December 2017

ISSN 1650-3414

Volume 28 Number 4

Communications and Publications Division (CPD) of the IFCC
Editor-in-chief : Prof. Gábor L. Kovács, MD, PhD, DSc
Department of Laboratory Medicine, Faculty of Medicine, University of Pecs, Hungary
e-mail: ejifcc@ifcc.org

The
Journal of theInternational
Federation ofClinicalChemistry andLaboratory
MedicineIn this issue

Farewell message from the editor-in-chief
Gábor L. Kovács

241

Foreword of the editor
Gábor L. Kovács

242

IFCC Task Force on Chronic Kidney Disease (Integrated Project)– (TF-CKD) special issue
Flavio F. Alcantara, Vanja Radišić Biljak

243

Task Force on CKD – we have come a long way
Graham R. D. Jones

246

Did creatinine standardization give benefits
to the evaluation of glomerular filtration rate?
L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye

251

Moving toward standardization of urine albumin measurements
Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann

258

Cystatin C is indispensable for evaluation of kidney disease
Anders Grubb

268

Novel filtration markers for GFR estimation
Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt

277

A pathway to national guidelines for laboratory diagnostics of chronic kidney disease
V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni

289

A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing
R. Ruiz-Arenas, R. Sierra-Amor, D. Seccombe, S. Raymondo, M. S. Graziani,
M. Panteghini, T. A. Adedeji, S. N. Kamatham, V. Radišić Biljak

302

Evaluation of correlation coefficient of polyethylene glycol treated
and direct prolactin results
Shyamali Pal

315

Letter: Lab test findings in the elderly
Barna Vásárhelyi, Lóránd A. Debreczeni

328

Erratum

333

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

Farewell message from the editor-in-chief

Gábor L. Kovács, M.D., Ph.D., DSc

Dear Colleagues,This is the last issue of the eJIFCC that I edit in mycapacity as the editor-in-chief of the journal. The
two terms, i.e. a six-year period of service I heldin the Communication and Publication Divisionof the IFCC, have elapsed rapidly. Looking back,a lot of things have happened to us including alarge variety of changes we were involved in,or we initiated. 14.000 laboratorians from 93
countries receive the journal mass-mailed totheir computers quarterly. Most importantly,
eJIFCC is now indexed by PubMed Central, adevelopment that has largely increased our visibility. A year ago, we submitted our application
to Web of Science as well as to Scopus indexing; these processes, however, take years. We
have also become member of the Committeeof Publication Ethics (COPE).
The Editorial Board has been extended considerably during this period. In addition to the“old” members, twenty new members accepted to volunteer for the journal’s editorial activities, all of them renowned laboratory scientists
from different continents. Thanks to them, I
almost never received a reviewer invitation rejected. The editorial board members were also
most helpful in reviewing the presentations of

IFCC’s e-Academy, a new and highly successful
product of the Communication and Publication
Division. I specially thank the help of my two associate editors, Dr. Harjit Pal Bhattoa (Hungary),
responsible for the linguistic editing, and Dr.
Reinhard B. Raggam (Austria), the case-report
editor of the journal.
The journal, the editorial board and the editor has always had the full support of Dr. Ellis
Jacobs and Dr. Khosrow Adeli, the two chairs of
the Communication and Publication Division,
who followed each other in my editorial period.
The professional help of Insoft Digital in the epublication of the journal needs to be acknowledged as well. They have greatly improved theprofessional appearance of the journal.
Last, but not least, I have to thank Mrs. Silvia
Colli-Lanzi at the IFCC Office in Milan. Without
her enthusiastic and highly professional assistance, neither the IFCC, nor the e-journal would
be the same.
I wish the new editor and especially the laboratorians out there on all the continents further
great success with the eJIFCC!
Pécs, November 13th, 2017
Gabor L. Kovacs

