

thrombosis"[tiab] OR "deep-vein thrombosis"[tiab] OR "deep venous 

thrombosis"[tiab] OR VTE[tiab] OR “pulmonary embolism”[tiab])) OR 

("factor v/genetics"[mh] OR ("factor v"[tiab] AND (leiden[tiab] OR 

g1691a[tiab] OR Arg506[tiab] OR R506Q[tiab] OR (ARG[tiab] AND 

506[tiab]))) OR "prothrombin/genetics"[mh] OR (prothrombin[tiab] AND 

(G20210A[tiab] OR 20210[tiab] OR 20210GA[tiab])))) AND 

1993:2008[dp] NOT (animals[mh] NOT humans[mh]) NOT (editorial[pt] 

OR comment[pt] OR letter[pt]) 

5/9/08 5704 

EMBASE (((Inherit*:ti,ab OR herit*:ti,ab OR genetic*:ti,ab OR genotype:ti,ab OR 

familial:ti,ab) AND (thrombophil*:ti,ab OR coagula*:ti,ab OR (venous:ti,ab 

AND (thromboemboli*:ti,ab OR thrombophili*:ti,ab)) OR "deep vein 

thrombosis":ti,ab OR "deep-vein thrombosis":ti,ab OR "deep venous 

thrombosis":ti,ab OR VTE:ti,ab OR “pulmonary embolism”:ti,ab)) OR 

(‘blood clotting factor v leiden’/exp OR ("factor v":ti,ab AND (leiden:ti,ab 

OR g1691a:ti,ab OR Arg506:ti,ab OR R506Q:ti,ab OR (ARG:ti,ab AND 

506:ti,ab))) OR ((prothrombin/exp OR prothrombin:ti,ab) AND 

(G20210A:ti,ab OR 20210:ti,ab OR 20210GA:ti,ab)))) AND [1993-

2008]/py NOT ([conference paper]/lim OR [editorial]/lim OR [erratum]/lim 

OR [letter]/lim OR [note]/lim) NOT ([animals]/lim NOT [humans]/lim) 

5/12/08 6297 

Cochrane 

Library 

(((Inherit*:ti,ab OR herit*:ti,ab OR genetic*:ti,ab OR genotype:ti,ab OR 

familial:ti,ab) AND (thrombophil*:ti,ab OR coagula*:ti,ab OR (venous:ti,ab 

AND (thromboemboli*:ti,ab OR thrombophili*:ti,ab)) OR "deep vein 

thrombosis":ti,ab OR "deep-vein thrombosis":ti,ab OR "deep venous 

thrombosis":ti,ab OR VTE:ti,ab OR “pulmonary embolism”:ti,ab)) OR 

(“factor v”:genetics[mh] OR ("factor v":ti,ab AND (leiden:ti,ab OR 

g1691a:ti,ab OR Arg506:ti,ab OR R506Q:ti,ab OR (ARG:ti,ab AND 

506:ti,ab))) OR "prothrombin:genetics"[mh] OR (prothrombin[tiab] AND 

(G20210A[tiab] OR 20210[tiab] OR 20210GA[tiab])))) , from 1993 to 

2008 

5/12/08 129 

Cochrane 

reviews 7 

DARE 3 

CENTRAL 

108 

Economic 

Evaluation

s 5 

PsycInfo (((AB Inherit* OR AB herit* OR AB genetic* OR AB genotype OR AB 

familial) AND (AB thrombophil* OR AB coagula* OR (AB venous AND 

(AB thromboemboli* OR AB thrombophili*)) OR AB "deep vein 

thrombosis" OR AB "deep-vein thrombosis" OR AB "deep venous 

thrombosis" OR AB VTE)) OR ((TI “factor V” OR AB "factor v") AND (AB 

leiden OR AB g1691a OR AB Arg506 OR AB R506Q OR (AB ARG AND 

AB 506))) OR ((TI prothrombin OR AB prothrombin) AND (AB G20210A 

OR AB 20210 OR AB 20210GA))) AND DT 199301-200812 NOT (PZ 

animal NOT PZ human) Not ((ZZ "BIBLIOGRAPHY") or (ZZ 

"CHAPTER") or (ZZ "COLUMN/OPINION") or (ZZ "COMMENT/REPLY") 

or (ZZ "EDITORIAL") or (ZZ "ENCYCLOPEDIA ENTRY") or (ZZ 

"LETTER") or (ZZ "OBITUARY") or (ZZ "PUBLICATION 

INFORMATION")) 

5/12/08 25 

CINAHL (((TX Inherit* OR TX herit* OR TX genetic* OR TX genotype OR TX 

familial) AND (TX thrombophil* OR TX coagula* OR (TX venous AND 

(TX thromboemboli* OR TX thrombophili*)) OR TX "deep vein 

thrombosis" OR TX "deep-vein thrombosis" OR TX "deep venous 

thrombosis" OR TX VTE)) OR ((TI “factor V” OR TX "factor v") AND (TX 

leiden OR TX g1691a OR TX Arg506 OR TX R506Q OR (TX ARG AND 

TX 506))) OR ((TI prothrombin OR TX prothrombin) AND (TX G20210A 

OR TX 20210 OR TX 20210GA))) AND (PY 1993-2008) AND (ZT 

"CLINICAL TRIAL" or ZT "JOURNAL ARTICLE" or ZT "RESEARCH" 

or ZT "REVIEW" or ZT "SYSTEMATIC REVIEW") 

5/12/08 660 
Appendix D: List of Excluded Articles 

 D-1 

Medical devices; hematology and pathology devices; 

classification of the Factor V Leiden DNA mutation 

detection systems devices. Final rule. Fed Regist 

2004;69(51):12271-3 

Other reason

Investigation and management of heritable thrombophilia. 

Br J Haematol 2001;114(3):512-28 

No original data

Comparison of thrombotic risk between 85 homozygotes 

and 481 heterozygotes carriers of the factor V Leiden 

mutation: retrospective analysis from the Procare Study. 

Blood Coagul Fibrinolysis 2000;11(6):511-8 

Doesn't address study question 

Adkins K K, Strom D A, Jacobson T E et al. Utilizing 

genomic DNA purified from clotted blood samples for 

single nucleotide polymorphism genotyping. Arch Pathol 

Lab Med 2002;126(3):266-70 

Evaluates only DNA extraction methods

Agnelli G. Long-term low-dose warfarin use is effective in 

the prevention of recurrent venous thromboembolism: No. 

J. Thromb. Haemost. 2004;2(7):1038-1040 

No original data

Aliev M A, Mierbekov E M, Sviatova G S et al. [Venous 

thromboembolic events in surgery: a role of genetic 

disorders]. Anesteziol Reanimatol 2008;(1):71-3 

Non-English article

Altes A, Souto J C, Mateo J et al. Activated protein C 

resistance assay when applied in the general population. 

Am J Obstet Gynecol 97;176(2):358-9 

Published before 2000

Amberger N, Masuhr F, Valdueza J M et al. A negative 

personal and family history for venous thrombotic events is 

not sufficient to exclude thrombophilia in patients with 

cerebral venous thrombosis. Eur J Neurol 2004;11(8):555-8 

Does not report results separately for FV Leiden and/or 

PT 20210A. 

Andersson H C, Krousel-Wood M A, Jackson K E et al. 

Medical genetic test reporting for cystic fibrosis 

(deltaF508) and factor V Leiden in North American 

laboratories. Genet Med 2002;4(5):324-7 

Doesn't address study question

Angelini A, Di Febbo C, Baccante G et al. Identification of 

three genetic risk factors for venous thrombosis using a 

multiplex allele-specific PCR assay: comparison of 

conventional and new alternative methods for the 

preparation of DNA from clinical samples. J Thromb 

Thrombolysis 2003;16(3):189-93 

Evaluates only DNA extraction methods

Angelini A, Di Febbo C, Rullo A et al. New method for the 

extraction of DNA from white blood cells for the detection 

of common genetic variants associated with thrombophilia. 

Pathophysiol Haemost Thromb 2002;32(4):180-3 

Evaluates only DNA extraction methods

Arruda V R, von Zuben P M, Annichino-Bizzachi J M et al. 

Rapid detection of factor V Leiden (FVQ506) by nonradioactive single strand conformation polymorphism 

(SSCP). Sangre (Barc) 96;41(5):379-81 

Published before 2000

Aslanidis C, Nauck M, Schmitz G. High-speed 

prothrombin G-->A 20210 and methylenetetrahydrofolate 

reductase C-->T 677 mutation detection using real-time 

fluorescence PCR and melting curves. Biotechniques 

99;27(2):234-6, 238 

Published before 2000

Aznar J, Vaya A, Estelles A et al. Risk of venous 

thrombosis in carriers of the prothrombin G20210A variant 

and factor V Leiden and their interaction with oral 

contraceptives. Haematologica 2000;85(12):1271-6 

Case-control or retrospective cohort of probands

Baglin C, Brown K, Luddington R et al. Risk of recurrent 

venous thromboembolism in patients with the factor V 

Leiden (FVR506Q) mutation: effect of warfarin and 

prediction by precipitating factors. East Anglian 

Thrombophilia Study Group. Br J Haematol 98;100(4):764-

8 

Other reason 

Bank I, Libourel E J, Middeldorp S et al. Prothrombin 

20210A mutation: a mild risk factor for venous 

thromboembolism but not for arterial thrombotic disease 

and pregnancy-related complications in a family study. 

Arch Intern Med 2004;164(17):1932-7 

Other reason 

Bathelier C, Champenois T, Lucotte G. ARMS test for 

diagnosis of factor V Leiden mutation and allele 

frequencies in France. Mol Cell Probes 98;12(2):121-3 

Published before 2000

Beauchamp N J, Daly M E, Cooper P C et al. Rapid twostage PCR for detecting factor V G1691A mutation. Lancet 

94;344(8923):694-5 

Published before 2000

Behn M, Schuermann M. Simple and reliable factor V 

genotyping by PNA-mediated PCR clamping. Thromb 

Haemost 98;79(4):773-7 

Published before 2000

Bennett C D, Campbell M N, Cook C J et al. The 

LightTyper: high-throughput genotyping using fluorescent 

melting curve analysis. Biotechniques 2003;34(6):1288-92, 

1294-5 

Other reason

Benson J M, Ellingsen D, Renshaw M A et al. Multiplex 
Appendix D: List of Excluded Articles 

 D-2 

analysis of mutations in four genes using fluorescence 

scanning technology. Thromb Res 99;96(1):57-64 

Published before 2000

Benson J M, Phillips D J, Holloway B P et al. 

Oligonucleotide ligation assay for detection of the factor V 

mutation (Arg506-->Gln) causing protein C resistance. 

Thromb Res 96;83(1):87-96 

Published before 2000

Bertina R M. Molecular risk factors for thrombosis. 

Thromb Haemost 99;82(2):601-9 

No original data

Biswas A, Bajaj J, Ranjan R et al. Factor V Leiden: Is it the 

chief contributor to activated protein C resistance in AsianIndian patients with deep vein thrombosis?. Clin Chim 

Acta 2008;392(1-2):21-4 

Doesn't address study question

Blasczyk R, Ritter M, Thiede C et al. Simple and rapid 

detection of factor V Leiden by allele-specific PCR 

amplification. Thromb Haemost 96;75(5):757-9 

Published before 2000

Blasczyk R, Wehling J, Ritter M et al. Allele-specific PCR 

amplification of factor V Leiden to identify patients at risk 

for thromboembolism. Beitr Infusionsther Transfusionsmed 

97;34236-41 

Published before 2000

Boda Z, Laszlo P, Rejto L et al. Low molecular weight 

heparin as thromboprophylaxis in familial thrombophilia 

during the whole period of pregnancy. Thromb Haemost 

96;76(1):128 

Doesn't address study question

Bokarewa M I, Bremme K, Blomback M. Arg506-Gln 

mutation in factor V and risk of thrombosis during 

pregnancy. Br J Haematol 96;92(2):473-8 

Other reason 

Bokarewa M I, Falk G, Sten-Linder M et al. Thrombotic 

risk factors and oral contraception. J Lab Clin Med 

95;126(3):294-8 

Does not report results separately for FV Leiden and/or 

PT 20210A

Bounameaux H. Factor V Leiden paradox: risk of deepvein thrombosis but not of pulmonary embolism. Lancet 

2000;356(9225):182-3 

Doesn't address study question

Bowen D J, Bowley S, John M et al. Factor V Leiden 

(G1691A), the prothrombin 3'-untranslated region variant 

(G20210A) and thermolabile methylenetetrahydrofolate 

reductase (C677T): a single genetic test genotypes all three 

loci--determination of frequencies in the S. Wales 

population of the UK. Thromb Haemost 98;79(5):949-54 

Published before 2000

Bowen D J, Standen G R, Granville S et al. Genetic 

diagnosis of factor V Leiden using heteroduplex 

technology. Thromb Haemost 97;77(1):119-22 

Published before 2000

Brenner B, Zivelin A, Lanir N et al. Venous 

thromboembolism associated with double heterozygosity 

for R506Q mutation of factor V and for T298M mutation of 

protein C in a large family of a previously described 

homozygous protein C-deficient newborn with massive 

thrombosis. Blood 96;88(3):877-80 

Other reason 

Brouwer J L, Bijl M, Veeger N J et al. The contribution of 

inherited and acquired thrombophilic defects, alone or 

combined with antiphospholipid antibodies, to venous and 

arterial thromboembolism in patients with systemic lupus 

erythematosus. Blood 2004;104(1):143-8 

Does not report results separately for FV Leiden and/or 

PT 20210A

Buller H R, Davidson B L, Decousus H et al. Fondaparinux 

or enoxaparin for the initial treatment of symptomatic deep 

venous thrombosis: a randomized trial. Ann Intern Med 

2004;140(11):867-73 

Doesn't address study question

Buller H R, Davidson B L, Decousus H et al. Subcutaneous 

fondaparinux versus intravenous unfractionated heparin in 

the initial treatment of pulmonary embolism. N Engl J Med 

2003;349(18):1695-702 

Doesn't address study question

Caprini J A, Goldshteyn S, Glase C J et al. Thrombophilia 

testing in patients with venous thrombosis. Eur J Vasc 

Endovasc Surg 2005;30(5):550-5 

Evaluates less than 10 probands or 10 family members

Castaman G, Tosetto A, Ruggeri M et al. 

Pseudohomozygosity for activated protein C resistance is a 

risk factor for venous thrombosis. Br J Haematol 

99;106(1):232-6 

Other reason 

Chakravarty A, Hansen T S, Horder M et al. A fast and 

robust dual-label nonradioactive oligonucleotide ligation 

assay for detection of factor V Leiden. Thromb Haemost 

97;78(4):1234-6 

Published before 2000

Charakidas A, Brouzas D, Andrioti E et al. Papillophlebitis 

associated with coexisting factor V Leiden and 

prothrombin G20210A mutations. Retina 2002;22(2):239-

40 

Doesn't address study question

Cibula D, Kvasnicka J, Salaj P et al. [Informed consent for 

factor V Leiden mutation screening before recommending 

use of combined hormonal contraceptives or hormone 

replacement therapy ]. Ceska Gynekol 2003;68(3):212 

Non-English article
Appendix D: List of Excluded Articles 

 D-3 

Clayton E W. Ethical, legal, and social implications of 

genomic medicine. N Engl J Med 2003;349(6):562-9 

Doesn't address study question

Cleary-Goldman J, Bettes B, Robinson J N et al. 

Thrombophilia and the obstetric patient. Obstet Gynecol 

2007;110(3):669-74 

Does not study probands or family members

Conard J, Plu-Bureau G, Bahi N et al. Progestogen-only 

contraception in women at high risk of venous 

thromboembolism. Contraception 2004;70(6):437-41 

Evaluates less than 10 probands or 10 family members

Coppens M, van de, Poel M H et al. A prospective cohort 

study on the absolute incidence of venous 

thromboembolism and arterial cardiovascular disease in 

asymptomatic carriers of the prothrombin 20210A 

mutation. Blood 2006;108(8):2604-7 

Other reason 

Corral J, Iniesta J A, Gonzalez-Conejero R et al. Detection 

of factor V Leiden from a drop of blood by PCR-SSCP. 

Thromb Haemost 96;76(5):735-7 

Published before 2000

Corral J, Zuazu-Jausoro I, Rivera J et al. Clinical and 

analytical relevance of the combination of prothrombin 

20210A/A and factor V Leiden: results from a large family. 

Br J Haematol 99;105(2):560-3 

No objective confirmation of VTE event

Correa M F, Rezende S M. A very rapid multiplex 

polymerase chain reaction for simultaneous detection of 

factor V Leiden and G20210A prothrombin mutation. Clin 

Lab Haematol 2004;26(3):239-40 

Evaluates less than 10 samples

Coude M, Pineau-Vincent F. Prothombin gene: 

Comparative study between PCR-RFLP/Allele specific 

PCR: ETUDE DU GENE DE LA PROTHROMBINE: 

ETUDE COMPARATIVE DE DEUX TECHNIQUES 

PCR-RFLP/PCR ALLELES SPECIFIQUES. ImmunoAnal. Biol. Spec. 2004;19(3):177-179 

Non-English article

Couturaud F, Leroyer C, Mottier D. Risk factors and 

clinical presentation of venous thromboembolism 

according to the age of relatives of patients with factor V 

Leiden. Thromb Haemost 2008;99(4):793-794 

Case-control or retrospective cohort of family members

Dahlback B. Procoagulant and anticoagulant properties of 

coagulation factor V: factor V Leiden (APC resistance) 

causes hypercoagulability by dual mechanisms. J Lab Clin 

Med 99;133(5):415-22 

No original data

Danneberg J, Abbes A P, Bruggeman B J et al. Reliable 

genotyping of the G-20210-A mutation of coagulation 

factor II (prothrombin). Clin Chem 98;44(2):349-51 

Published before 2000

David D, Ribeiro S, Ferrao L et al. Molecular basis of 

inherited antithrombin deficiency in Portuguese families: 

identification of genetic alterations and screening for 

additional thrombotic risk factors. Am J Hematol 

2004;76(2):163-71 

Case-control or retrospective cohort of probands

De Bel A V, Van der, Cruyssen G A et al. A 2-year 

retrospective analysis of laboratory testing for activated 

protein C resistance with a factor V-corrected activated 

partial thromboplastin time-based method. Blood Coagul 

Fibrinolysis 2006;17(2):155-60 

Evaluates only APC resistance

De Stefano V. Inherited thrombophilia and life-time risk of 

venous thromboembolism: Is the burden reducible?. J. 

Thromb. Haemost. 2004;2(9):1522-1525 

No original data

De Stefano V, Martinelli I, Mannucci P M et al. The risk of 

recurrent deep venous thrombosis among heterozygous 

carriers of both factor V Leiden and the G20210A 

prothrombin mutation. N Engl J Med 99;341(11):801-6 

Other reason 

De Stefano V, Martinelli I, Mannucci P M et al. The risk of 

recurrent venous thromboembolism among heterozygous 

carriers of the G20210A prothrombin gene mutation. Br J 

Haematol 2001;113(3):630-5 

Other reason 

De Stefano V, Martinelli I, Rossi E et al. The risk of 

recurrent venous thromboembolism in pregnancy and 

puerperium without antithrombotic prophylaxis. Br J 

Haematol 2006;135(3):386-91 

Other reason 

Dietrich J E, Hertweck S P, Perlman S E. Efficacy of 

family history in determining thrombophilia risk. J Pediatr 

Adolesc Gynecol 2007;20(4):221-4 

No objective confirmation of VTE event

Djordjevic V, Rakicevic L, Gagic M et al. Simultaneous 

detection of factor V Leiden and factor II G20210A 

variants by a multiplex PCR on whole blood. Balk. J. Med. 

Genet. 2001;4(1-2):15-17 

Does not report concordance, discordance, or 

reproducibility 

Dordevic V, Rakicevic L, Miljic P et al. Impact of acquired 

and genetic factors on thrombophilic phenotype in FV 

Leiden mutation carriers. Jugosl. Med. Biohem. 

2005;24(2):141-146 

Case-control or retrospective cohort of probands

Douketis J D, Foster G A, Crowther M A et al. Clinical risk 

factors and timing of recurrent venous thromboembolism 

during the initial 3 months of anticoagulant therapy. Arch 

Intern Med 2000;160(22):3431-6 
Appendix D: List of Excluded Articles 

 D-4 

Doesn't address study question

Dubreuil Lastrucci R M, Dawson D A, Bowden J H et al. 

Development of a simple multiplex polymerase chain 

reaction for the simultaneous detection of factor V Leiden 

and prothrombin 20210A mutations. Mol Diagn 

99;4(3):247-50 

Published before 2000

Dulicek P, Maly J, Safarova M. Risk of thrombosis in 

patients homozygous and heterozygous for factor V Leiden 

in the East Bohemian region. Clin Appl Thromb Hemost 

2000;6(2):87-9 

Doesn't address study question

Duperat V G, Fauchon M, Nurden A T et al. Simple and 

rapid combined genetic diagnosis of mutation (1691 G--

>A) of the factor V gene and (20210 G-->A) of the 

prothrombin gene. Blood Coagul Fibrinolysis 98;9(6):549-

51 

Published before 2000

Dzhafarov T A, Rustamov R S h. [Simultaneous 

determination of mutations 1691 G-A of the blood 

coagulation system factor V gene and 20210 G-A gene, 

coding for prothrombin, by means of complex PCR from 

whole blood using one restriction enzyme (HindIII)]. Mol 

Biol (Mosk) 2000;34(3):486-7 

Non-English article

Eichinger S, Pabinger I, Stumpflen A et al. The risk of 

recurrent venous thromboembolism in patients with and 

without factor V Leiden. Thromb Haemost 97;77(4):624-8 

Other reason 

Eichinger S, Weltermann A, Minar E et al. Symptomatic 

pulmonary embolism and the risk of recurrent venous 

thromboembolism. Arch Intern Med 2004;164(1):92-6 

Other reason 

Eikelboom J W, Ivey L, Ivey J et al. Familial thrombophilia 

and the prothrombin 20210A mutation: association with 

increased thrombin generation and unusual thrombosis. 

Blood Coagul Fibrinolysis 99;10(1):1-5 

Evaluates less than 10 probands or 10 family members

Emmerich J, Alhenc-Gelas M, Aillaud M F et al. Clinical 

features in 36 patients homozygous for the ARG 506--

>GLN factor V mutation. Thromb Haemost 97;77(4):620-3 

Other reason 

Eriksson H, Lundstrom T, Wahlander K et al. Prognostic 

factors for recurrence of venous thromboembolism (VTE) 

or bleeding during long-term secondary prevention of VTE 

with ximelagatran. Thromb Haemost 2005;94(3):522-7 

Doesn't address study question

Faioni E M, Franchi F, Bucciarelli P et al. Coinheritance of 

the HR2 haplotype in the factor V gene confers an 

increased risk of venous thromboembolism to carriers of 

factor V R506Q (factor V Leiden). Blood 99;94(9):3062-6 

Other reason 

Favaloro E J, Bonar R, Sioufi J et al. Multilaboratory 

testing of thrombophilia: current and past practice in 

Australasia as assessed through the Royal College of 

Pathologists of Australasia Quality Assurance Program for 

Hematology. Semin Thromb Hemost 2005;31(1):49-58 

Evaluates only APC resistance

Folsom A R, Cushman M, Tsai M Y et al. Prospective 

study of the G20210A polymorphism in the prothrombin 

gene, plasma prothrombin concentration, and incidence of 

venous thromboembolism. Am J Hematol 2002;71(4):285-

90 

Case-control or retrospective cohort of probands

Franco R F, Middeldorp S, Meinardi J R et al. Factor XIII 

Val34Leu and the risk of venous thromboembolism in 

factor V Leiden carriers. Br J Haematol 2000;111(1):118-

21 

Other reason 

Friedline J A, Ahmad E, Garcia D et al. Combined factor V 

Leiden and prothrombin genotyping in patients presenting 

with thromboembolic episodes. Arch Pathol Lab Med 

2001;125(1):105-11 

No objective confirmation of VTE event

Gadisseur A P, Christiansen S C, VAN D E R et al. The 

quality of oral anticoagulant therapy and recurrent venous 

thrombotic events in the Leiden Thrombophilia Study. J 

Thromb Haemost 2007;5(5):931-6 

Does not report results separately for FV Leiden and/or 

PT 20210A

Gancberg D, Corbisier P, Meeus N et al. Certification of 

reference materials for detection of the human prothrombin 

gene G20210A sequence variant. Clin Chem Lab Med 

2008;46(4):463-9 

Evaluates only DNA extraction methods

Gandrille S, Alhenc-Gelas M, Aiach M. A rapid screening 

method for the factor V Arg506-->Gln mutation. Blood 

Coagul Fibrinolysis 95;6(3):245-8 

Published before 2000

Gandrille S, Remones V, Aiach M. [Validation of a 

diagnostic kit for factor V Leiden mutation]. Ann Biol Clin 

(Paris) 2003;61(6):723-7 

Non-English article

Garcia-Fuster, M. J., Forner, M. J., Fernandez, C., Gil, J., 

Vaya, A., and Maldonado, L., Long-term prospective study 

of recurrent venous thromboembolism in patients younger 

than 50 years. Pathophysiol Haemost Thromb. 

2005;34(1):6-12 

Other reason 

Garcia-Hernandez M C, Romero Casanova A, Marco Vera 

P. [Clinical comments on genetic marker prevalence (factor 

V Leiden, prothrombin 20210A and homozygous 
Appendix D: List of Excluded Articles 

 D-5 

methylenetetrahydrofolate reductase form. Rev Clin Esp 

2007;207(1):26-8 

Non-English article

Gardiner C, Cooper P C, Makris M et al. An evaluation of 

screening tests for defects in the protein C pathway: 

commercial kits lack sensitivity and specificity. Blood 

Coagul Fibrinolysis 2002;13(2):155-63 

Evaluates only APC resistance

Geisel J, Walz T, Bodis M et al. Fluorescence-based SSCP 

analysis with automatic allele detection demonstrated for 

the factor V Leiden mutation. Clin Chem 98;44(9):1986-7 

Published before 2000

Ghosh K, Shetty S, Madkaikar M et al. Venous 

thromboembolism in young patients from western India: a 

study. Clin Appl Thromb Hemost 2001;7(2):158-65 

Case-control or retrospective cohort of probands

Girolami A, Simioni P, Girolami B et al. G to A 20210 

prothrombin polymorphism and venous thrombosis: simple 

association or causal relationship?. Clin Appl Thromb 

Hemost 2000;6(3):135-8 

No original data

Girolami A, Simioni P, Girolami B et al. Low incidence of 

venous thrombosis in homozygous patients with NT 20210 

G to a prothrombin polymorphism. Clin Appl Thromb 

Hemost 99;5(4):205-7 

No original data

Girolami A, Tormene D, Gavasso S et al. Long term use of 

oral contraceptives without thrombosis in patients with FV 

Leiden polymorphism: a study of 37 patients (2 

homozygous and 35 heterozygous). J Thromb 

Thrombolysis 2004;17(2):145-9 

Other reason 

Glynn R J, Ridker P M, Goldhaber S Z et al. Effects of 

random allocation to vitamin E supplementation on the 

occurrence of venous thromboembolism: report from the 

Women's Health Study. Circulation 2007;116(13):1497-

503 

Doesn't address study question

Golomb M R, Garg B P, Walsh L E et al. Perinatal stroke 

in baby, prothrombotic gene in mom: does this affect 

maternal health insurance?. Neurology 2005;65(1):13-6 

Other reason

Gomez E, van der, Poel S C et al. Rapid simultaneous 

screening of factor V Leiden and G20210A prothrombin 

variant by multiplex polymerase chain reaction on whole 

blood. Blood 98;91(6):2208-9 

Published before 2000

Gonzalez C M, Diaz-Golpe V, Martin S et al. Combination 

of congenital coagulation disorders: Factor II gene 

mutation G20210A, Factor V Leiden gene mutation 

G1691A and protein S deficiency. a family study. 

Haematologica 2003;88(6):ECR20 

Evaluates less than 10 probands or 10 family members

Grand'Maison A, Bates S M, Johnston M et al. "ProC 

Global": a functional screening test that predicts recurrent 

venous thromboembolism. Thromb Haemost 

2005;93(3):600-4 

Evaluates only APC resistance

Gray E, Hawkins J R, Morrison M et al. Establishment of 

the 1st International Genetic Reference Panel for Factor V 

Leiden, human gDNA. Thromb Haemost 2006;96(2):215-9 

Evaluates less than 10 samples

Greaves M, Baglin T. Laboratory testing for heritable 

thrombophilia: impact on clinical management of 

thrombotic disease annotation. Br J Haematol 

2000;109(4):699-703 

Doesn't address study question

Greengard J S, Xu X, Gandrille S et al. Alternative PCR 

method for diagnosis of mutation causing activated protein 

C resistant Gln506-factor V. Thromb Res 95;80(5):441-3 

Published before 2000

Guillerm C, Lellouche F, Darnige L et al. Rapid detection 

of the G1691A mutation of coagulation factor V by PCRmediated site-directed mutagenesis. Clin Chem 

96;42(2):329 

Published before 2000

Hanguehard A, Morelle M, Turchetto L et al. Genotyping 

in routine laboratory for detection of factor V Leiden 

(1691G>A) and prothrombin (20210G>A) mutations with 

the Invader(registered trademark) assay technology: 

EVALUATION EN ROUTINE D'UNE NOUVELLE 

METHODE POUR LE GENOTYPAGE DE LA 

MUTATION 1691G>A DU FACTEUR V LEIDEN ET 

DU VARIANT 20210G>A DU FACTEUR II PAR LA 

TECHNOLOGIE INVADER(registered trademark). 

Immuno-Anal. Biol. Spec. 2004;19(2):110-116 

Non-English article

Hansson P O, Sorbo J, Eriksson H. Recurrent venous 

thromboembolism after deep vein thrombosis: incidence 

and risk factors. Arch Intern Med 2000;160(6):769-74 

Doesn't address study question

Happich D, Schwaab R, Hanfland P et al. Allelic 

discrimination of factor V Leiden using a 5' nuclease assay. 

Thromb Haemost 99;82(4):1294-6 

Published before 2000

Hatzaki A, Anagnostopoulou E, Metaxa-Mariatou V et al. 

The impact of heterozygosity for the factor V Leiden and 

factor II G20210A mutations on the risk of thrombosis in 

Greek patients. Int Angiol 2003;22(1):79-82 

Doesn't address study question

Heinemann L A, Dominh T, Assmann A et al. VTE Risk 

assessment - a prognostic Model: BATER Cohort Study of 

young women. Thromb J 2005;3(1):5 
Appendix D: List of Excluded Articles 

 D-6 

Other reason

Heldal M, Seem E, Sandset P M et al. Deep vein 

thrombosis: a 7-year follow-up study. J Intern Med 

93;234(1):71-5 

Doesn't address study question

Hellmann E A, Leslie N D, Moll S. Knowledge and 

educational needs of individuals with the factor V Leiden 

mutation. J Thromb Haemost 2003;1(11):2335-9 

Less than 80% of the probands has had VTE

Herrington D M, Vittinghoff E, Howard T D et al. Factor V 

Leiden, hormone replacement therapy, and risk of venous 

thromboembolic events in women with coronary disease. 

Arterioscler Thromb Vasc Biol 2002;22(6):1012-7 

Other reason 

Hezard N, Cornillet P, Droulle C et al. Factor V Leiden: 

detection in whole blood by ASA PCR using an additional 

mismatch in antepenultimate position. Thromb Res 

97;88(1):59-66 

Published before 2000

Hezard N, Cornillet-Lefebvre P, Gillot L et al. Multiplex 

ASA PCR for a simultaneous determination of factor V 

Leiden gene, G-->A 20210 prothrombin gene and C-->T 

677 MTHFR gene mutations. Thromb Haemost 

98;79(5):1054-5 

Published before 2000

Holmberg K, Persson M-L, Uhlen M et al. Pyrosequencing 

analysis of thrombosis-associated risk markers. Clin. 

Chem. 2005;51(8):1549-1552 

Does not report concordance, discordance, or 

reproducibility

Hron G, Kollars M, Binder B R et al. Identification of 

patients at low risk for recurrent venous thromboembolism 

by measuring thrombin generation. JAMA 

2006;296(4):397-402 

Other reason 

Hudson M, Herr A L, Rauch J et al. The presence of 

multiple prothrombotic risk factors is associated with a 

higher risk of thrombosis in individuals with anticardiolipin 

antibodies. J Rheumatol 2003;30(11):2385-91 

Doesn't address study question

Hull R D, Pineo G F, Brant R F et al. Long-term lowmolecular-weight heparin versus usual care in proximalvein thrombosis patients with cancer. Am J Med 

2006;119(12):1062-72 

Other reason

Hunault M, Marsh-Scott C, Jou C et al. Automated 

detection of the factor V Leiden mutation using the LCx 

microparticle enzyme immunoassay. Clin Chem 

99;45(1):41-6 

Published before 2000

Huxtable L M, Tafreshi M J, Ondreyco S M. A protocol for 

the use of enoxaparin during pregnancy: Results from 85 

pregnancies including 13 multiple gestation pregnancies. 

Clin. Appl. Thromb. Hemost. 2005;11(2):171-181 

Doesn't address study question

Ivanov P, Komsa-Penkova R, Kovacheva K et al. Impact of 

thrombophilic genetic factors on pulmonary embolism: 

early onset and recurrent incidences. Lung 2008;186(1):27-

36 

Other reason 

Jastrzebska M, Balszan-Kowalska I, Ciechanowicz A et al. 

[Activated protein C resistance, Leiden mutation, 

anticoagulant proteins and fibrinogen levels in patients with 

deep venous thrombosis]. Pol Arch Med Wewn 

2003;109(6):579-88 

Non-English article

Kearon C, Ginsberg J S, Anderson D R et al. Comparison 

of 1 month with 3 months of anticoagulation for a first 

episode of venous thromboembolism associated with a 

transient risk factor. J Thromb Haemost 2004;2(5):743-9 

Other reason 

Keenan C, Livingstone W J, White B et al. Prevalence of 

the prothrombin G20210A mutation in the Irish 

populations: use of a novel polymerase chain reaction 

approach. Blood Coagul Fibrinolysis 2000;11(7):669-72 

Doesn't address study question

Kirschbaum N E, Foster P A. The polymerase chain 

reaction with sequence specific primers for the detection of 

the factor V mutation associated with activated protein C 

resistance. Thromb Haemost 95;74(3):874-8 

Published before 2000

Klein C L, Marki-Zay J, Corbisier P et al. Reference 

materials (RMs) for analysis of the human factor II 

(prothrombin) gene G20210A mutation. Clin Chem Lab 

Med 2005;43(8):862-8 

Does not report concordance, discordance, or 

reproducibility 

Klingler K R, Grote S, Holzem G et al. Activated protein C 

(APC)-resistance: automated detection of the point 

mutation at position 1691 in the factor V gene. Clin Chem 

Lab Med 99;37(2):101-7 

Published before 2000

Klingler K R, Holzem G, Wielckens K. APC-Resistance: 

Automated detection of the point mutation at position 1691 

in the factor V gene: APC-RESISTENZ: 

AUTOMATISIERTER NACHWEIS DER 

PUNKTMUTATION AN DER POSITION 1691 IM 

FAKTOR V GEN. LaboratoriumsMedizin 99;23(11):606-

611 

Non-English article

Klingler K R, Junold T, Wielckens K. Activated protein C 

resistance: automated detection of the factor V Leiden 

mutation by mismatch hybridization. Clin Chem 

99;45(11):1925-31 
Appendix D: List of Excluded Articles 

 D-7 

Published before 2000

Koizumi M, Morita K, Takagi M et al. SNP genotyping by 

allele-specific PCR using ENA primers. Nucleic Acids 

Symp Ser (Oxf) 2005;(49):47-8 

Does not report concordance, discordance, or 

reproducibility

Kornblihtt L I, Heller P G, Correa G et al. Associated 

thrombophilic defects in essential thrombocythaemia: their 

relationship with clinical manifestations. Thromb Res 

2003;112(3):131-5 

Did not study venous thrombosis

Krawczak M, Cooper D N, Schmidtke J. Estimating the 

efficacy and efficiency of cascade genetic screening. Am J 

Hum Genet 2001;69(2):361-70 

Doesn't address the study question

Krousel-Wood M, Andersson H C, Rice J et al. Physicians' 

perceived usefulness of and satisfaction with test reports for 

cystic fibrosis (DeltaF508) and factor V Leiden. Genet Med 

2003;5(3):166-71 

Doesn't address the study question

Kwiatkowski R W, Lyamichev V, de Arruda M et al. 