Page 241

eJIFCC2017Vol28No4pp241-241

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

Foreword of the editor

Editor-in-Chief: Gábor L. Kovács, MD, PhD, DSc

This special issue has been dedicated to thelaboratory diagnosis of chronic kidney diseases.
The IFCC Task Force on Chronic Kidney Disease
sheds light on several aspects of the field, frombasic research to daily clinical practices, uniting
many IFCC member countries working in different aspects of the laboratory in chronic kidney care. Dr. Flavio Ferraz de Paes e Alcantara(Brazil), the chair of the task force and Dr. Vanja
Radišić Biljak (Croatia), a member of the task
force, were asked to guest-edit the issue.
Dr. Flavio Ferraz de Paes e Alcantara graduatedin medicine in Santos, Brazil. He earned thespecialization degree in clinical pathology after
finishing medical residency at the clinical hospital of the University of Sao Paulo. Worked asa post-doctoral fellow at The Scripps ResearchInstitute (USA), from 1996 to 2001. In 2001,
he came back to Brazil as associated director of a medium size private clinical laboratory (IACS), working there part time, and as of2012 became the leading director of IACS. Dr.
Alcantara also works part time at the Universityof São Paulo. Initially as a research fellow, since2006 he holds a tenure position as assistantphysician at the Central Laboratory Division Molecular Biology Section, the largest public
hospital in South America. Dr. Alcantara is anactive member of the Brazilian Society for
Clinical Pathology and Laboratory Medicine,where he is frequently invited for lectures andmeetings. In 2010, Dr. Alcantara started a termat the IFCC-WASPaLM Task Force on Chronic
Kidney Diseases, and became the chair person
for the 2016-2018 term.
Dr. Vanja Radišić Biljak, PhD, from Zagreb,
Croatia, studied medical biochemistry at
the Faculty of Pharmacy and Biochemistry,
University of Zagreb (1999-2004). In 2005,
she started her postgraduate doctoral study in
medical biochemistry and defended her doctoral thesis „Chronic obstructive pulmonary
disease and glutathione cycle” in 2010, gaining
her PhD degree. Since 2010 she has been employed in Merkur University Hospital where she
started her residency in Medical Biochemistry
and Laboratory Medicine and graduated in
2014. Her major interest shifted towards nephrology, diabetes, and medical informatics.
In 2010, she got a scholarship for EASD Young
Scientists Training Course “Reactive metabolites in late diabetic complications” (University
Hospital Heidelberg, Dept. of Medicine I and
Clinical Chemistry) in Heidelberg, Germany. In
2013, she received a travel grant for the EFLM
Postgraduate Course in Clinical Chemistry andLaboratory Medicine New Trends in Diagnosis
and Monitoring using POC Instruments. In
2016, she was awarded as the best young scientist for 2015. The award was presented by
the Croatian Society for Medical Biochemistry
and Laboratory Medicine.
Page 242

eJIFCC2017Vol28No4pp242-242

n Chroni special
Kidney Diseas– Integrated
Pro ect
TF-CKDissue: Thelaboratory
diagnostics of chronic kidney diseases

IFCC Task Force on Chronic Kidney Disease(Integrated Project) – (TF-CKD) special issue
Guest editors: Flavio F. Alcantara1, Vanja Radišić Biljak2
1
2

Central Laboratory Division, Clinical Hospital, University of São Paulo, Medical School, Brazil
Department of Medical Biochemistry and Laboratory Medicine, Merkur University Hospital,
Zagreb, Croatia

ARTICLE INFOEDITORIAL

Corresponding author:
Flavio F. Alcantara
Hospital das Clínicas
Av Eneas Carvalho Aguiar
155. Bloco 4, 2o andar
Sao Paulo 05403-000 SP
BrazilPhone: +55 11 2661 7275
E-mail: flavio.alcantara@hc.fm.usp.br

This series of articles is a milestone not only for theInternational Federation of Clinical Chemistry andLaboratory Medicine (IFCC) and the World Associationof Societies of Pathology and Laboratory Medicine(WASPaLM) Task Force on Chronic Kidney Disease (TFCKD) integrated project, but also an important outline
on the role of the laboratory on assessment of therenal function in different scenarios. It sheds lighton several aspects of the field, from basic research
to daily clinical practices, uniting many IFCC member
countries working with different aspects of the laboratory in CKD care.