Clinical, genetic, and pharmacogenetic applications of the 

Invader assay. Mol Diagn 99;4(4):353-64 

Published before 2000

Kwon H J, Peiper S C, Kang K A. Fiber optic 

immunosensors for cardiovascular disease diagnosis: 

quantification of Protein C, Factor V Leiden, and cardiac 

Troponin T in plasma. Adv Exp Med Biol 2003;510115-9 

Does not report concordance, discordance, or 

reproducibility

Kyrle P A, Eichinger S. The risk of recurrent venous 

thromboembolism: the Austrian Study on Recurrent 

Venous Thromboembolism. Wien Klin Wochenschr 

2003;115(13-14):471-4 

No original data

Laczkovics C, Grafenhofer H, Kaider A et al. Risk of 

recurrence after a first venous thromboembolic event in 

young women. Haematologica 2007;92(9):1201-7 

Other reason 

Lapham E V, Kozma C, Weiss J O. Genetic discrimination: 

perspectives of consumers. Science 96;274(5287):621-4 

Doesn't address study question

Lechner D, Wiener C, Weltermann A et al. Comparison 

between idiopathic deep vein thrombosis of the upper and 

lower extremity regarding risk factors and recurrence. J 

Thromb Haemost 2008;6(4):1269-74. Epub 2008 Apr 26 

Does not report results separately for FV Leiden and/or 

PT 20210A

Lengyel T, Sasvari-Szekely M, Guttman A. Highthroughput genotyping of factor V Leiden mutation by 

ultrathin-layer agarose gel electrophoresis. J Chromatogr A 

99;853(1-2):519-25 

Published before 2000

Lensen R P, Bertina R M, de Ronde H et al. Venous 

thrombotic risk in family members of unselected 

individuals with factor V Leiden. Thromb Haemost 

2000;83(6):817-21 

No objective confirmation of VTE event

Lewandowski K, Rozek M, Zawilska K et al. An 

alternative method for identifying the factor V gene Leiden 

mutation. Thromb Res 97;85(2):105-13 

Published before 2000

Libourel E J, Bank I, Meinardi J R et al. Co-segregation of 

thrombophilic disorders in factor V Leiden carriers; the 

contributions of factor VIII, factor XI, thrombin activatable 

fibrinolysis inhibitor and lipoprotein(a) to the absolute risk 

of venous thromboembolism. Haematologica 

2002;87(10):1068-73 

Other reason 

Lim W, Eikelboom J W, Ginsberg J S. Inherited 

thrombophilia and pregnancy associated venous 

thromboembolism. BMJ 2007;334(7607):1318-21 

No original data

Lindqvist P G, Dahlback B. Reactions to awareness of 

activated protein C resistance carriership: a descriptive 

study of 270 women. Acta Obstet Gynecol Scand 

2003;82(5):467-70 

Other reason

Lindqvist P G, Olofsson P, Dahlback B. Use of selective 

factor V Leiden screening in pregnancy to identify 

candidates for anticoagulants. Obstet Gynecol 

2002;100(2):332-6 

Evaluates less than 10 probands or 10 family members

Linfert D R, Rezuke W N, Tsongalis G J. Rapid multiplex 

analysis for the factor V Leiden and prothrombin G20210A 

mutations associated with hereditary thrombophilia. Conn 

Med 98;62(9):519-25 

Published before 2000

Lloyd F J, Reyna V F, Whalen P. Accuracy and ambiguity 

in counseling patients about genetic risk. Arch Intern Med 

2001;161(20):2411-3 

Doesn't address study question

Lopaciuk S, Bielska-Falda H, Noszczyk W et al. Low 

molecular weight heparin versus acenocoumarol in the 

secondary prophylaxis of deep vein thrombosis. Thromb 

Haemost 99;81(1):26-31 

Doesn't address study question

Ludemann P, Nabavi D G, Junker R et al. Factor V Leiden 

mutation is a risk factor for cerebral venous thrombosis: a 

case-control study of 55 patients. Stroke 98;29(12):2507-10 

Case-control or retrospective cohort of probands
Appendix D: List of Excluded Articles 

 D-8 

Lutz C T, Foster P A, Noll W W et al. Multicenter 

evaluation of PCR methods for the detection of factor V 

Leiden (R506Q) genotypes. Clin Chem 98;44(6 Pt 1):1356-

8 

Published before 2000

Lyondagger E, Millsondagger A, Phan T et al. Detection 

and Identification of Base Alterations Within the Region of 

Factor V Leiden by Fluorescent Melting Curves. Mol 

Diagn 98;3(4):203-209 

Published before 2000

Mahadevan M S, Benson P V. Factor V null mutation 

affecting the Roche LightCycler factor V Leiden assay. 

Clin Chem 2005;51(8):1533-5 

Evaluates less than 10 samples

Maher C, Crowley D, Cullen C et al. Double fluorescentamplification refractory mutation detection (dF-ARMS) of 

the factor V Leiden and prothrombin mutations. Thromb 

Haemost 99;81(1):76-80 

Published before 2000

Makris M, Preston F E, Beauchamp N J et al. Coinheritance of the 20210A allele of the prothrombin gene 

increases the risk of thrombosis in subjects with familial 

thrombophilia. Thromb Haemost 97;78(6):1426-9 

Does not report results separately for FV Leiden and/or 

PT 20210A

Marcucci R, Liotta A A, Cellai A P et al. Increased plasma 

levels of lipoprotein(a) and the risk of idiopathic and 

recurrent venous thromboembolism. Am J Med 

2003;115(8):601-5 

Does not report results separately for FV Leiden and/or 

PT 20210A

Margaglione M, D'Andrea G, Colaizzo D et al. Coexistence 

of factor V Leiden and Factor II A20210 mutations and 

recurrent venous thromboembolism. Thromb Haemost 

99;82(6):1583-7 

Other reason 

Marteau T M, Croyle R T. The new genetics. Psychological 

responses to genetic testing. BMJ 98;316(7132):693-6 

Doesn't address study question

Martinelli I, Battaglioli T, Bucciarelli P et al. Risk factors 

and recurrence rate of primary deep vein thrombosis of the 

upper extremities. Circulation 2004;110(5):566-70 

Does not report results separately for FV Leiden and/or 

PT 20210A

Martinelli I, Mannucci P M, De Stefano V et al. Different 

risks of thrombosis in four coagulation defects associated 

with inherited thrombophilia: a study of 150 families. 

Blood 98;92(7):2353-8 

Other reason 

McColl M D, Walker I D, Greer I A. A mutation in the 

prothrombin gene contributing to venous thrombosis during 

pregnancy. Br J Obstet Gynaecol 98;105(8):923-5 

Doesn't address study question

McKenzie S B, Clare C N, Smith L A et al. Laboratory test 

utilization in the diagnosis of hypercoagulability. Clin Lab 

Sci 2000;13(4):215-21 

Less than 80% of the probands has the FV Leiden or 

PT 20210A mutation

Medina P, Navarro S, Estelles A et al. Influence of the 

4600A/G and 4678G/C polymorphisms in the endothelial 

protein C receptor (EPCR) gene on the risk of venous 

thromboembolism in carriers of factor V Leiden. Thromb 

Haemost 2005;94(2):389-94 

Case-control or retrospective cohort of probands

Meinardi J R, Middeldorp S, de Kam P J et al. Risk of 

venous thromboembolism in carriers of factor V Leiden 

with a concomitant inherited thrombophilic defect: a 

retrospective analysis. Blood Coagul Fibrinolysis 

2001;12(8):713-20 

Other reason 

Meltzer M E, Lisman T, Doggen C J et al. Synergistic 

Effects of Hypofibrinolysis and Genetic and Acquired Risk 

Factors on the Risk of a First Venous Thrombosis. PLoS 

Med 2008;5(5):e97 

Case-control or retrospective cohort of probands

Meyer M, Kutscher G, Vogel G. Simultaneous genotyping 

for factor V Leiden and prothrombin G20210A variant by a 

multiplex PCR-SSCP assay on whole blood. Thromb 

Haemost 99;81(1):162-3 

Published before 2000

Minano A, Ordonez A, Espana F et al. AB0 blood group 

and risk of venous or arterial thrombosis in carriers of 

factor V Leiden or prothrombin G20210A polymorphisms. 

Haematologica 2008;93(5):729-34 

Case-control or retrospective cohort of probands

Misgav M, Eldor A, Berliner S. [A new mutation in the 

prothrombin gene (G20210A) and the risk for venous and 

arterial thromboembolism]. Harefuah 2000;139(1-2):51-6 

Non-English article

Mitterer M, Lanthaler A J, Mair W et al. Simultaneous 

detection of FV Q506 and prothrombin 20210 A variation 

by allele-specific PCR. Haematologica 99;84(3):204-7 

Published before 2000

Moll S, Escobar M. Prothrombin and factor V mutations in 

women with a history of thrombosis during pregnancy and 

the puerperium. N Engl J Med 2000;342(26):1996-7 

Doesn't address study question

Muriel G, Christine B, Philippe J et al. An even easier 

method for one-step detection of both FV Leiden and FII 

G20210A transition. Blood 98;92(9):3478-9 

Published before 2000

Murphy K M, Eshleman J R. Simultaneous sequencing of 

multiple polymerase chain reaction products and combined 
Appendix D: List of Excluded Articles 

 D-9 

polymerase chain reaction with cycle sequencing in single 

reactions. Am J Pathol 2002;161(1):27-33 

Does not report concordance, discordance, or 

reproducibility

Murphy K M, Berg K D, Eshleman J R. Sequencing of 

genomic DNA by combined amplification and cycle 

sequencing reaction. Clin Chem 2005;51(1):35-9 

Evaluates only DNA extraction methods

Mustafa S, Mannhalter C, Rintelen C et al. Clinical features 

of thrombophilia in families with gene defects in protein C 

or protein S combined with factor V Leiden. Blood Coagul 

Fibrinolysis 98;9(1):85-9 

No objective confirmation of VTE event

Nagy P L, Schrijver I, Zehnder J L. Molecular Diagnosis of 

Hypercoagulable States. Lab. Med. 2004;35(4):214-221 

No original data

Naudziunas A, Miliauskas S. Factor V Leiden and post 

thromboembolic pulmonary hypertension. Medicina 

(Kaunas) 2003;39(12):1171-4 

Did not study venous thrombosis

Navarro S, Medina P, Mira Y et al. Haplotypes of the 

EPCR gene, prothrombin levels, and the risk of venous 

thrombosis in carriers of the prothrombin G20210A 

mutation. Haematologica 2008;93(6):885-91. Epub 2008 

Apr 9 

Case-control or retrospective cohort of probands

Neoh S H, Brisco M J, Firgaira F A et al. Rapid detection 

of the factor V Leiden (1691 G > A) and 

haemochromatosis (845 G > A) mutation by fluorescence 

resonance energy transfer (FRET) and real time PCR. J 

Clin Pathol 99;52(10):766-9 

Published before 2000

Noboa S, Le Gal G, Lacut K et al. Family history as a risk 

factor for venous thromboembolism. Thromb Res 

2008;122(5):624-9. Epub 2008 Feb 20 

Does not report results separately for FV Leiden and/or 

PT 20210A

Oguzulgen I K, Demirtas S, Erkekol F O et al. The Role of 

Plasminogen Activator Inhibitor-1 Polymorphism, FactorV-Leiden, and Prothrombin-20210 Mutations in Pulmonary 

Thromboembolism. Clin Appl Thromb Hemost 2007;[Epub 

ahead of print] 

Case-control or retrospective cohort of probands

Oguzulgen I K, Ekim N N, Akar N et al. The role of 

thrombophilic risk factors in the severity of pulmonary 

thromboembolism. Eur Respir J 2002;19(4):709-11 

Doesn't address study question

Olds R J, Fitches A C, Geary C P. The multigenic basis for 

venous thrombosis. Br J Haematol 2000;109(3):508-11 

No original data

Orum H, Jakobsen M H, Koch T et al. Detection of the 

factor V Leiden mutation by direct allele-specific 

hybridization of PCR amplicons to photoimmobilized 

locked nucleic acids. Clin Chem 99;45(11):1898-905 

Published before 2000

Otterson G A, Monahan B P, Harold N et al. Clinical 

significance of the FV:Q506 mutation in unselected 

oncology patients. Am J Med 96;101(4):406-12 

Evaluates less than 10 probands or 10 family members

Pabinger I, Grafenhofer H, Kaider A et al. Risk of 

pregnancy-associated recurrent venous thromboembolism 

in women with a history of venous thrombosis. J Thromb 

Haemost 2005;3(5):949-54 

Other reason 

Palareti G, Cosmi B, Legnani C et al. D-dimer testing to 

determine the duration of anticoagulation therapy. N Engl J 

Med 2006;355(17):1780-9 

Other reason

Palareti G, Legnani C, Cosmi B et al. Poor anticoagulation 

quality in the first 3 months after unprovoked venous 

thromboembolism is a risk factor for long-term recurrence. 

J Thromb Haemost 2005;3(5):955-61 

Doesn't address study question

Paolo, G., Marco, C., Silvia, M., Alessandra, V., Fabbio, 

M., Nicoletta, C., Giorgio, F., Carlo Federico, P., and 

Sergio, B. Impact of storage conditions on genetic analysis 

or viral load determination in clinical specimens. Clin 

Chem Lab Med, 2007;[Epub ahead of print] 

Doesn't address study question 

Paschoa A F, Guillaumon A T. Impact of screening on 

thrombophilia for patients with venous thrombosis. Int 

Angiol 2006;25(1):52-9 

Other reason

Patri S, Salmeron S, Caillon M et al. Multiplex PCR for 

one-step determination of the G20210A variation and the 

factor V Leiden mutation by denaturing gradient gel 

electrophoresis (DGGE). Thromb Haemost 99;81(2):313-4 

Published before 2000

Patrushev L I, Zykova E S, Kayushin A L et al. New DNA 

diagnostic system for detection of factor V Leiden. Thromb 

Res 98;92(6):251-9 

Published before 2000

Pecheniuk N M, Marsh N A, Walsh T P et al. Use of first 

nucleotide change technology to determine the frequency 

of factor V Leiden in a population of Australian blood 

donors. Blood Coagul Fibrinolysis 97;8(8):491-5 

Published before 2000

Peng F, Triplett D, Barna L. Direct PCR of leukocytes 

treated with microwave irradiation to detect factor V 

Leiden mutation. Thromb Res 96;82(2):193-7 

Published before 2000

Pernod G, Mossuz P, Polack B. Optimized factor V gene 
Appendix D: List of Excluded Articles 

 D-10 

mutation detection using buffy-coat direct PCR. 

Biotechniques 97;22(5):837, 840, 841 

Published before 2000

Pettila V, Kaaja R, Leinonen P et al. Thromboprophylaxis 

with low molecular weight heparin (dalteparin) in 

pregnancy. Thromb Res 99;96(4):275-82 

Doesn't address study question

Pini M, Aiello S, Manotti C et al. Low molecular weight 

heparin versus warfarin in the prevention of recurrences 

after deep vein thrombosis. Thromb Haemost 94;72(2):191-

7 

Doesn't address study question

Prandoni P, Lensing A W, Piccioli A et al. Recurrent 

venous thromboembolism and bleeding complications 

during anticoagulant treatment in patients with cancer and 

venous thrombosis. Blood 2002;100(10):3484-8 

Doesn't address study question

Prandoni P, Lensing A W, Prins M H et al. Residual venous 

thrombosis as a predictive factor of recurrent venous 

thromboembolism. Ann Intern Med 2002;137(12):955-60 

Doesn't address study question

Press R D. Thrombophilic mutations impart a high risk of 

pregnancy-related venous thrombosis. Mol Diagn 

2000;5(2):158 

Doesn't address study question

Quehenberger P, Handler S, Mannhalter C et al. The Factor 

V (Leiden) test: evaluation of an assay based on dilute 

Russell Viper Venom Time for the detection of the Factor 

V Leiden mutation. Thromb Res 99;96(2):125-33 

Published before 2000

Raoul M, Mathonnet F, Peltier J Y et al. An improved 

method for the detection of the G20210A transition in the 

prothrombin gene. Thromb Res 97;88(5):441-3 

Published before 2000

Reitsma P H, van der, Velden P A et al. Use of the direct 

RNA amplification technique NASBA to detect factor V 

Leiden, a point mutation associated with APC resistance. 

Blood Coagul Fibrinolysis 96;7(6):659-63 

Published before 2000

Ren, Y., Rezania, S., and Kang, K. A. Biosensor for 

diagnosing Factor V Leiden, a single amino acid mutated 

abnormality of Factor V. Adv Exp Med Biol 2008;614:245-

52 

Other reason 

Ridker P M, Goldhaber S Z, Danielson E et al. Long-term, 

low-intensity warfarin therapy for the prevention of 

recurrent venous thromboembolism. N Engl J Med 

2003;348(15):1425-34 

Other reason 

Rintelen C, Mannhalter C, Lechner K et al. No evidence for 

an increased risk of venous thrombosis in patients with 

factor V Leiden by the homozygous 677 C to T mutation in 

the methylenetetrahydrofolate-reductase gene. Blood 

Coagul Fibrinolysis 99;10(2):101-5 

Does not report results separately for FV Leiden and/or 

PT 20210A

Rintelen C, Pabinger I, Knobl P et al. Probability of 

recurrence of thrombosis in patients with and without factor 

V Leiden. Thromb Haemost 96;75(2):229-32 

Other reason 

Robert A, Aillaud M F, Eschwege V et al. ABO blood 

group and risk of venous thrombosis in heterozygous 

carriers of factor V Leiden. Thromb Haemost 

2000;83(4):630-1 

Doesn't address study question

Rodeghiero F, Tosetto A. The epidemiology of inherited 

thrombophilia: the VITA Project. Vicenza Thrombophilia 

and Atherosclerosis Project. Thromb Haemost 

97;78(1):636-40 

Doesn't address study question

Ruggeri M, Gisslinger H, Tosetto A et al. Factor V Leiden 

mutation carriership and venous thromboembolism in 

polycythemia vera and essential thrombocythemia. Am J 

Hematol 2002;71(1):1-6 

Other reason

Russeva M G, Janakiev P J, Kirov S A et al. A simple 

method for detection of factor V R506Q (Leiden) mutation 

in dried blood spots. Clin Chim Acta 99;284(1):89-92 

Published before 2000

Ryan D, Nuccie B, Arvan D. Non-PCR-dependent 

detection of the factor V Leiden mutation from genomic 

DNA using a homogeneous invader microtiter plate assay. 

Mol Diagn 99;4(2):135-44 

Published before 2000

Sabbagh A S, Ibrahim G, Kanaan Z et al. Prevalence of the 

prothrombin G20210A polymorphism in the Lebanese 

population: use of a reverse hybridization strip assay 

approach. Mol Biol Rep 2007;[Epub ahead of print] 

Does not report concordance, discordance, or 

reproducibility

Saffroy R, Lemoine A, Haas P et al. Rapid automated 

simultaneous screening of (G1691A) Factor V, (G20210A) 

prothrombin, and (C677T) methylenetetrahydrofolate 

reductase variants by multiplex PCR using fluorescence 

scanning technology. Genet Test 2002;6(3):233-6 

Does not report concordance, discordance, or 

reproducibility

Samama M M, Rached R A, Conard J et al. [Pregnancyassociated venous thrombosis in women with hereditary 

heterozygous factor V Leiden and/or factor II gene 

mutations]. Bull Acad Natl Med 2004;188(8):1377-93; 

discussion 1393-6 

Non-English article
Appendix D: List of Excluded Articles 

 D-11 

Samama M-M, Rached R A, Conard J et al. Pregnancyassociated venous thrombosis in women with hereditary 

heterozygous factor V Leiden and/or factor II gene 

mutations: RISQUE DE THROMBOSE LIE A LA 

GROSSESSE CHEZ LES FEMMES PORTEUSES D'UNE 

MUTATION HETEROZYGOTE DU FACTEUR V 

LEIDEN, DU FACTEUR II OU DE LEUR 

ASSOCIATION. Bull. Acad. Natl. Med. 

2004;188(8):1377-1396 

Non-English article

Santamaria A, Mateo J, Oliver A et al. Risk of thrombosis 

associated with oral contraceptives of women from 97 

families with inherited thrombophilia: high risk of 

thrombosis in carriers of the G20210A mutation of the 

prothrombin gene. Haematologica 2001;86(9):965-71 

Other reason 

Sasvari-Szekely M, Gerstner A, Ronai Z et al. Rapid 

genotyping of factor V Leiden mutation using single-tube 

bidirectional allele-specific amplification and automated 

ultrathin-layer agarose gel electrophoresis. Electrophoresis 

2000;21(4):816-21 

Does not report concordance, discordance, or 

reproducibility

Scharnhorst V, Gijselhart-Nivillac N J F, Goorhuis A D et 

al. Detection of factor V Leiden and factor II mutations by 

using duplex PCR: DETECTIE VAN DE FACTOR-VLEIDEN- EN FACTOR-II-MUTATIES MET BEHULP 

VAN EEN DUPLEX-PCR. Ned. Tijdschr. Klin. Chem. 

Lab. Geneeskd. 2004;29(1):33-35 

Non-English article

Schattner A, Kasher I, Berrebi A. Causes and outcome of 

deep-vein thrombosis in otherwise-healthy patients under 

50 years. QJM 97;90(4):283-7 

Does not report results separately for FV Leiden and/or 

PT 20210A

Schramm W, Heinemann L A, Spannagl M et al. [The 

Bavarian Thromboembolic Risk Cohort Study (BATER). 

Study protocol, state of the investigation and first results]. 

Dtsch Med Wochenschr 2000;125(1-2):2-6 

Non-English article

Schroder W, Koesling M, Wulff K et al. Large-scale 

screening for factor V Leiden mutation in a north-eastern 

German population. Haemostasis 96;26(5):233-6 

Published before 2000

Schulman S, Granqvist S, Holmstrom M et al. The duration 

of oral anticoagulant therapy after a second episode of 

venous thromboembolism. The Duration of 

Anticoagulation Trial Study Group. N Engl J Med 

97;336(6):393-8 

Doesn't address study question

Schulman S, Lindmarker P, Holmstrom M et al. Postthrombotic syndrome, recurrence, and death 10 years after 

the first episode of venous thromboembolism treated with 

warfarin for 6 weeks or 6 months. J Thromb Haemost 

2006;4(4):734-42 

Other reason 

Schulman S, Wahlander K, Lundstrom T et al. Secondary 

prevention of venous thromboembolism with the oral direct 

thrombin inhibitor ximelagatran. N Engl J Med 

2003;349(18):1713-21 

Doesn't address study question

Scobie G A, Ho S T, Dolan G et al. Development of a rapid 

DNA screening procedure for the Factor V Leiden 

mutation. Clin Mol Pathol 96;49(6):M361-M363 

Published before 2000

Shrivastava S, Ridker P M, Glynn R J et al. D-dimer, factor 

VIII coagulant activity, low-intensity warfarin and the risk 

of recurrent venous thromboembolism. J Thromb Haemost 

2006;4(6):1208-14 

Doesn't address study question

Simioni P, Prandoni P, Lensing A W et al. The risk of 

recurrent venous thromboembolism in patients with an 

Arg506-->Gln mutation in the gene for factor V (factor V 

Leiden). N Engl J Med 97;336(6):399-403 

Other reason 

Simkova M, Batorova A, Dostalova K et al. Factor V 

Leiden in patients with venous thrombosis in Slovak 

population. Gen Physiol Biophys 2004;23(4):435-42 

Doesn't address study question

Sinclair G D, Low S, Poon M C. A hemi-nested, allele 

specific, whole blood PCR assay for the detection of the 

factor V Leiden mutation. Thromb Haemost 97;77(6):1154-

5 

Published before 2000

Sitbon G, Hurtig M, Palotie A et al. A colorimetric 

minisequencing assay for the mutation in codon 506 of the 

coagulation factor V gene. Thromb Haemost 97;77(4):701-

3 

Published before 2000

Snyder T M, McGown L B. Multiplex single strand 

conformation polymorphism analysis by capillary 

electrophoresis with on-the-fly fluorescence lifetime 

detection. Appl Spectrosc 2005;59(3):335-9 

Does not report concordance, discordance, or 

reproducibility

Solem C A, Loftus E V, Tremaine W J et al. Venous 

thromboembolism in inflammatory bowel disease. Am J 

Gastroenterol 2004;99(1):97-101 

Doesn't address study question

Souto J C, Mateo J, Soria J M et al. Homozygotes for 

prothrombin gene 20210 A allele in a thrombophilic family 

without clinical manifestations of venous 

thromboembolism. Haematologica 99;84(7):627-32 

Did not study venous thrombosis 
Appendix D: List of Excluded Articles 

 D-12 

Sproul A M, Chalmers E A. Multiplex PCR for the 

detection of the factor V Leiden and prothrombin 20210A 

mutations. Methods Mol Med 2004;9179-87 

Does not report concordance, discordance, or 

reproducibility 

Srur A E, Vargas R C, Salas F S et al. Primary 

thombophilia. Report of 93 cases and 12 asymptomatic 

relatives: TROMBOFILIA PRIMARIA: DETECCION Y 

MANIFESTACION CLINICA EN 105 CASOS. Rev. Med. 

Chile 2004;132(12):1466-1473 

Non-English article

Stegnar M, Peternel P, Uhrin P et al. Fibrinolysis in 

patients with the 1691 G->A mutation in factor V gene and 

history of deep vein thrombosis. FIBRINOLYSIS 

PROTEOLYSIS 97;11(4):201-207 

Doesn't address study question

Strobl F J, Hoffman S, Huber S et al. Activated protein C 

resistance assay performance: improvement by sample 

dilution with factor V-deficient plasma. Arch Pathol Lab 

Med 98;122(5):430-3 

Published before 2000

Tirado I, Mateo J, Soria J M et al. Contribution of 

prothrombin 20210A allele and factor V Leiden mutation to 

thrombosis risk in thrombophilic families with other 

hemostatic deficiencies. Haematologica 2001;86(11):1200-

8 

Other reason 

Tormene D, Fortuna S, Tognin G et al. The incidence of 

venous thromboembolism in carriers of antithrombin, 

protein C or protein S deficiency associated with the HR2 

haplotype of factor V: a family cohort study. J Thromb 

Haemost 2005;3(7):1414-20 

Doesn't address study question

Tormene D, Simioni P, Prandoni P et al. The risk of fetal 

loss in family members of probands with factor V Leiden 

mutation. Thromb Haemost 99;82(4):1237-9 

Published before 2000

Tosetto A, Missiaglia E, Frezzato M et al. The VITA 

project: prothrombin G20210A mutation and venous 

thromboembolism in the general population. Thromb 

Haemost 99;82(5):1395-8 

Case-control or retrospective cohort of probands

Toulon P, Lequerrec A, Piquet P et al. Significant influence 

of the instrument on the result of the ProC Global assay. A 

multicenter evaluation using lyophilized plasmas and 

frozen plasma samples from carriers and non-carriers of the 

factor V Leiden mutation. Thromb Res 2002;107(3-4):181-

8 

Evaluates only APC resistance

Tran M, Spencer F A. Thromboepidemiology: identifying 

patients with heritable risk for thrombin-mediated 

thromboembolic events. Am Heart J 2005;149(1 Suppl):S9-

18 

No original data

Uitte de, Willige S, De Visser M C et al. Selectin 

haplotypes and the risk of venous thrombosis: influence of 

linkage disequilibrium with the factor V Leiden mutation. J 

Thromb Haemost 2008;6(3):478-85 

Case-control or retrospective cohort of probands

Ulvik A, Ren J, Refsum H et al. Simultaneous 

determination of methylenetetrahydrofolate reductase 

C677T and factor V G1691A genotypes by mutagenically 

separated PCR and multiple-injection capillary 

electrophoresis. Clin Chem 98;44(2):264-9 

Published before 2000

van Boven H H, Vandenbroucke J P, Briet E et al. Genegene and gene-environment interactions determine risk of 

thrombosis in families with inherited antithrombin 

deficiency. Blood 99;94(8):2590-4 

Does not report results separately for FV Leiden and/or 

PT 20210A

van de, Locht L T, Kuypers A W et al. Semi-automated 

detection of the factor V mutation by allele specific 

amplification and capillary electrophoresis. Thromb 

Haemost 95;74(5):1276-9 

Published before 2000

van der, Meer F J, Koster T et al. The Leiden 

Thrombophilia Study (LETS). Thromb Haemost 

97;78(1):631-5 

No original data

van Hylckama, Vlieg A, Baglin C A et al. Proof of 

principle of potential clinical utility of multiple SNP 

analysis for prediction of recurrent venous thrombosis. J 

Thromb Haemost 2008;6(5):751-4 

Other reason 

Van Rooijen M, Bremme K. A family history can display a 

synergistic effect of atherogenic and prothrombotic risk in 

pregnancy. Acta Obstet Gynecol Scand 2001;80(9):873-4 

Doesn't address study question

Vandenbroucke J P, Briet E, Helmerhorst F et al. [Oral 

contraception increases the chance of venous thrombosis in 

factor V Leiden mutation]. Ned Tijdschr Geneeskd 

95;139(21):1067-71 

Non-English article

Varga Elizabeth A, Moll Stephan. Prothrombin 20210 

Mutation (Factor II Mutation). Circulation 

2004;110(3):e15-18 

No original data

Vatan R, Delpeu K, Vergnes C et al. [Clinicobiological 

study of 100 symptomatic patients with factor V Leiden 

mutation]. Rev Med Interne 2000;21(5):465-6 

Non-English article

Veiga F, Escriba A, Maluenda M P et al. Low molecular 

weight heparin (enoxaparin) versus oral anticoagulant 
Appendix D: List of Excluded Articles 

 D-13 

therapy (acenocoumarol) in the long-term treatment of deep 

venous thrombosis in the elderly: a randomized trial. 

Thromb Haemost 2000;84(4):559-64 

Doesn't address study question

Voelkerding K V, Huber S, Strobl F et al. Resistance to 

Activated Protein C: Comparison of Three Different PCR 

Methods for Detection FV R506Q. Mol Diagn 96;1(4):297-

304 

Published before 2000

von Ahsen N, Oellerich M, Schutz E. A method for 

homogeneous color-compensated genotyping of factor V 

(G1691A) and methylenetetrahydrofolate reductase 

(C677T) mutations using real-time multiplex fluorescence 

PCR. Clin Biochem 2000;33(7):535-9 

Does not report concordance, discordance, or 

reproducibility

von Ahsen N, Oellerich M, Armstrong V W et al. 

Application of a thermodynamic nearest-neighbor model to 

estimate nucleic acid stability and optimize probe design: 

prediction of melting points of multiple mutations of 

apolipoprotein B-3500 and factor V with a hybridization 

probe genotyping assay on the LightCycler. Clin Chem 

99;45(12):2094-101 

Published before 2000

von Ahsen N, Schutz E, Armstrong V W et al. Rapid 

detection of prothrombotic mutations of prothrombin 

(G20210A), factor V (G1691A), and 

methylenetetrahydrofolate reductase (C677T) by real-time 

fluorescence PCR with the LightCycler. Clin Chem 

99;45(5):694-6 

Does not report concordance, discordance, or 

reproducibility

Walz T, Geisel J, Bodis M et al. Fluorescence-based singlestrand conformation polymorphism analysis of mutations 

by capillary electrophoresis. Electrophoresis 

2000;21(2):375-9 

Does not report concordance, discordance, or 

reproducibility

Welbourn J T, Maiti S, Paley J. Factor V Leiden detection 

by polymerase chain reaction-restriction fragment length 

polymorphism with mutagenic primers in a multiplex 

reaction with Pro G20210A--a novel technique. 

Hematology 2003;8(2):73-5 

Does not report concordance, discordance, or 

reproducibility

Weltermann A, Eichinger S, Bialonczyk C et al. The risk of 

recurrent venous thromboembolism among patients with 

high factor IX levels. J Thromb Haemost 2003;1(1):28-32 

Other reason 

White R H, Chan W S, Zhou H et al. Recurrent venous 

thromboembolism after pregnancy-associated versus 

unprovoked thromboembolism. Thromb Haemost 

2008;100(2):246-52 

Doesn't address study question

Wilde J T, O'Sullivan J J, Roper J L et al. A novel ELISAbased primer extension assay for the detection of the factor 

V Leiden mutation. Br J Haematol 99;106(2):427-30 

Published before 2000

Wisotzkey J D, Bell T, Monk J S. Simultaneous 

polymerase chain reaction restriction fragment length 

polymorphism identification of the factor V Leiden allele 

and the prothrombin 20210A mutation. Diagn Mol Pathol 

98;7(3):180-3 

Published before 2000

Wong T, Nightingale J, Winter M et al. Spontaneous 

venous thrombosis in inflammatory bowel disease: 

relevance of factor V Leiden and the prothrombin gene 

mutation. J Thromb Haemost 2003;1(6):1326-8 

Doesn't address study question

Xu X, Bauer K A, Griffin J H. Two multiplex PCR-based 

DNA assays for the thrombosis risk factors prothrombin 

G20210A and coagulation factor V G1691A 

polymorphisms. Thromb Res 99;93(6):265-9 

Does not report concordance, discordance, or 

reproducibility

Yandava C N, Zappulla D C, Korf B R et al. ARMS test for 

diagnosis of factor VLeiden mutation, a common cause of 

inherited thrombotic tendency. J Clin Lab Anal 

96;10(6):414-7 

Published before 2000

Young L, Ockelford P, Milne D et al. Post-treatment 

residual thrombus increases the risk of recurrent deep vein 

thrombosis and mortality. J Thromb Haemost 

2006;4(9):1919-24 

Doesn't address study question

Zehnder J L, Benson R C, Cheng S. A microplate allelespecific oligonucleotide hybridization assay for detection of 

factor V Leiden. Diagn Mol Pathol 97;6(6):347-52 

Published before 2000

Zehnder J, Van Atta R, Jones C et al. Cross-linking 

hybridization assay for direct detection of factor V Leiden 

mutation. Clin Chem 97;43(9):1703-8 

Published before 2000

Zotz R B, Maruhn-Debowski B, Scharf R E. Mutation in 

the gene coding for coagulation factor V and resistance to 

activated protein C: detection of the genetic mutation by 

oligonucleotide ligation assay using a semi-automated 

system. Thromb Haemost 96;76(1):53-5 

Published before 2000 
Impact of Testing for Factor V Leiden (G1691A) and Prothrombin (G20210A) 

Mutations on Health Outcomes in Adults with a History of Venous 

Thromboembolic Events and Their Adult Family Members

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Reviewer Name: _____________________________ 

Please use this form to enter your comments on the sections assigned to you. In addition to reviewing the 

sections assigned to you, we will accept any additional comments you include. 

1 Structured Abstract: 

2 Executive Summary :

3 Chapter 1: Introduction : 

. 
Impact of Testing for Factor V Leiden (G1691A) and Prothrombin (G20210A) 

Mutations on Health Outcomes in Adults with a History of Venous 

Thromboembolic Events and Their Adult Family Members

Feedback Form 

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4 Chapter 2: Methods 

 

5 Chapter 3: Results 

 

 Comments concerning description of results: Key Questions #3a 

What is the evidence that the presence of FVL alone or prothrombin G20210A alone or the two in 

combination predicts risk of recurrent VTE in individuals (probands) who have had VTE and predicts the 

risk of VTE in the probands’ family members who have been tested? Does the testing add predictive 

information beyond clinical data?: 
Impact of Testing for Factor V Leiden (G1691A) and Prothrombin (G20210A) 

Mutations on Health Outcomes in Adults with a History of Venous 

Thromboembolic Events and Their Adult Family Members

Feedback Form 

Page 3 of 5 

 Comments concerning description of results: Key Questions #3b 

What is the evidence that demographic or clinical factors modify the relationship between the presence of 

FVL or prothrombin G20210A and the risk of VTE?: 

 Comments concerning description of results: Key Question #4a 

What is the evidence that clinicians manage patients differently based on the results of testing for FVL or 

prothrombin G20210A? How do clinicians manage anticoagulation of individuals who have had testing 

compared to those who have not had testing? What other diagnostic tests do clinicians order or not order 

based on testing results? What recommendations do clinicians make regarding other therapies and 

exposures based on testing results?: 

 Comments concerning description of results: Key Question #4b 

What is the evidence that testing, and the resultant management, reduces VTE related-outcomes or has 

other benefits in individuals who have had VTE or in the probands’ family members who have been 

tested?: 

 Comments concerning description of results: Key Question #4c 

What is the evidence of harms to individuals with VTE or to the probands’ family members who are tested 

for FVL or prothrombin G20210A as a result of testing or as a result of changed management based on the 

test results?: 
Impact of Testing for Factor V Leiden (G1691A) and Prothrombin (G20210A) 

Mutations on Health Outcomes in Adults with a History of Venous 

Thromboembolic Events and Their Adult Family Members

Feedback Form 

Page 4 of 5 

 Comments concerning description of results: Key Question #4d 

What is the evidence that testing for FVL alone, prothrombin G20210A alone, or the two tests in 

combination is a cost-effective strategy when caring for a patient with VTE or a family member of a 

proband?: 

6 Chapter 4: Discussion 

7 Figures, Tables, and Appendix materials including Evidence Tables: 

As above 

8 Specific comments about text (e.g., typos, unclear sentence) 

 Chapter Page Line Comment 

 
Impact of Testing for Factor V Leiden (G1691A) and Prothrombin (G20210A) 

Mutations on Health Outcomes in Adults with a History of Venous 

Thromboembolic Events and Their Adult Family Members

Feedback Form 

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9 General comments: 

Thank you for your feedback. Please e-mail to Renee F. Wilson 

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Appendix F 
Appendix G 
Appendix G, Evidence Table 1

Page 33

Evidence Table 1. Quality of studies addressing Analytic Validity of Tests to Identify Factor V Leiden and Prothrombin G20210A Mutations 

Reference List 

 1. Hessner MJ, Budish MA, Friedman KD. Genotyping of factor V G1691A 

(Leiden) without the use of PCR by invasive cleavage of oligonucleotide 

probes. Clin Chem 2000; 46(8 Pt 1):1051-6. 

 2. Kowalski A, Radu D, Gold B. Colorimetric microwell plate detection of 

the factor V Leiden mutation. Clin Chem 2000; 46(8 Pt 1):1195-8. 

 3. Ledford M, Friedman KD, Hessner MJ, Moehlenkamp C, Williams TM, 

Larson RS. A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay. J Mol Diagn 2000; 2(2):97-104. 

 4. Nauck M, Marz W, Wieland H. Evaluation of the Roche diagnostics 

LightCycler-Factor V Leiden Mutation Detection Kit and the LightCyclerProthrombin Mutation Detection Kit. Clin Biochem 2000; 33(3):213-6. 

 5. Pecheniuk NM, Marsh NA, Walsh TP. Multiple analysis of three common 

genetic alterations associated with thrombophilia. Blood Coagul Fibrinolysis 2000; 11(2):183-9. 

 6. Sanders Sevall J. Factor V Leiden genotyping using real-time fluorescent 

polymerase chain reaction. Mol Cell Probes 2000; 14(4):249-53. 