Key words:
Chronic Kidney Disease (CKD),IFCC TF-CKD, KDIGO 2012 guidelines

In the initial article “We have come a long way” (1),a brief history of the Task Force is narrated. The IFCC
leadership with WASPaLM partnership formulated a
TF which amalgated the two major International scientific societies in Laboratory Medicine. The initialidea of “forging a global consensus” has shifted onto
“helping to create and implement national guidelines
in each country through corresponding members”, andlater using the KDIGO 2012 guidelines (2) as a frame
document.
IFCC has succeeded in gaining acceptance as a global
partner for CKD care (3).
Page 243

eJIFCC2017Vol28No4pp243-245

Flavio F. Alcantara, Vanja Radišić Biljak
IFCC Task Force on Chronic Kidney Disease (Integrated Project) – (TF-CKD) special issue

KDIGO 2012 guidelines classifies CKD based on
cause, Glomerular Filtration Rate (GFR) category,
and albuminuria category (CGA) (2), thus emphasizing the role of laboratory medicine in management of CKD. One of the major laboratory tests
involved in CKD management is certainly creatinine and consequently estimation of GFR via
estimating equations (2). The group of authors
on behalf of the Société Française de Biologie
Clinique in the article “Did creatinine standardization give benefits to the evaluation of glomerular filtration rate” (4) evaluate some limitationsof creatinine and emphasize the importance on
using IDMS traceable enzymatic assays and thereporting of eGFR.
Regarding albuminuria measurement, the article “Moving Toward Standardization of Urine
Albumin Measurements” (5) reports on thecontinuing effort undertaken by the NKDEP
Laboratory Working Group following their success on the standardization of creatinine measurement. It mentions their work on pre-analytical issues, the current state of measurements
evaluating their precision and accuracy, the strategy undertaken defining a candidate reference
method and for producing certified referencematerials, including an evaluation of several albumin methods as previously published (6).
KDIGO 2012 guidelines also recognize the valueof estimating GFR using Cystatin C measurements
as a biomarker alternative to creatinine (2). In thearticle “Cystatin C is indispensable for evaluationof kidney disease” (7) a good case is made forusing Cystatin C instead of other biomarkers for
GFR. In fact, given their expertise, the Swedish
have realize the importance of using botheGFRcrea and eGFRcysC, providing not only the best
estimate GFR, but more importantly, yielding themean eGFRcrea and eGFRcysC value. This eGFRmean,
when in close agreement with each of the single
eGFR results, is the best evaluation of GFR; insituations where the disagreement is above~1/3 (eGFRcysC ≤ 60% eGFRcrea), the decreased

cystatin C filtration signals the presence of therecently described “Shrunken Pore Syndrome”,
predicting increase in mortality and morbidity
while elucidating key pathophysiologic aspects
on kidney diseases.
Nevertheless, neither creatinine nor Cystatine C
present the ideal marker for estimating GFR. The“Novel Filtration Markers for GFR estimation”(8) article includes an update on the past andpresent research on two Glomerular Filtration
Rate markers: the 11.8 kDa Beta Trace Protein(BTP) and the 23-29 kDa Beta 2 Microglobulin(B2M), some equations designed for their use
in GFR estimation and the experience in specific patient cohorts using these markers, comments on approaches using panels of markers
such as eGFRcre, eGFRcys, eGFRBTP, and eGFRB2M.
Additionally, there is a glimpse on the use of
metabolomic on studies searching for markers
associated with eGFRcre.
“A pathway to national guidelines for laboratory
diagnostics of chronic kidney disease – examples
from diverse European countries” take us on
the path travelled by several countries toward
improvement of CKD care (9). Various scenarios
on developing and implementing national CKD
guidelines are described, ranging from as earlyas 2002 when the Sociéte Francaise de Biologie
Clinique (SFBC) formed the “Creatinine Working
Group”, later joined by Sociéte de Nephrologie,
until 2017 and releasing the newest recommendations from Croatian Working Group forLaboratory Diagnostic of CKD.
The last article of the series “A summary ofworldwide activities in chronic kidney disease(CKD) testing” (10) includes examples of countries with different settings. National CKD activities from almost every continent are presented,
which makes the very first step in achieving thenational CKD guidelines as a final goal.
A world of CKD has been depicted and different
activities have been summarized. Unfortunately,