 7. Benson JM, Ellingsen D, El-Jamil M et al. Factor V Leiden and factor V 

R2 allele: high-throughput analysis and association with venous thromboembolism. Thromb Haemost 2001; 86(5):1188-92. 

 8. Crockett AO, Wittwer CT. Fluorescein-labeled oligonucleotides for realtime pcr: using the inherent quenching of deoxyguanosine nucleotides. Anal 

Biochem 2001; 290(1):89-97. 

 9. Humeny A, Bonk T, Berkholz A, Wildt L, Becker CM. Genotyping of 

thrombotic risk factors by MALDI-TOF mass spectrometry. Clin Biochem 2001; 34(7):531-6. 

 10. Parks SB, Popovich BW, Press RD. Real-time polymerase chain reaction 

with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes. Am J Clin Pathol 2001; 115(3):439-47. 

 11. Potter CG, Liu YT, Rees DC. Factor V Leiden mutation screened by PCR 

and detected with lanthanide-labeled probes. Genet Test 2001; 5(4):291-7. 

 12. Reyes AA, Ugozzoli LA, Lowery JD et al. Linked linear amplification: a 

new method for the amplification of DNA. Clin Chem 2001; 47(1):31-40. 

 13. Rhodes RB, Lewis K, Shultz J et al. Analysis of the factor V Leiden 

mutation using the READIT Assay. Mol Diagn 2001; 6(1):55-61. 

 14. Tsongalis GJ, Rainey BJ, Hodges KA. READIT: a novel technology used 

in the interrogation of nucleic acid sequences for single-nucleotide polymorphisms. Exp Mol Pathol 2001; 71(3):222-5. 

 15. Evans JG, Lee-Tataseo C. Determination of the factor V Leiden singlenucleotide polymorphism in a commercial clinical 

NanoChip microelectronic array technology. Clin 

16. Huang TJ, Liu M, Knight LD, Grody WW, M

electrochemical detection scheme for identificatiopolymorphisms using hairpin-forming probes. Nuc30(12):e55. 17. Hung K, Sun X, Ding H, Kalafatis M, Simionassisted laser desorption/ionization time-of-flight bthe 1691G --> A mutation in the factor V gene. Bl2002; 13(2):117-22. 18. Ozkan D, Erdem A, Kara P et al. Allele-spec

factor V Leiden mutation from polymerase chain rlabel-free electrochemical genosensor. Anal Chem 

19. Alsmadi OA, Bornarth CJ, Song W et al. Hig

directly from genomic DNA using a rolling circle BMC Genomics 2003; 4(1):21. 20. Cooper PC, Cooper SM, Smith JM, Kitchen Sthe Roche LightCycler: a simple and rapid method V Leiden and prothrombin G20210A genotypes frthe need for DNA extraction. Blood Coagul Fibrin

21. El Housni H, Heimann P, Parma J, Vassart G

polymorphism genotyping by melting analysis of dexamples using factor V Leiden and prothrombin Chem 2003; 49(10):1669-72. 22. Moser MJ, Marshall DJ, Grenier JK et al. Exrecognition of an unnatural base pair to develop a system. Clin Chem 2003; 49(3):407-14. 23. Murphy K, Hafez M, Philips J, Yarnell K, Guof temperature gradient capillary electrophoresis fLeiden mutation: coincident identification of a novV. Mol Diagn 2003; 7(1):35-40. 24. Ozsoz M, Erdem A, Kerman K et al. Electroc

colloidal gold nanoparticles for the detection of Fausing disposable pencil graphite electrodes. Anal C

25. Schroell-Metzger B, Dicato M, Bosseler M, B

standard PCR and the LightCycler technique to demutations: an efficiency and cost study. Clin Chem 5. 26. Simundic AM, Topic E, Stefanovic M. DetecPCR-SSCP using GMA precast Elchrom scientific 
Appendix G, Evidence Table 1

Page 34

Hemost 2003; 9(3):227-31. 

 27. Vaughn CP, Elenitoba-Johnson KS. Hybridization-induced dequenching 

of fluorescein-labeled oligonucleotides: a novel strategy for PCR detection and genotyping. Am J Pathol 2003; 163(1):29-35. 

 28. Behrens M, Lange R. A highly reproducible and economically competitive 

SNP analysis of several well characterized human mutations. Clin Lab 2004; 50(5-6):305-16. 

 29. French C, Li C, Strom C et al. Detection of the factor V Leiden mutation 

by a modified photo-cross-linking oligonucleotide hybridization assay. Clin Chem 2004; 50(2):296-305. 

 30. Liew M, Pryor R, Palais R et al. Genotyping of single-nucleotide 

polymorphisms by high-resolution melting of small amplicons. Clin Chem 2004; 50(7):1156-64. 

 31. Louis M, Dekairelle AF, Gala JL. Rapid combined genotyping of factor V, 

prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction. Clin Chem Lab Med 2004; 42(12):1364-9. 

 32. Angeline T, Bentley HA, Hawk AB et al. Prevalence of the Factor V 

G1691A and the Factor II/prothrombin G20210A gene polymorphisms among 

Tamilians. Exp Mol Pathol 2005; 79(1):9-13. 

 33. Castley A, Higgins M, Ivey J, Mamotte C, Sayer DC, Christiansen FT. 

Clinical applications of whole-blood PCR with real-time instrumentation. Clin Chem 2005; 51(11):2025-30. 

 34. Mammo L, Sheereen A, Saour T, Saour J. Comparative study between the 

Light Cycler and the PCR-restriction fragment length polymorphism in detecting factor V Leiden and factor II 20210G>A mutations. Clin Biochem 2006; 39(7):767-9. 

 35. Bravo-Osorio M, Bydlowski SP. Detection of methylenetetrahydrofolate 

reductase (MTHFR) C677T and prothrombin G20210A mutations: second restriction site for digestion control of PCR products. Clin Chim Acta 2000; 301(1-2):219-23. 

 36. Happich D, Madlener K, Schwaab R, Hanfland P, Potzsch B. Application 

of the TaqMan-PCR for genotyping of the prothrombin G20210A mutation and of the thermolabile methylenetetrahydrofolate reductase mutation. Thromb Haemost 2000; 84(1):144-5. 

 37. Meyer M, Kutscher G, Vogel G. The prothrombin nt20210 A allele as a 

risk factor for venous thromboembolism: detection of heterozygous and homozygous carriers by alternative methods. Thromb Res 2000; 97(5):359-63. 

 38. Gellings A, Holzem G, Wielckens K, Klingler KR. Prothrombin mutation: 

Employing the electrochemiluminescence technology of the elecsys(registered trademark) system for the detection of the point mutation at position 20210 in the 3' untranslated region of the prothrombin gene: PROTHROMBIN MUTATION: VERWENDUNG DER 

ELEKTROCHEMILUMINESZENZTECHNOLOELECSYS(registered trademark) SYSTEMS ZUR PUNKTMUTATION AN DER POSITION 20210 UNTRANSLATIERTEN. LaboratoriumsMedizin 

 39. Gilchrist A, Solomon N, Erickson D et al. Au

G20210A prothrombin mutation using the LCx miimmunoassay. Clin Chim Acta 2001; 314(1-2):249

 40. Hessner MJ, Friedman KD, Voelkerding KV 

genotyping of the factor II (prothrombin) G20210A assay. Clin Chem 2001; 47(11):2048-50. 

 41. Huber S, McMaster KJ, Voelkerding KV. An

engineered multiplex polymerase chain reaction-repolymorphism for detection of factor V Leiden anMol Diagn 2000; 2(3):153-7. 

 42. Leyte A, Smits PH, van Straalen JP, van Doo

Automated, simultaneous detection of the factor V 

(G20210A) variants using multiplex PCR and a linHaemost 2000; 83(2):354-5. 

 43. van den Bergh FA, van Oeveren-Dybicz AM

genotyping of the factor V Leiden and prothrombiPCR using dual-color detection. Clin Chem 2000; 

 44. DelRio-LaFreniere SA, McGlennen RC. Sim

amplification: a strategy using modified primer-temdetection--application to prothrombin 20210A (fac(1691A) gene mutations. Mol Diagn 2001; 6(3):20

 45. Endler G, Kyrle PA, Eichinger S, Exner M, M

mutagenically separated PCR: simultaneous singleV R506Q (G1691A), the prothrombin G20210A, amethylenetetrahydrofolate reductase A223V (C672001; 47(2):333-5. 

 46. Ameziane N, Lamotte M, Lamoril J et al. Co

(G1691A) and prothrombin (G20210A) genotypinpolymerase chain reaction using fluorescent resonhybridization probes on the Rotor-Gene 2000. Blo

14(4):421-4. 

 47. Erali M, Schmidt B, Lyon E, Wittwer C. Eva

microarrays for genotyping factor V, factor II, and 49(5):732-9. 

 48. Lucotte G, Champenois T. Duplex PCR-RFL

of factor V Leiden and prothrombin G20210A. Mo17(5):267-9. 

 49. Neitzel B, Matern C, Holinski-Feder E. Easy

screening for SNPs by ion pair/reverse phase HPLfactor V G1691A, prothrombin G20210A and met
Appendix G, Evidence Table 1

Page 35

reductase C677T variants. Clin Lab 2003; 49(7-8):313-8. 

 50. Schrijver I, Lay MJ, Zehnder JL. Diagnostic single nucleotide 

polymorphism analysis of factor V Leiden and prothrombin 20210G > A. A comparison of the Nanogen Eelectronic Microarray with restriction enzyme digestion and the Roche LightCycler. Am J Clin Pathol 2003; 119(4):490-6. 

 51. Baris I, Koksal V, Etlik O. Multiplex PCR-RFLP assay for detection of 

factor V Leiden and prothrombin G20210A. Genet Test 2004; 8(4):381-3. 

 52. Carmi N, Cohen D, Zvang E, Naparstek E, Deutsch V. Pronto 

ThromboRisk--a novel primer-extension ELISA based assay for the detection 

of mutations associated with increased risk for thrombophilia. J Clin Lab Anal 2004; 18(5):259-64. 

 53. Patnaik M, Dlott JS, Fontaine RN et al. Detection of genomic 

polymorphisms associated with venous thrombosis using the invader biplex assay. J Mol Diagn 2004; 6(2):137-44. 

 54. Ugozzoli LA, Hamby K. Four-color multiplex 5' nuclease assay for the 

simultaneous detection of the factor V Leiden and the prothrombin G20210A mutations. Mol Cell Probes 2004; 18(3):161-6. 

 55. Hobson-Peters J, Toye P. A whole-blood homogeneous assay for the 

multiplex detection of the factor V G1691A and thmutations. Mol Cell Probes 2005; 19(4):290-7. 

 56. Koksal V, Baris I, Etlik O. Primer-engineered 

detection of MTHFR C677T, prothrombin G20210mutations. Exp Mol Pathol 2007; 83(1):1-3. 

 57. Lopez M, Giraldo P, Alvarez P et al. Multipl

of thrombophilia: a method for routine clinical car21(6):349-55. 

 58. Hertzberg M, Neville S, Favaloro E, McDona

assurance of DNA testing for thrombophilia mutat123(2):189-93. 

 59. Jennings I, Kitchen S, Woods TA, Preston FE

thrombophilia through the United Kingdom NationAssessment Scheme (Blood Coagulation) Quality 

Thromb Hemost 2005; 31(1):66-72. 

 60. Tripodi A, Chantarangkul V, Menegatti M, T

Performance of clinical laboratories for DNA analmutations. Clin Chem 2005; 51(7):1310-1. 
Appendix G, Evidence Table 2

Page 25

Evidence Table 2. Results of tests for detection of factor V leiden. 

Reference List 

 1. Hertzberg M, Neville S, Favaloro E, McDonald D. External quality 

assurance of DNA testing for thrombophilia mutations. Am J Clin Pathol 2005; 123(2):189-93. 

 2. Jennings I, Kitchen S, Woods TA, Preston FE. Multilaboratory testing in 

thrombophilia through the United Kingdom National External Quality Assessment Scheme (Blood Coagulation) Quality Assurance Program. Semin 

Thromb Hemost 2005; 31(1):66-72. 

 3. Angeline T, Bentley HA, Hawk AB et al. Prevalence of the Factor V 

G1691A and the Factor II/prothrombin G20210A gene polymorphisms among 

Tamilians. Exp Mol Pathol 2005; 79(1):9-13. 

 5. Nauck M, Marz W, Wieland H. Evaluation of the Roche diagnostics 

LightCycler-Factor V Leiden Mutation Detection Kit and the LightCyclerProthrombin Mutation Detection Kit. Clin Biochem 2000; 33(3):213-6. 

 6. Parks SB, Popovich BW, Press RD. Real-time polymerase chain reaction 

with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes. Am J Clin Pathol 2001; 115(3):439-47. 

 7. Ledford M, Friedman KD, Hessner MJ, Moehlenkamp C, Williams TM, 

Larson RS. A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay. J Mol Diagn 2000; 2(2):97-104. 

 8. Moser MJ, Marshall DJ, Grenier JK et al. Exploiting the enzymatic 

recognition of an unnatural base pair to develop a universal genetic analysis 

system. Clin Chem 2003; 49(3):407-14. 

 9. Castley A, Higgins M, Ivey J, Mamotte C, Sayer DC, Christiansen FT. 

Clinical applications of whole-blood PCR with real-time instrumentation. Clin 

Chem 2005; 51(11):2025-30. 

 10. Hessner MJ, Budish MA, Friedman KD. Genotyping of factor V G1691A 

(Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes. Clin Chem 2000; 46(8 Pt 1):1051-6. 

 11. Cooper PC, Cooper SM, Smith JM, Kitchen S, Makris M. Evaluation of the 

Roche LightCycler: a simple and rapid method for direct detection of factor V Leiden and prothrombin G20210A genotypes from blood samples without the need for DNA extraction. Blood Coagul Fibrinolysis 2003; 14(5):499-503. 

 12. Schroell-Metzger B, Dicato M, Bosseler M, Berchem G. Comparison of 

standard PCR and the LightCycler technique to determine the thrombophilic mutations: an efficiency and cost study. Clin Chem Lab Med 2003; 41(4):482-5. 

 13. Liew M, Pryor R, Palais R et al. Genotyping of 

polymorphisms by high-resolution melting of sma2004; 50(7):1156-64. 

 14. Sanders Sevall J. Factor V Leiden genotyping u

polymerase chain reaction. Mol Cell Probes 2000; 

 15. Behrens M, Lange R. A highly reproducible and 

SNP analysis of several well characterized human 50(5-6):305-16. 

 16. Louis M, Dekairelle AF, Gala JL. Rapid combin

prothrombin and methylenetetrahydrofolate reducpolymorphisms using minor groove binding DNA probes) and real-time polymerase chain reaction. C42(12):1364-9. 

 17. Benson JM, Ellingsen D, El-Jamil M et al. Fact

allele: high-throughput analysis and association w

thromboembolism. Thromb Haemost 2001; 86(5):

 18. Murphy K, Hafez M, Philips J, Yarnell K, Guts

temperature gradient capillary electrophoresis for 

Leiden mutation: coincident identification of a novV. Mol Diagn 2003; 7(1):35-40. 

 19. Ozkan D, Erdem A, Kara P et al. Allele-specific 

V Leiden mutation from polymerase chain reactiofree electrochemical genosensor. Anal Chem 2002

 20. Huang TJ, Liu M, Knight LD, Grody WW, Mill

electrochemical detection scheme for identificatiopolymorphisms using hairpin-forming probes. Nuc30(12):e55. 

 21. Ozsoz M, Erdem A, Kerman K et al. Electroche

colloidal gold nanoparticles for the detection of Fausing disposable pencil graphite electrodes. Anal C

 22. Humeny A, Bonk T, Berkholz A, Wildt L, Beck

thrombotic risk factors by MALDI-TOF mass spec2001; 34(7):531-6. 

 23. Hung K, Sun X, Ding H, Kalafatis M, Simioni P

laser desorption/ionization time-of-flight based me1691G --> A mutation in the factor V gene. Blood 13(2):117-22. 

 25. Pecheniuk NM, Marsh NA, Walsh TP. Multiple 

genetic alterations associated with thrombophilia. 2000; 11(2):183-9. 
Appendix G, Evidence Table 2

Page 26

 26. Potter CG, Liu YT, Rees DC. Factor V Leiden mutation screened by PCR 

and detected with lanthanide-labeled probes. Genet Test 2001; 5(4):291-7. 

 27. Mammo L, Sheereen A, Saour T, Saour J. Comparative study between the 

Light Cycler and the PCR-restriction fragment length polymorphism in detecting factor V Leiden and factor II 20210G>A mutations. Clin Biochem 2006; 39(7):767-9. 

 28. El Housni H, Heimann P, Parma J, Vassart G. Single-nucleotide 

polymorphism genotyping by melting analysis of dual-labeled probes: examples using factor V Leiden and prothrombin 20210A mutations. Clin Chem 2003; 49(10):1669-72. 

 29. Alsmadi OA, Bornarth CJ, Song W et al. High accuracy genotyping directly 

from genomic DNA using a rolling circle amplification based assay. BMC Genomics 2003; 4(1):21. 

 30. Tsongalis GJ, Rainey BJ, Hodges KA. READIT: a novel technology used in 

the interrogation of nucleic acid sequences for single-nucleotide polymorphisms. Exp Mol Pathol 2001; 71(3):222-5. 

 31. Kowalski A, Radu D, Gold B. Colorimetric microwell plate detection of the 

factor V Leiden mutation. Clin Chem 2000; 46(8 Pt 1):1195-8. 

 32. Simundic AM, Topic E, Stefanovic M. Detection of factor V Leiden by 

PCR-SSCP using GMA precast Elchrom scientific gels. Clin Appl Thromb Hemost 2003; 9(3):227-31. 

 33. Reyes AA, Ugozzoli LA, Lowery JD et al. Linked linear amplification: a 

new method for the amplification of DNA. Clin Chem 2001; 47(1):31-40. 

 34. Evans JG, Lee-Tataseo C. Determination of the factor V Leiden singlenucleotide polymorphism in a commercial clinical laboratory by use of 

NanoChip microelectronic array technology. Clin Chem 2002; 48(9):1406-11. 

 35. Crockett AO, Wittwer CT. Fluorescein-labeled oligonucleotides for real-time 

pcr: using the inherent quenching of deoxyguanosine nucleotides. Anal Biochem 2001; 290(1):89-97. 

 36. Vaughn CP, Elenitoba-Johnson KS. Hybridization-induced dequenching of 

fluorescein-labeled oligonucleotides: a novel strategy for PCR detection and genotyping. Am J Pathol 2003; 163(1):29-35. 

 37. Tripodi A, Chantarangkul V, Menegatti M, Tagliabue L, Peyvandi F. 

Performance of clinical laboratories for DNA analyses to detect thrombophilia mutations. Clin Chem 2005; 51(7):1310-1. 

 38. French C, Li C, Strom C et al. Detection of the factor V Leiden mutation by 

a modified photo-cross-linking oligonucleotide hybridization assay. Clin Chem 2004; 50(2):296-305. 