Page 244

eJIFCC2017Vol28No4pp243-245

Flavio F. Alcantara, Vanja Radišić Biljak
IFCC Task Force on Chronic Kidney Disease (Integrated Project) – (TF-CKD) special issue

a lot has not been told and several fundamental
authors and settings were not included, due totime and space constraints. Still, different levelsof maturity on CKD care can be grasped upon. In
laboratory, the saying goes that “quality is not
an end point but a journey”, we hope the roadahead may now have some additional marks forthe travelers.

4. Piéroni L, Bargnoux AS, Cristol JP, Cavalier E, Delanaye
P. Did creatinine standardization give benefits to the evaluation of glomerular filtration rate? eJIFCC.

REFERENCES

7. Grubb A. Cystatin C is indispensable for evaluation of
kidney disease. eJIFCC.

1. Jones GRD. Task Force on Chronic Kidney Disease – We
have come a long way. eJIFCC

8. Karger AB, Inker LA, Coresh J, Levey AS and Eckfeldt JH.
Novel filtration markers for GFR estimation. eJIFCC.

2. Kidney Disease: Improving Global Outcomes (KDIGO)CKD Work Group. KDIGO 2012 clinical practice guideline
for the evaluation and management of chronic kidneydisease. Kidney Int Suppl. 2013;3(1):1-150.

9. Radišić Biljak V, Momberg Aakre K, Yucel D, Bargnoux
AS, Cristol JP, Pieroni L. A pathway to national guidelines
for diagnosis of chronic kidney disease – examples fromdiverse European countries. eJIFCC.

3. Levin A, et at. Global kidney health 2017 and beyond:a roadmap for closing gaps in care, research, and policy.
Lancet 2017; 390: 1888–917.

5. Seegmiller J, Miller WG, Bachman LM. Moving towards standardization of urinary albumin measurements.
eJIFCC.
6. Bachmann LM, Nilsson G, Bruns DE, McQueen MJ,
Lieske JC, Zakowski JJ, and Miller WG. State of the Art for
Measurement of Urine Albumin: Comparison of Routine
Measurement Procedures to Isotope Dilution Tandem
Mass Spectrometry Clin Chem 2014;60(3):471–480.

10. Ruiz-Arenas R, Sierra-Amor R, Seccombe D, Raymondo S, Graziani MS, Panteghini M, Adedeji TA, Kamatham
SN, and Radišić Biljak V. A summary of national activities
in Chronic Kidney Disease CKD testing. eJIFCC.

Page 245

eJIFCC2017Vol28No4pp243-245

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

Task Force on CKD – we have come a long way
Graham R. D. Jones

Department of Chemical Pathology, SydPath, St Vincent’s Hospital, Sydney, Australia

ARTICLE INFOABSTRACT

Corresponding author:
Graham R. D. Jones
Department of Chemical Pathology,
SydPath, St Vincent’s Hospital
Victoria St, Darlinghurst
NSW 2010, Sydney
Australia
E-mail: Graham.jones@svha.org.au

Chronic Kidney Disease (CKD) is an important medical
condition where diagnosis, staging and monitoring islargely based on routine laboratory tests. During thelast 15 years there have been many important changes in the clinical management of CKD described in
key international guidelines. In order to successfully
implement these guidelines, laboratories must collaborate with clinicians to provide a co-ordinated service, including accurate measurements and of creatinine and urine albumin and reporting of an estimatedglomerular filtration rate (eGFR). The IFCC/WASPaLM
Task Force on Chronic Kidney Disease (TF-CKD) wasestablished in 2008 and since that time has worked to
improve laboratory testing in CKD. Key aspects of thework of the TF-CKD include supporting national laboratory medicine organisations to develop CKD testing
guidelines, recognition of the vital role of collaboration between laboratory and clinical organisations,the importance of accurate measurements, and endorsement of the KDIGO 2012 CKD guidelines. A key
function of the TF-CKD has been to facilitate sharing
and learning between countries to provide the best
outcomes.

Key words:
urine albumin, reference measurement
procedure, standardization, calibration

Page 246

eJIFCC2017Vol28No4pp246-250

Graham R. D. Jones
Task Force on CKD – we have come a long way

INTRODUCTION

A BRIEF HISTORY OF THE TF-CKD

One of the great ways that progress is made isthrough the power of people working together.
The current practices for laboratory testing for
Chronic Kidney Disease (CKD) in many countries
is the product of many different collaborations.
The players involved in these collaborations include laboratory scientists, chemical pathologists, nephrologists, general practitioners, researchers, diagnostic manufacturers and manyothers. Often the mechanism for these collaborations is through professional societies and other organised structures.

The TF-CKD was formed in 2008 on the initiativeof then IFCC President Mathias Müller who recommended the formation of a Working Groupon Screening for Chronic Kidney Disease (WGCKD). The members of the WG included laboratory scientists, nephrologists and a chemical pathologist. In 2009 the terminology waschanged to “Task Force” (TF-CKD) and an invitation was extended to the World Association ofSocieties of Pathology and Laboratory Medicine(WASPaLM) to be a joint sponsor of the TF, recognising the importance of pathologists as wellas laboratory scientists and with the aim of getting the widest professional and organisational
coverage. The invitation was accepted and theinitial full membership is shown in Table 1.

I believe the IFCC Task Force on Chronic Kidney
Disease (TF-CKD) has played an important role inpromoting good laboratory practice in this field
through collaboration on a range of levels. In
this paper I outline the activities of the TF-CKD
and its role in laboratory testing for CKD.
For this purpose I will consider three separate
aspects: the formation and early years; the recommended approach to organising CKD testing;and the effects of sharing.
Table 1

These members had experience with developing and implementing CKD testing guidelines, inresearch in the field of CKD testing, of the measurement of serum creatinine and in the clinicalapplication of the laboratory tests. Importantly
the membership also had key roles in clinical, research and guideline organisations outside the

WG-CKD initial membership
IFCC Nominees

Graham JONES (AUS)

Joe CORESH (USA)

Edmund LAMB (UK)

Andy NARVA (USA)David SECCOMBE (CAN)

Mauro PANTEGHINI (IT)Joris DELANGHE (BEL)
WASPaLM Nominees

John ECKFELDT (USA)

Adagmar ANDRIOLO (BRA)(replaced by Flavio ALCANTARA (BRA) during 2010)
Page 247

eJIFCC2017Vol28No4pp246-250

Graham R. D. Jones
Task Force on CKD – we have come a long way

laboratory medicine community allowing communication and collaboration. The membership
remained very similar for the first six years andsome original members remain active today.
The following Terms of Reference were adopted at the first meeting:•To achieve global consensus on the laboratory strategy (including reporting) for theidentification, diagnosis, and monitoring ofchronic kidney disease.

•

To collaborate in the preparation of international diagnostic guidelines with relevant
clinical organisations by providing guidanceon laboratory aspects of chronic kidney disease testing.

•To facilitate the guideline implementation
within IFCC member organizations and reach
improvement over the current situation.