 39. Rhodes RB, Lewis K, Shultz J et al. Analysis of the factor V Leiden 

mutation using the READIT Assay. Mol Diagn 2001; 6(1):55-61. 
Appendix G, Evidence Table 3

Page 13

Evidence Table 3. Results of tests for detection of prothrombin G20210A. 

Reference List 

 1. Hertzberg M, Neville S, Favaloro E, McDonald D. External quality 

assurance of DNA testing for thrombophilia mutations. Am J Clin Pathol 2005; 123(2):189-93. 

 2. Jennings I, Kitchen S, Woods TA, Preston FE. Multilaboratory testing in 

thrombophilia through the United Kingdom National External Quality Assessment Scheme (Blood Coagulation) Quality Assurance Program. Semin 

Thromb Hemost 2005; 31(1):66-72. 

 3. Angeline T, Bentley HA, Hawk AB et al. Prevalence of the Factor V 

G1691A and the Factor II/prothrombin G20210A gene polymorphisms among 

Tamilians. Exp Mol Pathol 2005; 79(1):9-13. 

 4. Bravo-Osorio M, Bydlowski SP. Detection of methylenetetrahydrofolate 

reductase (MTHFR) C677T and prothrombin G20210A mutations: second restriction site for digestion control of PCR products. Clin Chim Acta 2000; 301(1-2):219-23. 

 6. Nauck M, Marz W, Wieland H. Evaluation of the Roche diagnostics 

LightCycler-Factor V Leiden Mutation Detection Kit and the LightCyclerProthrombin Mutation Detection Kit. Clin Biochem 2000; 33(3):213-6. 

 7. Parks SB, Popovich BW, Press RD. Real-time polymerase chain reaction 

with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes. Am J Clin Pathol 2001; 115(3):439-47. 

 8. Hessner MJ, Friedman KD, Voelkerding KV et al. Multisite study for 

genotyping of the factor II (prothrombin) G20210A mutation by the invader assay. Clin Chem 2001; 47(11):2048-50. 

 9. Moser MJ, Marshall DJ, Grenier JK et al. Exploiting the enzymatic 

recognition of an unnatural base pair to develop a universal genetic analysis 

system. Clin Chem 2003; 49(3):407-14. 

 10. Castley A, Higgins M, Ivey J, Mamotte C, Sayer DC, Christiansen FT. 

Clinical applications of whole-blood PCR with real-time instrumentation. Clin 

Chem 2005; 51(11):2025-30. 

 11. Cooper PC, Cooper SM, Smith JM, Kitchen S, Makris M. Evaluation of the 

Roche LightCycler: a simple and rapid method for direct detection of factor V Leiden and prothrombin G20210A genotypes from blood samples without the need for DNA extraction. Blood Coagul Fibrinolysis 2003; 14(5):499-503. 

 12. Schroell-Metzger B, Dicato M, Bosseler M, Berchem G. Comparison of 

standard PCR and the LightCycler technique to determine the thrombophilic mutations: an efficiency and cost study. Clin Chem Lab Med 2003; 41(4):482-5. 

 13. Liew M, Pryor R, Palais R et al. Genotyping of single-nucleotide 

polymorphisms by high-resolution melting of small amplicons. Clin Chem 2004; 50(7):1156-64. 

 14. Behrens M, Lange R. A highly reproducible and 

SNP analysis of several well characterized human 50(5-6):305-16. 

 15. Louis M, Dekairelle AF, Gala JL. Rapid combin

prothrombin and methylenetetrahydrofolate reducpolymorphisms using minor groove binding DNA probes) and real-time polymerase chain reaction. C42(12):1364-9. 

 16. Happich D, Madlener K, Schwaab R, Hanfland 

the TaqMan-PCR for genotyping of the prothrombof the thermolabile methylenetetrahydrofolate reduHaemost 2000; 84(1):144-5. 

 17. Gellings A, Holzem G, Wielckens K, Klingler K

Employing the electrochemiluminescence technolotrademark) system for the detection of the point mthe 3' untranslated region of the prothrombin gene

MUTATION: VERWENDUNG DER ELEKTROCHEMILUMINESZENZTECHNOLOELECSYS(registered trademark) SYSTEMS ZUR PUNKTMUTATION AN DER POSITION 20210 UNTRANSLATIERTEN. LaboratoriumsMedizin 

 18. Humeny A, Bonk T, Berkholz A, Wildt L, Beck

thrombotic risk factors by MALDI-TOF mass spec2001; 34(7):531-6. 

 19. Gilchrist A, Solomon N, Erickson D et al. Auto

G20210A prothrombin mutation using the LCx miimmunoassay. Clin Chim Acta 2001; 314(1-2):249

 21. Pecheniuk NM, Marsh NA, Walsh TP. Multiple 

genetic alterations associated with thrombophilia. 2000; 11(2):183-9. 

 22. Mammo L, Sheereen A, Saour T, Saour J. Com

Light Cycler and the PCR-restriction fragment lendetecting factor V Leiden and factor II 20210G>A 2006; 39(7):767-9. 

 23. El Housni H, Heimann P, Parma J, Vassart G. S

polymorphism genotyping by melting analysis of dexamples using factor V Leiden and prothrombin Chem 2003; 49(10):1669-72. 

 24. Alsmadi OA, Bornarth CJ, Song W et al. High a

from genomic DNA using a rolling circle amplificGenomics 2003; 4(1):21. 
Appendix G, Evidence Table 3

Page 14

 25. Tripodi A, Chantarangkul V, Menegatti M, Tagliabue L, Peyvandi F. 

Performance of clinical laboratories for DNA analyses to detect thrombophilia mutations. Clin Chem 2005; 51(7):1310-1. 

 26. Meyer M, Kutscher G, Vogel G. The prothrombin nt20210 A allele as a risk 

factor for venous thromboembolism: detection of heterozygous and homozygous carriers by alternative methods. Thromb Res 2000; 97(5):359-63. 
Appendix G, Evidence Table 4

Page 9

Evidence Table 4. Results of tests for detection of simultaneous mutations. 

Reference List 

 1. Neitzel B, Matern C, Holinski-Feder E. Easy, accurate and reliable screening 

for SNPs by ion pair/reverse phase HPLC: simultaneous detection of factor V G1691A, prothrombin G20210A and methylenetetrahydrofolate reductase C677T variants. Clin Lab 2003; 49(7-8):313-8. 

 2. Huber S, McMaster KJ, Voelkerding KV. Analytical evaluation of primer 

engineered multiplex polymerase chain reaction-restriction fragment length 

polymorphism for detection of factor V Leiden and prothrombin G20210A. J 

Mol Diagn 2000; 2(3):153-7. 

 3. Lucotte G, Champenois T. Duplex PCR-RFLP for simultaneous detection of 

factor V Leiden and prothrombin G20210A. Mol Cell Probes 2003; 17(5):267-9. 

 4. Koksal V, Baris I, Etlik O. Primer-engineered multiplex PCR-RFLP for 

detection of MTHFR C677T, prothrombin G20210A and factor V Leiden mutations. Exp Mol Pathol 2007; 83(1):1-3. 

 5. Baris I, Koksal V, Etlik O. Multiplex PCR-RFLP assay for detection of 

factor V Leiden and prothrombin G20210A. Genet Test 2004; 8(4):381-3. 

 6. DelRio-LaFreniere SA, McGlennen RC. Simultaneous allele-specific 

amplification: a strategy using modified primer-template mismatches for SNP detection--application to prothrombin 20210A (factor II) and factor V Leiden (1691A) gene mutations. Mol Diagn 2001; 6(3):201-9. 

 7. Ameziane N, Lamotte M, Lamoril J et al. Combined factor V leiden 

(G1691A) and prothrombin (G20210A) genotyping by multiplex real-time polymerase chain reaction using fluorescent resonance energy transfer hybridization probes on the Rotor-Gene 2000. Blood Coagul Fibrinolysis 2003; 

14(4):421-4. 

 8. Hobson-Peters J, Toye P. A whole-blood homogeneous assay for the 

multiplex detection of the factor V G1691A and the prothrombin G20210A mutations. Mol Cell Probes 2005; 19(4):290-7. 

 9. van den Bergh FA, van Oeveren-Dybicz AM, Bon MA. Rapid single-tube 

genotyping of the factor V Leiden and prothrombin mutations by real-time 

PCR using dual-color detection. Clin Chem 2000; 46(8 Pt 1):1191-5. 

 10. Patnaik M, Dlott JS, Fontaine RN et al. Detection of genomic 

polymorphisms associated with venous thrombosis using the invader biplex assay. J Mol Diagn 2004; 6(2):137-44. 

 11. Carmi N, Cohen D, Zvang E, Naparstek E, Deutsch V. Pronto ThromboRisk-

-a novel primer-extension ELISA based assay for the detection of mutations associated with increased risk for thrombophilia. J Clin Lab Anal 2004; 18(5):259-64. 

 12. Lopez M, Giraldo P, Alvarez P et al. Multiplex assay for genetic testing of 

thrombophilia: a method for routine clinical care. J Clin Lab Anal 2007; 21(6):349-55. 

 13. Ugozzoli LA, Hamby K. Four-color multiplex 5

simultaneous detection of the factor V Leiden and mutations. Mol Cell Probes 2004; 18(3):161-6. 

 14. Endler G, Kyrle PA, Eichinger S, Exner M, Ma

mutagenically separated PCR: simultaneous singleV R506Q (G1691A), the prothrombin G20210A, amethylenetetrahydrofolate reductase A223V (C672001; 47(2):333-5. 

 15. Leyte A, Smits PH, van Straalen JP, van Doorn 

simultaneous detection of the factor V Leiden and variants using multiplex PCR and a line probe assa83(2):354-5. 

 16. Schrijver I, Lay MJ, Zehnder JL. Diagnostic sin

analysis of factor V Leiden and prothrombin 2021Nanogen Eelectronic Microarray with restriction e

Roche LightCycler. Am J Clin Pathol 2003; 119(4

 17. Erali M, Schmidt B, Lyon E, Wittwer C. Evalua

microarrays for genotyping factor V, factor II, and 49(5):732-9. 

 18. Verri A, Focher F, Tettamanti G, Grazioli V. Tw

thrombophilia by pyrosequencing. Clin Chem 200
Appendix G, Evidence Table 5 

Page 23 

Evidence Table 5. Quality and applicability of studies addressing Factor V Leiden and Recurrent Venous Thrombosis in Probands 

Reference List 

 1. Ridker PM, Miletich JP, Stampfer MJ, Goldhaber SZ, Lindpaintner K, 

Hennekens CH. Factor V Leiden and risks of recurrent idiopathic venous thromboembolism. Circulation 1995; 92(10):2800-2. 

 2. Kearon C, Gent M, Hirsh J et al. A comparison of three months of 

anticoagulation with extended anticoagulation for a first episode of idiopathic venous thromboembolism. N Engl J Med 1999; 340(12):901-7. 

 3. Lindmarker P, Schulman S, Sten-Linder M, Wiman B, Egberg N, Johnsson 

H. The risk of recurrent venous thromboembolism in carriers and non-carriers of the G1691A allele in the coagulation factor V gene and the G20210A allele 

in the prothrombin gene. DURAC Trial Study Group. Duration of Anticoagulation. Thromb Haemost 1999; 81(5):684-9. 

 4. Simioni P, Prandoni P, Lensing AW et al. Risk for subsequent venous 

thromboembolic complications in carriers of the prothrombin or the factor V gene mutation with a first episode of deep-vein thrombosis. Blood 2000; 96(10):3329-33. 

 5. Hoibraaten E, Mowinckel MC, de Ronde H, Bertina RM, Sandset PM. 

Hormone replacement therapy and acquired resistance to activated protein C: results of a randomized, double-blind, placebo-controlled trial. Br J Haematol 2001; 115(2):415-20. 

 6. Miles JS, Miletich JP, Goldhaber SZ, Hennekens CH, Ridker PM. G20210A 

mutation in the prothrombin gene and the risk of recurrent venous thromboembolism. J Am Coll Cardiol 2001; 37(1):215-8. 

 7. Eichinger S, Weltermann A, Mannhalter C et al. The risk of recurrent venous 

thromboembolism in heterozygous carriers of factor V Leiden and a first spontaneous venous thromboembolism. Arch Intern Med 2002; 162(20):2357-60. 

 8. Baglin T, Luddington R, Brown K, Baglin C. Incidence of recurrent venous 

thromboembolism in relation to clinical and thrombophilic risk factors: prospective cohort study. Lancet 2003; 362(9383):523-6. 

 9. Is recurrent venous thromboembolism more frequent in homozygous patients 

for the factor V Leiden mutation than in heterozygous patients? Blood Coagul Fibrinolysis 2003; 14(6):523-9. Notes: CORPORATE NAME: Procare Group 

 10. Palareti G, Legnani C, Cosmi B et al. Predictive value of D-dimer test for 

recurrent venous thromboembolism after anticoagulation withdrawal in subjects with a previous idiopathic event and in carriers of congenital thrombophilia. Circulation 2003; 108(3):313-8. 

 11. Baarslag HJ, Koopman MM, Hutten BA et al. Long-term follow-up of 

patients with suspected deep vein thrombosis of the upper extremity: survival, 

risk factors and post-thrombotic syndrome. Eur J I7. 

 12. Kyrle PA, Minar E, Bialonczyk C, Hirschl M, W

The risk of recurrent venous thromboembolism in Med 2004; 350(25):2558-63. 

 13. Christiansen SC, Cannegieter SC, Koster T, Van

FR. Thrombophilia, clinical factors, and recurrent 

JAMA 2005; 293(19):2352-61. 

 14. Mansilha A, Araujo F, Severo M, Sampaio SM, 

Genetic polymorphisms and risk of recurrent deep people: prospective cohort study. Eur J Vasc Endo

 15. Santamaria MG, Agnelli G, Taliani MR et al. T

and recurrence of venous thromboembolism in patstandardized anticoagulant treatment. Thromb Res Notes: CORPORATE NAME: Warfarin Optimal D(WODIT) Investigators 

 16. Vossen CY, Walker ID, Svensson P et al. Recur

thrombosis in patients with familial thrombophiliaBiol 2005; 25(9):1992-7. 

 17. Gonzalez-Porras JR, Garcia-Sanz R, Alberca I e

thrombosis in patients with G20210A mutation in factor V Leiden mutation. Blood Coagul Fibrinoly

 18. Hron G, Eichinger S, Weltermann A et al. Fami

thromboembolism and the risk for recurrence. Am 

 19. Wahlander K, Eriksson H, Lundstrom T et al. R

thromboembolism or bleeding in relation to thrompatients receiving ximelagatran or placebo for longof venous thromboembolism. Br J Haematol 2006Notes: CORPORATE NAME: THRIVE III Invest

 20. Prandoni P, Noventa F, Ghirarduzzi A et al. The 

thromboembolism after discontinuing anticoagulatproximal deep vein thrombosis or pulmonary embstudy in 1,626 patients. Haematologica 2007; 92(2

 21. Strandberg K, Svensson PJ, Ohlin AK. Venous 

of the Factor V Leiden mutation and in patients wirisk factor; prediction of recurrence and APC-PCI and/or soluble thrombomodulin antigen and activi121(2):145-51. 

 22. Kearon C, Julian JA, Kovacs MJ et al. Influence 
Appendix G, Evidence Table 5 

Page 24 

recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 

 23. Eichinger S, Minar E, Hirschl M et al. The risk of early recurrent venous 

thromboembolism after oral anticoagulant therapy in patients with the G20210A transition in the prothrombin gene. Thromb Haemost 1999; 81(1):14-7. 
Appendix G, Evidence Table 6 

Page 4 

Evidence Table 6. Results for Probands Heterozygous for the Factor V Leiden Mutation 

Reference List 

1. Gonzalez-Porras JR, Garcia-Sanz R, Alberca I et al. Risk of recurrent venous 

thrombosis in patients with G20210A mutation in the prothrombin gene or factor V Leiden mutation. Blood Coagul Fibrinolysis 2006; 17(1):23-8. 

 2. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

 3. Is recurrent venous thromboembolism more frequent in homozygous patients 

for the factor V Leiden mutation than in heterozygous patients? Blood Coagul Fibrinolysis 2003; 14(6):523-9. Notes: CORPORATE NAME: Procare Group 

 4. Palareti G, Legnani C, Cosmi B et al. Predictive value of D-dimer test for 

recurrent venous thromboembolism after anticoagulation withdrawal in subjects with a previous idiopathic event and in carriers of congenital 

thrombophilia. Circulation 2003; 108(3):313-8. 

 5. Eichinger S, Weltermann A, Mannhalter C et al. The risk of recurrent venous 

thromboembolism in heterozygous carriers of factor V Leiden and a first spontaneous venous thromboembolism. Arch Intern Med 2002; 162(20):2357-60. 

 6. Hoibraaten E, Mowinckel MC, de Ronde H, Bertina RM, Sandset PM. 

Hormone replacement therapy and acquired resistance to activated protein C: results of a randomized, double-blind, placebo-controlled trial. Br J Haematol 2001; 115(2):415-20. 

 7. Miles JS, Miletich JP, Goldhaber SZ, Hennekens CH, Ridker PM. G20210A 

mutation in the prothrombin gene and the risk of recurrent venous thromboembolism. J Am Coll Cardiol 2001; 37(1):215-8. 

 8. Simioni P, Prandoni P, Lensing AW et al. Risk for subsequent venous 

thromboembolic complications in carriers of the prothrombin or the factor V gene mutation with a first episode of deep-vein thrombosis. Blood 2000; 

96(10):3329-33. 

 9. Lindmarker P, Schulman S, Sten-Linder M, Wiman B, Egberg N, Johnsson 

H. The risk of recurrent venous thromboembolism in carriers and non-carriers of the G1691A allele in the coagulation factor V gene and the G20210A allele 

in the prothrombin gene. DURAC Trial Study Group. Duration of Anticoagulation. Thromb Haemost 1999; 81(5):684-9. 

 10. Ridker PM, Miletich JP, Stampfer MJ, Goldhaber SZ, Lindpaintner K, 

Hennekens CH. Factor V Leiden and risks of recurrent idiopathic venous thromboembolism. Circulation 1995; 92(10):2800-2. 

 11. Prandoni P, Noventa F, Ghirarduzzi A et al. The risk of recurrent venous 

thromboembolism after discontinuing anticoagulatproximal deep vein thrombosis or pulmonary embstudy in 1,626 patients. Haematologica 2007; 92(2

 12. Kearon C, Gent M, Hirsh J et al. A comparison 

anticoagulation with extended anticoagulation for 

venous thromboembolism. N Engl J Med 1999; 34

 13. Kearon C, Julian JA, Kovacs MJ et al. Influence 

recurrent venous thromboembolism while on warfrandomized trial. Blood 2008. 