EARLY ACTIVITIES OF THE TF-CKD
While the initial plans for the TF-CKD were aimed
at preparing a global guidance document, thefirst major activity was a survey of current practice in laboratory testing related to CKD. At thattime the latest international guidelines were
the United States National Kidney Foundation
Kidney Disease Outcome Quality Initiative (K/
DOQI) 2002 guidelines which provided, amongst
many items, a clear definition of CKD and also
recommended routine reporting of an eGFR
with serum creatinine (1). The survey was conducted in 2010 and distributed amongst IFCC
and WASPaLM member organisations with 25
responses with the aim to assess uptake of the
K-DOQI recommendations. It is likely that the results were skewed to countries with an interestin the topic. Of the respondents 42% had national guidelines on eGFR reporting, with the guidelines being produced either by, or in collaboration with a renal medicine organisation. Fewerthan half the responding countries estimatedthat over 80% of laboratories routinely reported
an eGFR and many were using creatinine assays
which were not aligned to the reference method (isotope dilution mass spectrometry, IDMS).
A key feature was a strong positive reaction toquestions about willingness to share experienceand to receive assistance in this area.
Members of the Task Force were also independently active in developing guidelines in theirown countries, speaking at national and international meetings, and with involvement in research on laboratory and clinical issues. For example, in 2011 Task Force members presented atmeetings in Berlin, China, Malaysia and Mexico.
ORGANISING CKD TESTING
As stated above, the original goals of the TFCKD included “to achieve global consensus on
the laboratory strategy (including reporting) for
the identification, diagnosis, and monitoring ofchronic kidney disease.” However over time it
became apparent to the membership that CKD
testing programs are best organised at the national rather than global level. The examples ofstructured CKD testing that were in place wereorganised at the national rather than the international level. For example by 2010 the survey showed that at least nine countries were
known to have national CKD testing programs.
Other organisational categories may be regional(a number of countries acting together) or at astate or provincial level within a country. There
are many reasons for thinking that way. Firstly
the available resources, including laboratory facilities, doctors, medicine, are often very different in different parts of the world. Importantly
an organisational structure is required to formulate then implement change. The relevant
structures include laboratory and clinical professional organisations, medical education (preand post-graduate), medical and laboratory
funding and governments.

Page 248

eJIFCC2017Vol28No4pp246-250

Graham R. D. Jones
Task Force on CKD – we have come a long way

Thus the recommendation of the TF-CKD became to assist countries (or regions or states) todevelop and implement CKD testing programs,
as opposed to recommending the same approach for everyone. The issues that need tobe considered can be addressed by, and owned
by, local organisations and people. The role of
the TF-CKD then is to support these national
activities.
The other major international event in thefield of CKD was the publication of the Kidney
Disease Improving Global Outcomes (KDIGO)
2012 Guideline on the diagnosis and management of CKD (2). It is hard to overestimate thequality and importance of this document in thefield of CKD testing and management. Buildingon the 2002 KDOQI guideline, it contains a balanced and evidence based-approach to achieving the aims in the document title. Importantly
it also describes key aspects of laboratory testing including creatinine standardisation, eGFR
reporting and interpretation and urine albuminmeasurement. The document also provides away of approaching the data, for example recommending the use of the CKD-EPI formula for
estimation of GFR, unless there is evidence thatan alternative formula can improve the accuracyof the result. The use of a common guideline tosupport both clinical and laboratory activities
ensures that laboratory testing is supportive of
the clinical goals in caring for patients with kidney disease.
In response to the publication of this document,
the TF-CKD formally recommended that any
CKD testing programs should be based on thisdocument. To put these last two items together,
the TF-CKD, in 2013, recommended that CKD
testing programs should be organised nationally, using the 2012 KDIGO guidelines as a basiswith changes as required for local adoption.

SUPPORT FOR NATIONAL ACTIVITIESThe original membership of the TF-CKD waslimited to individuals with known expertise in
the area. In 2012 the concept was raise of inviting “corresponding members” from as manymember organisations as wished to join. A corresponding member needed to have an interestin the field and the support of the relevant national biochemistry or pathology organisation.
The effect of this was to markedly expand themembership and include people with an active
interest, but possibly limited specific knowledge
in the field.
One assessment of this expansion is that it has
produced the greatest effects of the TF-CKD. By
becoming a member and participating at meetings and in e-mail discussions, there was an opportunity to learn and then facilitate activities
in the home country. A key example of this approach in action was the TF open meeting heldat the Paris IFCC congress in 2015. The formatof the meeting was presentations from members about the state of progress in CKD testingin their home country. There were presentations
from thirteen countries from six continents enabling a period of sharing experiences and creating new contacts. The countries presented
were at many stages of the process of developing or implementing CKD guidelines. Following
on from this meeting members have played key
roles in the development of CKD guidelines in
Croatia (3) and Turkey (4).
In an offshoot from the TF-CKD, a similar process
has started under the auspices of the Asia PacificFederation of Clinical Biochemistry (APFCB).
At their regional meeting in Taiwan in 2016, a
meeting of national representatives of seven
Asian countries again shared experiences andchallenges in the area.