 14. Christiansen SC, Cannegieter SC, Koster T, Van

FR. Thrombophilia, clinical factors, and recurrent JAMA 2005; 293(19):2352-61. 
Appendix G, Evidence Table 7 

Page 3 

Evidence Table 7. Results for probands homozygous for Factor V Leiden mutation 

Reference List 

 1. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

 2. Is recurrent venous thromboembolism more frequent in homozygous patients 

for the factor V Leiden mutation than in heterozygous patients? Blood Coagul Fibrinolysis 2003; 14(6):523-9. Notes: CORPORATE NAME: Procare Group 

 3. Palareti G, Legnani C, Cosmi B et al. Predictive value of D-dimer test for 

recurrent venous thromboembolism after anticoagulation withdrawal in subjects with a previous idiopathic event and in carriers of congenital 

thrombophilia. Circulation 2003; 108(3):313-8. 

 4. Hoibraaten E, Mowinckel MC, de Ronde H, Bertina RM, Sandset PM. 

Hormone replacement therapy and acquired resistance to activated protein C: 

results of a randomized, double-blind, placebo-controlled trial. Br J Haematol 

2001; 115(2):415-20. 

 5. Lindmarker P, Schulman S, Sten-Linder M, Wim

H. The risk of recurrent venous thromboembolism of the G1691A allele in the coagulation factor V gin the prothrombin gene. DURAC Trial Study GroAnticoagulation. Thromb Haemost 1999; 81(5):68

 6. Christiansen SC, Cannegieter SC, Koster T, Van

FR. Thrombophilia, clinical factors, and recurrent 

JAMA 2005; 293(19):2352-61. 

 7. Kearon C, Gent M, Hirsh J et al. A comparison 

anticoagulation with extended anticoagulation for 

venous thromboembolism. N Engl J Med 1999; 34

 8. Kearon C, Julian JA, Kovacs MJ et al. Influenc

recurrent venous thromboembolism while on warfrandomized trial. Blood 2008. 
Appendix G, Evidence Table 8

Page 3

Evidence Table 8. Results for probands with unspecified Factor V Leiden mutations 

Reference List 

 1. Strandberg K, Svensson PJ, Ohlin AK. Venous thromboembolism in carriers 

of the Factor V Leiden mutation and in patients without known thrombophilic risk factor; prediction of recurrence and APC-PCI complex concentration and/or soluble thrombomodulin antigen and activity. Thromb Res 2007; 121(2):145-51. 

 2. Hron G, Eichinger S, Weltermann A et al. Family history for venous 

thromboembolism and the risk for recurrence. Am J Med 2006; 119(1):50-3. 

 3. Mansilha A, Araujo F, Severo M, Sampaio SM, Toledo T, Albuquerque R. 

Genetic polymorphisms and risk of recurrent deep venous thrombosis in young people: prospective cohort study. Eur J Vasc Endovasc Surg 2005; 30(5):545-9. 

 4. Santamaria MG, Agnelli G, Taliani MR et al. Thrombophilic abnormalities 

and recurrence of venous thromboembolism in patients treated with 

standardized anticoagulant treatment. Thromb Res 2005; 116(4):301-6. Notes: CORPORATE NAME: Warfarin Optimal Duration Italian Trial 

(WODIT) Investigators 

 5. Vossen CY, Walker ID, Svensson P et al. Recur

thrombosis in patients with familial thrombophilia

Biol 2005; 25(9):1992-7. 

 6. Baarslag HJ, Koopman MM, Hutten BA et al. L

patients with suspected deep vein thrombosis of thrisk factors and post-thrombotic syndrome. Eur J I7. 

 7. Kyrle PA, Minar E, Bialonczyk C, Hirschl M, W

The risk of recurrent venous thromboembolism in Med 2004; 350(25):2558-63. 

 8. Baglin T, Luddington R, Brown K, Baglin C. In

thromboembolism in relation to clinical and thromprospective cohort study. Lancet 2003; 362(9383)
Appendix G, Evidence Table 9

Page 2

Evidence Table 9. Results for probands double heterozygous for Factor V Leiden mutation and prothrombin G20210A mutation 

Reference List 

1. Vossen CY, Walker ID, Svensson P et al. Recurrence rate after a first venous 

thrombosis in patients with familial thrombophilia. Arterioscler Thromb Vasc Biol 2005; 25(9):1992-7. 2. Palareti G, Legnani C, Cosmi B et al. Predictive value of D-dimer test for recurrent venous thromboembolism after anticoagulation withdrawal in subjects with a previous idiopathic event and in carriers of congenital thrombophilia. Circulation 2003; 108(3):313-8. 

3. Miles JS, Miletich JP, Goldhaber SZ, Hennekens Cmutation in the prothrombin gene and the risk of recurrthromboembolism. J Am Coll Cardiol 2001; 37(1):215-4. Simioni P, Prandoni P, Lensing AW et al. Risk for 

thromboembolic complications in carriers of the prothrmutation with a first episode of deep-vein thrombosis. B
Appendix G, Evidence Table 10

Page 3

Evidence Table 10. Results for probands heterozygous for prothrombin G20210A 

Reference List 

 1. Gonzalez-Porras JR, Garcia-Sanz R, Alberca I et al. Risk of recurrent venous 

thrombosis in patients with G20210A mutation in the prothrombin gene or factor V Leiden mutation. Blood Coagul Fibrinolysis 2006; 17(1):23-8. 

 2. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

 3. Mansilha A, Araujo F, Severo M, Sampaio SM, Toledo T, Albuquerque R. 

Genetic polymorphisms and risk of recurrent deep venous thrombosis in young people: prospective cohort study. Eur J Vasc Endovasc Surg 2005; 30(5):545-9. 

 4. Palareti G, Legnani C, Cosmi B et al. Predictive value of D-dimer test for 

recurrent venous thromboembolism after anticoagulation withdrawal in subjects with a previous idiopathic event and in carriers of congenital 

thrombophilia. Circulation 2003; 108(3):313-8. 

 5. Miles JS, Miletich JP, Goldhaber SZ, Hennekens CH, Ridker PM. G20210A 

mutation in the prothrombin gene and the risk of recurrent venous thromboembolism. J Am Coll Cardiol 2001; 37(1):215-8. 

 6. Simioni P, Prandoni P, Lensing AW et al. Risk for subsequent venous 

thromboembolic complications in carriers of the prothrombin or the factor V gene mutation with a first episode of deep-vein thrombosis. Blood 2000; 96(10):3329-33. 

 7. Lindmarker P, Schulman S, Sten-Linder M, Wiman B, Egberg N, Johnsson 

H. The risk of recurrent venous thromboembolism in carriers and non-carriers of the G1691A allele in the coagulation factor V gene and the G20210A allele 

in the prothrombin gene. DURAC Trial Study Group. Duration of Anticoagulation. Thromb Haemost 1999; 81(5):684-9. 

 8. Prandoni P, Noventa F, Ghirarduzzi A et al. The risk of recurrent venous 

thromboembolism after discontinuing anticoagulation in patients with acute proximal deep vein thrombosis or pulmonary embolism. A prospective cohort study in 1,626 patients. Haematologica 2007; 92(2):199-205. 

 9. Kearon C, Gent M, Hirsh J et al. A comparison of three months of 

anticoagulation with extended anticoagulation for a first episode of idiopathic 

venous thromboembolism. N Engl J Med 1999; 340(12):901-7. 

 10. Kearon C, Julian JA, Kovacs MJ et al. Influence of thrombophilia on risk of 

recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 
Reference List 

 1. Hron G, Eichinger S, Weltermann A et al. Family history for venous 

thromboembolism and the risk for recurrence. Am J Med 2006; 119(1):50-3. 

 2. Santamaria MG, Agnelli G, Taliani MR et al. Thrombophilic abnormalities 

and recurrence of venous thromboembolism in patients treated with standardized anticoagulant treatment. Thromb Res 2005; 116(4):301-6. 

 3. Baarslag HJ, Koopman MM, Hutten BA et al. Long-term follow-up of 

patients with suspected deep vein thrombosis of the upper extremity: 

survival, risk factors and post-thrombotic syndrome. Eur J Intern Med 2004; 15(8):503-7. 

 4. Kyrle PA, Minar E, Bialonczyk C, Hirschl M, Weltermann A, Eichinger S. 

The risk of recurrent venous thromboembolism in men and women. N Engl J Med 2004; 350(25):2558-63. 

 5. Baglin T, Luddington R, Brown K, Baglin C. Incidence of recurrent venous 

thromboembolism in relation to clinical and thrombophilic risk factors: prospective cohort study. Lancet 2003; 362(9383):523-6 . 

 6. Christiansen SC, Cannegieter SC, Koster T, Vandenbroucke JP, Rosendaal 

FR. Thrombophilia, clinical factors, and recurrent venous thrombotic events. 

JAMA 2005; 293(19):2352-61. 

 7. Eichinger S, Minar E, Hirschl M et al. The risk of early recurrent venous 

thromboembolism after oral anticoagulant therapy in patients with the G20210A transition in the prothrombin gene. Thromb Haemost 1999; 81(1):14-7. 
Appendix G, Evidence Table 12

Page 2

Evidence Table 12. Results for probands homozygous for prothrombin G20210A 

Reference List 

1. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 2. Kearon C, Julian JA, Kovacs MJ et al. Influence of thrombophilia on risk of recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 
Appendix G, Evidence Table 13 

Page 7 

Evidence Table 13. Quality of studies addressing Factor V Leiden and Prothrombin G20210A predicting events in family members 

Reference List 

 1. Samama MM, Simon D, Horellou MH, Trossaert M, Elalamy I, Conard J. 

Diagnosis and clinical characteristics of inherited activated protein C resistance. Haemostasis 1996; 26 Suppl 4:315-30. 

 2. Middeldorp S, Henkens CM, Koopman MM et al. The incidence of venous 

thromboembolism in family members of patients with factor V Leiden mutation and venous thrombosis. Ann Intern Med 1998; 128(1):15-20. 

 3. Le Cam-Duchez V, Gandrille S, Tregouet D et al. Influence of three 

potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation. Br J Haematol 1999; 106(4):889-97. 

 4. Simioni P, Sanson BJ, Prandoni P et al. Incidence of venous 

thromboembolism in families with inherited thrombophilia. Thromb Haemost 1999; 81(2):198-202. 

 5. Simioni P, Prandoni P, Girolami A. Low rate of venous thromboembolism in 

asymptomatic relatives of probands with factor V Leiden mutation. Ann Intern Med 1999; 130(6):538. 

 6. Lensen R, Rosendaal F, Vandenbroucke J, Bertina R. Factor V Leiden: the 

venous thrombotic risk in thrombophilic families. Br J Haematol 2000; 110(4):939-45. 

 7. Martinelli I, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, 

Mannucci PM. The risk of venous thromboembolism in family members with mutations in the genes of factor V or prothrombin or both. Br J Haematol 

2000; 111(4):1223-9. 

 8. Simioni P, Tormene D, Luni S, Caldato M, Girolami A. Clinical and 

laboratory expression of associated thrombophilic conditions (homozygous/heterozygous factor V Leiden mutation and heterozygous 

prothrombin variant 20210A) in an Italian family. Blood Coagul Fibrinolysis 2000; 11(4):379-84. 

 9. Lensen R, Bertina RM, Vandenbroucke JP, Rosendaal FR. High factor VIII 

levels contribute to the thrombotic risk in families with factor V Leiden. Br J Haematol 2001; 114(2):380-6. 

 10. Middeldorp S, Meinardi JR, Koopman MM et al. A prospective study of 

asymptomatic carriers of the factor V Leiden mutation to determine the incidence of venous thromboembolism. Ann Intern Med 2001; 135(5):322-7. 

 11. Middeldorp S, Libourel EJ, Hamulyak K, Van der Meer J, Buller HR. The 

risk of pregnancy-related venous thromboembolism in women who are homozygous for factor V Leiden. Br J Haematol 2001; 113(2):553-5. 

 12. Rintelen C, Pabinger I, Bettelheim P et al. Impact of the factor II: G20210A 

variant on the risk of venous thromboembolism in relatives from families with the factor V: R506Q mutation. Eur J Haematol 2001; 67(3):165-9. 

 13. Meinardi JR, Middeldorp S, de Kam PJ et al. The incidence of recurrent 

venous thromboembolism in carriers of factor V Lconcomitant thrombophilic disorders. Br J Haema

 14. Simioni P, Tormene D, Prandoni P et al. Inciden

thromboembolism in asymptomatic family membeV Leiden: a prospective cohort study. Blood 2002

 15. Vossen CY, Conard J, Fontcuberta J et al. Fami

lifetime risk of venous thrombosis. J Thromb Haem

 16. Vossen CY, Conard J, Fontcuberta J et al. Risk 

event in carriers of a familial thrombophilic defectCohort on Thrombophilia (EPCOT). J Thromb Ha

 17. Couturaud F, Kearon C, Leroyer C et al. Inciden

thromboembolism in first-degree relatives of patie

thromboembolism who have factor V Leiden. Thro96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de Occidentale (G.E.T.B.O) 

 18. Castaman G, Tosetto A, Cappellari A, Ruggeri 

A20210 allele in the prothrombin gene enhances thin carriers of inherited protein S deficiency. Blood 11(4):321-6. 

 19. Martinelli I, Legnani C, Bucciarelli P, Grandone 

PM. Risk of pregnancy-related venous thrombosis inherited thrombophilia. Thromb Haemost 2001; 8

 20. Tormene D, Simioni P, Prandoni P et al. Factor 

risk of venous thromboembolism in pregnant wom86(12):1305-9. 

 21. Tormene D, De Stefano V, Grandone E et al. Th

variant and the risk of venous thromboembolism owomen: a family study. J Thromb Haemost 2007; 

 22. Martinelli I, Battaglioli T, De Stefano V et al. T

thromboembolism during pregnancy and puerperiufor factor V Leiden and prothrombin G20210A. J 6(3):494-8. Notes: CORPORATE NAME: GIT (Gruppo Italia
Appendix G, Evidence Table 14

Page 4

Evidence Table 14. FVL predicting events in family members, study characteristics 

Reference List 

 1. Samama MM, Simon D, Horellou MH, Trossaert M, Elalamy I, Conard J. 

Diagnosis and clinical characteristics of inherited activated protein C resistance. Haemostasis 1996; 26 Suppl 4:315-30. 

 2. Middeldorp S, Henkens CM, Koopman MM et al. The incidence of venous 

thromboembolism in family members of patients with factor V Leiden mutation and venous thrombosis. Ann Intern Med 1998; 128(1):15-20. 

 3. Le Cam-Duchez V, Gandrille S, Tregouet D et al. Influence of three 

potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation. Br J Haematol 1999; 106(4):889-97. 

 4. Simioni P, Sanson BJ, Prandoni P et al. Incidence of venous 

thromboembolism in families with inherited thrombophilia. Thromb Haemost 1999; 81(2):198-202. 

 5. Simioni P, Prandoni P, Girolami A. Low rate of venous thromboembolism in 

asymptomatic relatives of probands with factor V Leiden mutation. Ann Intern Med 1999; 130(6):538. 

 6. Lensen R, Rosendaal F, Vandenbroucke J, Bertina R. Factor V Leiden: the 

venous thrombotic risk in thrombophilic families. Br J Haematol 2000; 110(4):939-45. 

 7. Martinelli I, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, 

Mannucci PM. The risk of venous thromboembolism in family members with mutations in the genes of factor V or prothrombin or both. Br J Haematol 

2000; 111(4):1223-9. 

 8. Simioni P, Tormene D, Luni S, Caldato M, Girolami A. Clinical and 

laboratory expression of associated thrombophilic conditions (homozygous/heterozygous factor V Leiden mutation and heterozygous 

prothrombin variant 20210A) in an Italian family. Blood Coagul Fibrinolysis 2000; 11(4):379-84. 

 9. Lensen R, Bertina RM, Vandenbroucke JP, Rosendaal FR. High factor VIII 

levels contribute to the thrombotic risk in families with factor V Leiden. Br J Haematol 2001; 114(2):380-6. 

 10. Middeldorp S, Meinardi JR, Koopman MM et al. A prospective study of 

asymptomatic carriers of the factor V Leiden mutation to determine the incidence of venous thromboembolism. Ann Intern Med 2001; 135(5):322-7. 

 11. Rintelen C, Pabinger I, Bettelheim P et al. Impact of the factor II: G20210A 

variant on the risk of venous thromboembolism in relatives from families with the factor V: R506Q mutation. Eur J Haematol 2001; 67(3):165-9. 

 12. Meinardi JR, Middeldorp S, de Kam PJ et al. The incidence of recurrent 

venous thromboembolism in carriers of factor V Leiden is related to 

concomitant thrombophilic disorders. Br J Haematol 2002; 116(3):625-31. 

 13. Simioni P, Tormene D, Prandoni P et al. Incidence of venous 

thromboembolism in asymptomatic family members who are carriers of factor 

V Leiden: a prospective cohort study. Blood 2002

 14. Vossen CY, Conard J, Fontcuberta J et al. Fami

lifetime risk of venous thrombosis. J Thromb Haem

 15. Vossen CY, Conard J, Fontcuberta J et al. Risk 

event in carriers of a familial thrombophilic defectCohort on Thrombophilia (EPCOT). J Thromb Ha

 16. Couturaud F, Kearon C, Leroyer C et al. Inciden

thromboembolism in first-degree relatives of patiethromboembolism who have factor V Leiden. Thro96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de Occidentale (G.E.T.B.O) 
Appendix G, Evidence Table 15

Page 15

Evidence Table 15. Results of studies of family members with Factor V Leiden 

Reference List 

 1. Samama MM, Simon D, Horellou MH, Trossaert M, Elalamy I, Conard J. 

Diagnosis and clinical characteristics of inherited activated protein C resistance. Haemostasis 1996; 26 Suppl 4:315-30. 

 2. Middeldorp S, Henkens CM, Koopman MM et al. The incidence of venous 

thromboembolism in family members of patients with factor V Leiden mutation and venous thrombosis. Ann Intern Med 1998; 128(1):15-20. 

 3. Le Cam-Duchez V, Gandrille S, Tregouet D et al. Influence of three 

potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation. Br J Haematol 1999; 106(4):889-97. 

 4. Simioni P, Sanson BJ, Prandoni P et al. Incidence of venous 

thromboembolism in families with inherited thrombophilia. Thromb Haemost 1999; 81(2):198-202. 

 5. Simioni P, Prandoni P, Girolami A. Low rate of venous thromboembolism in 

asymptomatic relatives of probands with factor V Leiden mutation. Ann Intern Med 1999; 130(6):538. 

 6. Lensen R, Rosendaal F, Vandenbroucke J, Bertina R. Factor V Leiden: the 

venous thrombotic risk in thrombophilic families. Br J Haematol 2000; 110(4):939-45. 

 7. Martinelli I, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, 

Mannucci PM. The risk of venous thromboembolism in family members with mutations in the genes of factor V or prothrombin or both. Br J Haematol 

2000; 111(4):1223-9. 

 8. Simioni P, Tormene D, Luni S, Caldato M, Girolami A. Clinical and 

laboratory expression of associated thrombophilic conditions (homozygous/heterozygous factor V Leiden mutation and heterozygous 

prothrombin variant 20210A) in an Italian family. Blood Coagul Fibrinolysis 2000; 11(4):379-84. 

 9. Lensen R, Bertina RM, Vandenbroucke JP, Ros

levels contribute to the thrombotic risk in families Haematol 2001; 114(2):380-6. 

 10. Middeldorp S, Meinardi JR, Koopman MM et a

asymptomatic carriers of the factor V Leiden mutaincidence of venous thromboembolism. Ann Intern 

 11. Rintelen C, Pabinger I, Bettelheim P et al. Impa

variant on the risk of venous thromboembolism in the factor V: R506Q mutation. Eur J Haematol 200

 12. Meinardi JR, Middeldorp S, de Kam PJ et al. Th

venous thromboembolism in carriers of factor V Lconcomitant thrombophilic disorders. Br J Haema

 13. Simioni P, Tormene D, Prandoni P et al. Inciden

thromboembolism in asymptomatic family membeV Leiden: a prospective cohort study. Blood 2002

 14. Vossen CY, Conard J, Fontcuberta J et al. Fami

lifetime risk of venous thrombosis. J Thromb Haem

 15. Vossen CY, Conard J, Fontcuberta J et al. Risk 

event in carriers of a familial thrombophilic defectCohort on Thrombophilia (EPCOT). J Thromb Ha

 16. Couturaud F, Kearon C, Leroyer C et al. Inciden

thromboembolism in first-degree relatives of patiethromboembolism who have factor V Leiden. Thro96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de Occidentale (G.E.T.B.O) 
Appendix G, Evidence Table 16,

Page 2

Evidence Table 16. Characteristics of studies of prothrombin G20210A in family members 

Reference List 

1. Castaman G, Tosetto A, Cappellari A, Ruggeri M, Rodeghiero F. The A20210 

allele in the prothrombin gene enhances the risk of venous thrombosis in 

carriers of inherited protein S deficiency. Blood Coagul Fibrinolysis 2000; 

11(4):321-6. 

2. Martinelli I, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, 

Mannucci PM. The risk of venous thromboembolism in family members with 

mutations in the genes of factor V or prothrombin or both. Br J Haematol 

2000; 111(4):1223-9. 

3. Simioni P, Tormene D, Luni S, Caldato M, Girolami A. Clinical and laboratory 

expression of associated thrombophilic conditions (homozygous/heterozygous 

factor V Leiden mutation and heterozygous prothrombin variant 20210A) in an 

Italian family. Blood Coagul Fibrinolysis 2000; 11

 4. Rintelen C, Pabinger I, Bettelheim P et al. Impact 

variant on the risk of venous thromboembolism in the factor V: R506Q mutation. Eur J Haematol 200

 5. Couturaud F, Kearon C, Leroyer C et al. Incidence 

thromboembolism in first-degree relatives of patiethromboembolism who have factor V Leiden. Thro96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de Occidentale (G.E.T.B.O) 
Appendix G, Evidence Table 17

Page 3

Evidence Table 17. Results of studies on prothrombin G20210A predicting events in family members. 

Reference List 

1. Castaman G, Tosetto A, Cappellari A, Ruggeri M, Rodeghiero F. The A20210 

allele in the prothrombin gene enhances the risk of venous thrombosis in 

carriers of inherited protein S deficiency. Blood Coagul Fibrinolysis 2000; 

11(4):321-6. 

2. Martinelli I, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, 

Mannucci PM. The risk of venous thromboembolism in family members with 

mutations in the genes of factor V or prothrombin or both. Br J Haematol 

2000; 111(4):1223-9. 

3. Simioni P, Tormene D, Luni S, Caldato M, Girolami A. Clinical and laboratory 

expression of associated thrombophilic conditions (homozygous/heterozygous 

factor V Leiden mutation and heterozygous prothrombin variant 20210A) in an 

Italian family. Blood Coagul Fibrinolysis 2000; 11(4):379-84. 

4. Rintelen C, Pabinger I, Bettelheim P et al. Impact of the factor II: G20210A 

variant on the risk of venous thromboembolism in relatives from families with the factor V: R506Q mutation. Eur J Haematol 2001; 67(3):165-9. 

5. Couturaud F, Kearon C, Leroyer C et al. Incidence of venous 

thromboembolism in first-degree relatives of patients with venous thromboembolism who have factor V Leiden. Thromb Haemost 2006; 

96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de la Thrombose de Bretagne Occidentale (G.E.T.B.O) 
Appendix G, Evidence Table 18

Page 2

Evidence Table 18. Characteristics of studies of Factor V Leiden and prothrombin G20210A in pregnant family members 

Reference List 

1. Martinelli I, Legnani C, Bucciarelli P, Grandone E, De Stefano V, Mannucci 

PM. Risk of pregnancy-related venous thrombosis in carriers of severe 

inherited thrombophilia. Thromb Haemost 2001; 86(3):800-3. 

2. Middeldorp S, Libourel EJ, Hamulyak K, Van der Meer J, Buller HR. The risk 

of pregnancy-related venous thromboembolism in women who are homozygous 

for factor V Leiden. Br J Haematol 2001; 113(2):553-5. 

3. Tormene D, Simioni P, Prandoni P et al. Factor V Leiden mutation and the risk 

of venous thromboembolism in pregnant women. Haematologica 2001; 86(12):1305-9. 

4. Couturaud F, Kearon C, Leroyer C et al. Incidence of venous 

thromboembolism in first-degree relatives of patients with venous thromboembolism who have factor V Leiden. Thromb Haemost 2006; 96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de la Thrombose de Bretagne Occidentale (G.E.T.B.O) 

5. Tormene D, De Stefano V, Grandone E et al. The G20210A prothrombin 

variant and the risk of venous thromboembolism or fetal loss in pregnant women: a family study. J Thromb Haemost 2007; 5(11):2193-6. 

6. Martinelli I, Battaglioli T, De Stefano V et al. The risk of first venous 

thromboembolism during pregnancy and puerperium in double heterozygotes for factor V Leiden and prothrombin G20210A. J Thromb Haemost 2008; 6(3):494-8. Notes: CORPORATE NAME: GIT (Gruppo Italiano Trombofilia) 
Appendix G, Evidence Table 19

Page 3

Evidence Table 19. Results of studies of Factor V Leiden and prothrombin G20210A in pregnant family members 

Reference List 

1. Martinelli I, Legnani C, Bucciarelli P, Grandone E, De Stefano V, Mannucci 

PM. Risk of pregnancy-related venous thrombosis in carriers of severe 

inherited thrombophilia. Thromb Haemost 2001; 86(3):800-3. 

2. Middeldorp S, Libourel EJ, Hamulyak K, Van der Meer J, Buller HR. The risk 

of pregnancy-related venous thromboembolism in women who are homozygous 

for factor V Leiden. Br J Haematol 2001; 113(2):553-5. 

3. Tormene D, Simioni P, Prandoni P et al. Factor V Leiden mutation and the risk 

of venous thromboembolism in pregnant women. Haematologica 2001; 86(12):1305-9. 

4. Couturaud F, Kearon C, Leroyer C et al. Incidence of venous 

thromboembolism in first-degree relatives of patients with venous thromboembolism who have factor V Leiden. Thromb Haemost 2006; 96(6):744-9. Notes: CORPORATE NAME: Groupe d'Etude de la Thrombose de Bretagne Occidentale (G.E.T.B.O) 

5. Tormene D, De Stefano V, Grandone E et al. The G20210A prothrombin 

variant and the risk of venous thromboembolism or fetal loss in pregnant women: a family study. J Thromb Haemost 2007; 5(11):2193-6. 

6. Martinelli I, Battaglioli T, De Stefano V et al. The risk of first venous 

thromboembolism during pregnancy and puerperium in double heterozygotes for factor V Leiden and prothrombin G20210A. J Thromb Haemost 2008; 6(3):494-8. Notes: CORPORATE NAME: GIT (Gruppo Italiano Trombofilia) 
Appendix G, Evidence Table 20

Page 5

Evidence Table 20. Quality of studies using survey designs included for effect of testing on clinicians’ management or effect of testing and resu

Reference List 

 1. Heshka J, Palleschi C, Wilson B et al. Cognitive and Behavioural Effects of 

Genetic Testing for Thrombophilia. J Genet Couns 2008. 

 2. Kaptein AA, van Korlaar IM, Cameron LD, Vossen CY, van der Meer FJ, 

Rosendaal FR. Using the common-sense model to predict risk perception and disease-related worry in individuals at increased risk for venous thrombosis. 

Health Psychol 2007; 26(6):807-12. 

 3. Rodger MA, Carrier M, Keely E et al. The management of thrombophilia 

during pregnancy: a Canadian survey. J Obstet Gynaecol Can 2002; 24(12):946-52. 

 4. Vossen CY, Walker ID, Svensson P et al. Recurrence rate after a first venous 

thrombosis in patients with familial thrombophilia. Arterioscler Thromb Vasc Biol 2005; 25(9):1992-7. 

 5. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

 6. Kearon C, Julian JA, Kovacs MJ et al. Influence of thrombophilia on risk of 

recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 

 7. Ridker PM, Goldhaber SZ, Danielson E et al. Long-term, low-intensity 

warfarin therapy for the prevention of recurrent venous thromboembolism. N 

Engl J Med 2003; 348(15):1425-34. Notes: CORPORATE NAME: PREVENT Investigators 

 8. Saukko PM, Ellard S, Richards SH, Shepherd MH, Campbell JL. Patients' 

understanding of genetic susceptibility testing in mainstream medicine: 

qualitative study on thrombophilia. BMC Health Serv Res 2007; 7:82. 

 9. Saukko PM, Richards SH, Shepherd MH, Campbell JL. Are genetic tests 

exceptional? Lessons from a qualitative study on thrombophilia. Soc Sci Med 2006; 63(7):1947-59. 

 10. Bank I, Scavenius MP, Buller HR, Middeldorp S. Social aspects of genetic 

testing for factor V Leiden mutation in healthy individuals and their importance for daily practice. Thromb Res 2004; 113(1):7-12. 
Appendix G, Evidence Table 21

Page 2

Evidence Table 21. Study characteristics for studies on effect of testing on clinicians’ management and the effect of testing and results on othe

Reference List 

 1. Rodger MA, Carrier M, Keely E et al. The management of thrombophilia 

during pregnancy: a Canadian survey. J Obstet Gynaecol Can 2002; 24(12):946-52. 

 2. Bank I, Scavenius MP, Buller HR, Middeldorp S. Social aspects of genetic 

testing for factor V Leiden mutation in healthy individuals and their importance for daily practice. Thromb Res 2004; 113(1):7-12. 

 3. Kaptein AA, van Korlaar IM, Cameron LD, Vossen CY, van der Meer FJ, 

Rosendaal FR. Using the common-sense model to predict risk perception and disease-related worry in individuals at increased risk for venous thrombosis. 

Health Psychol 2007; 26(6):807-12. 

4. Saukko PM, Ellard S, Richards SH, Shepherd MH, Campbell JL. Patients' 

understanding of genetic susceptibility testing in mainstream medicine: 

qualitative study on thrombophilia. BMC Health Serv Res 2007; 7:82. 

5. Saukko PM, Richards SH, Shepherd MH, Campbell JL. Are genetic tests 

exceptional? Lessons from a qualitative study on thrombophilia. Soc Sci Med 2006; 63(7):1947-59. 

6. Heshka J, Palleschi C, Wilson B et al. Cognitive and Behavioural Effects of 

Genetic Testing for Thrombophilia. J Genet Couns 2008. 
Appendix G, Evidence Table 22

Page 2

Evidence Table 22. Study characteristics for studies on the effect of testing on management 

Reference List 

 1. Vossen CY, Walker ID, Svensson P et al. Recurrence rate after a first venous 

thrombosis in patients with familial thrombophilia. Arterioscler Thromb Vasc Biol 2005; 25(9):1992-7. 

2. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

3. Kearon C, Julian JA, Kovacs MJ et al. Influence of thrombophilia on risk of 

recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 

4. Ridker PM, Goldhaber SZ, Danielson E et al. Long-term, low-intensity 

warfarin therapy for the prevention of recurrent venous thromboembolism. N 

Engl J Med 2003; 348(15):1425-34. Notes: CORPORATE NAME: PREVENT Investigators 
Appendix G, Evidence Table 23

Page 5

Evidence Table 23. Population characteristics for studies addressing other effects of testing 

Reference List 

1. Vossen CY, Walker ID, Svensson P et al. Recurrence rate after a first venous 

thrombosis in patients with familial thrombophilia. Arterioscler Thromb Vasc Biol 2005; 25(9):1992-7. 

2. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

3. Kearon C, Julian JA, Kovacs MJ et al. Influence of thrombophilia on risk of 

recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 

4. Ridker PM, Goldhaber SZ, Danielson E et al. Long-term, low-intensity 

warfarin therapy for the prevention of recurrent venous thromboembolism. N 

Engl J Med 2003; 348(15):1425-34. Notes: CORPORATE NAME: PREVENT Investigators 
Appendix G, Evidence Table 24

Page 10

Evidence Table 24. Results for articles addressing the effects of testing on VTE outcomes 

Reference List 

1. Vossen CY, Walker ID, Svensson P et al. Recurrence rate after a first venous 

thrombosis in patients with familial thrombophilia. Arterioscler Thromb Vasc Biol 2005; 25(9):1992-7. 

2. Wahlander K, Eriksson H, Lundstrom T et al. Risk of recurrent venous 

thromboembolism or bleeding in relation to thrombophilic risk factors in patients receiving ximelagatran or placebo for long-term secondary prevention of venous thromboembolism. Br J Haematol 2006; 133(1):68-77. Notes: CORPORATE NAME: THRIVE III Investigators 

3. Kearon C, Julian JA, Kovacs MJ et al. Influence of thrombophilia on risk of 

recurrent venous thromboembolism while on warfarin: Results from a randomized trial. Blood 2008. 

4. Ridker PM, Goldhaber SZ, Danielson E et al. Long-term, low-intensity 

warfarin therapy for the prevention of recurrent venous thromboembolism. N 

Engl J Med 2003; 348(15):1425-34. Notes: CORPORATE NAME: PREVENT Investigators 
Appendix G, Evidence Table 25

Page 19

Evidence Table 25. Description of Studies Evaluating the Cost-Effectiveness of Testing for Factor V Leiden or Prothrombin G2

Reference List 

1. Sarasin FP, Bounameaux H. Decision analysis model of prolonged oral anticoagulant treatment in factor V Leiden carriers with first episode of deep vein thrombosis. BMJ 1998; 316(7125):95-9. 2. Marchetti M, Quaglini S, Barosi G. Cost-effectiveness of screening and extended anticoagulation for carriers of both factor V Leiden and prothrombin G20210A. QJM 2001; 94(7):365-72. 3. Marchetti M, Pistorio A, Barosi G. Extended anticoagulation for prevention of recurrent venous thromboembolism in carriers of factor V Leiden--cost-effectiveness analysis. Thromb Haemost 2000; 84(5):752-7. 4. Eckman MH, Singh SK, Erban JK, Kao G. Testing for factor V Leiden in patients with pulmonary or venous thromboembolism: a costeffectiveness analysis. Med Decis Making 2002; 22(2):108-24. 5. Clark P, Twaddle S, Walker ID, Scott L, Greer IA. Cost-effectiveness of screening for the factor V Leiden mutation in pregnant women. Lancet 2002; 359(9321):1919-20. 6. Auerbach AD, Sanders GD, Hambleton J. Cost-effectiveness of testing for hypercoagulability and effects on treatment strategies in patients with deep vein thrombosis. Am J Med 2004; 116(12):816-28. 7. Wu O, Robertson L, Twaddle S et al. Screening for thrombophilia in high-risk situations: systematic review and cost-effectiveness analysis. The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. Health Technol Assess 2006; 10(11):1-110. 
Appendix G, Evidence Table 26

Page 27

Evidence Table 26. Quality Assessment of the Studies Evaluating the Cost-Effectiveness of Testing for Factor V Leiden or Prothrombin G202

Reference List 

 1. Sarasin FP, Bounameaux H. Decision analysis model of prolonged oral 

anticoagulant treatment in factor V Leiden carriers with first episode of deep vein thrombosis. BMJ 1998; 316(7125):95-9.  2. Marchetti M, Quaglini S, Barosi G. Cost-effectiveness of screening and extended anticoagulation for carriers of both factor V Leiden and prothrombin G20210A. QJM 2001; 94( 7):365-72.  3. Marchetti M, Pistorio A, Barosi G. Extended anticoagulation for prevention of recurrent venous thromboembolism in carriers of factor V Leiden--costeffectiveness analysis. Thromb Haemost 2000; 84(5):752-7.  4. Eckman MH, Singh SK, Erban JK, Kao G. Testing for factor V Leiden in patients with pulmonary or venous thromboembolism: a cost-effectiveness analysis. Med Decis Making 2002; 22(2):108-24.  5. Clark P, Twaddle S, Walker ID, Scott L, Greer IA. Cost-effectiveness of screening for the factor V Leiden mutation in pregnant women. Lancet 2002; 359(9321):1919-20.  6. Auerbach AD, Sanders GD, Hambleton J. Cost-effectiveness of testing for hypercoagulability and effects on treatment strategies in patients with deep vein thrombosis. Am J Med 2004; 116(12):816-28.  7. Wu O, Robertson L, Twaddle S et al. Screening for thrombophilia in high-risk 

situations: systematic review and cost-effectiveness analysis. The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. Health Technol Assess 2006; 10(11):1-110. 
Appendix G, Evidence Table 27 

Page 2 

Evidence Table 27. Applicability of the Studies Evaluating the Cost-Effectiveness of Testing for Factor V Leiden or Prothromb

Research Question 

Reference List 

1. Sarasin FP, Bounameaux H. Decision analysis model of prolonged oral anticoagulant treatment in factor V Leiden carriers with first episode of deep vein thrombosis. BMJ 1998; 316(7125):95-9. 2. Marchetti M, Quaglini S, Barosi G. Cost-effectiveness of screening and extended anticoagulation for carriers of both factor V Leiden and prothrombin G20210A. QJM 2001; 94(7):365-72. 3. Marchetti M, Pistorio A, Barosi G. Extended anticoagulation for prevention of recurrent venous thromboembolism in carriers of factor V Leiden--cost-effectiveness analysis. Thromb Haemost 2000; 84(5):752-7. 4. Eckman MH, Singh SK, Erban JK, Kao G. Testing for factor V Leiden in patients with pulmonary or venous thromboembolism: a costeffectiveness analysis. Med Decis Making 2002; 22(2):108-24. 5. Clark P, Twaddle S, Walker ID, Scott L, Greer IA. Cost-effectiveness of screening for the factor V Leiden mutation in pregnant women. Lancet 2002; 359(9321):1919-20. 6. Auerbach AD, Sanders GD, Hambleton J. Cost-effectiveness of testing for hypercoagulability and effects on treatment strategies in patients with deep vein thrombosis. Am J Med 2004; 116(12):816-28. 7. Wu O, Robertson L, Twaddle S et al. Screening for thrombophilia in high-risk situations: systematic review and cost-effectiveness analysis. The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. Health Technol Assess 2006; 10(11):1-110.