Page 249

eJIFCC2017Vol28No4pp246-250

Graham R. D. Jones
Task Force on CKD – we have come a long way

THE FUTURE OF THE TF-CKD
With the rise of the numbers of corresponding
members, as well as corporate representatives,in 2017 the TF includes 26 members representing 23 countries (5).
There remains much work to be done. A recent
international survey by the International Societyof Nephrology has indicated that measurementof serum creatinine with eGFR reporting was either not available or only rarely available in 63%
of countries worldwide, and creatinine alone either not available or only rarely available in 35%
of 119 countries assessed (6). A follow-up paper
has identified the IFCC as a partner organisation
for improvement in laboratory testing in CKD (7).
There also persists a need for activity to promote
improvements in assay quality. Specifically creatinine is the basis of GFR assessment in most of
the world and clinicians rely on laboratories for
quality results. While the quality of assays usedin the developed world has improved markedly,in the developing world it is often difficult fora laboratory scientist to even identify whether
a creatinine assay is traceable to international
standards (8).
I believe that the TF-CKD has been, and will continue to be an active force for change in improving the use of laboratory testing to identify andmanage patients with CKD. The mechanisms
are to promote the development of appropriate national programs through collaboration oflaboratory medicine and other organisations.
This assistance may be through providing a listof issues to address, partnering with individual
countries, advising on the processes or technicalissues, providing guest speakers or other ways.

It has been my pleasure to be involved in CKD
testing for over 12 years during which it is fairto say the world has changed (9). The improvement of laboratory medicine is an adventure weall should play a part in.
REFERENCES
1. K/DOQI clinical practice guidelines for chronic kidneydisease: evaluation, classification and stratification. Am J
Kidney Dis. 2002;39:S1-S246
2. Kidney Disease: Improving Global Outcomes (KDIGO)CKD Work Group. KDIGO 2012 clinical practice guideline
for the evaluation and management of chronic kidneydisease. Kidney Int Suppl. 2013;3(1):1-150.
3. Radišić Biljak V, Honovic L, Matica J, Krešić B, Šimić
Vojak S. The role of laboratory testing in detection andclassification of chronic kidney disease: national recommendations. Biochem Med 2017;27(1):153–76
4. Abuşoğlu S, Aydın I, Bakar F, Bekdemir T, Gülbahar O,İşlekel H, Özarda Y, Pektaş M, Pir K, Portakal O, Serdar M,
Turhan T, Yücel D, Zengi O. A short guideline on chronic
kidney disease for medical laboratory practice. Turkish
Journal of Biochemistry – Türk Biyokimya Dergisi; 2016;
41 (4): 292-301
5. Task Force on Chronic Kidney Disease (Integrated Project) - (TF-CKD). http://www.ifcc.org/executive-boardand-council/eb-task-forces/task-force-on-chronic-kidney-disease/ (accessed 30th October 2017)
6. Bello AK, Levin A, Tonelli M, Okpechi IG, Feehally J, Harris D et al. et al. Assessment of Global Kidney Health Carestatus. JAMA. 2017
7. Levin A, Tonelli M, Bonventre J, Coresh J, Donner J-A,
Fogo AB et al. Global kidney health 2017 and beyond: aroadmap for closing gaps in care, research, and policy.
Lancet 2017; 390: 1888–917
8. Radišić Biljak V, Honovic L, Matica J, Kneževic B,Šimić Vojak S. Laboratory diagnostics of chronic kidney disease in Croatia: state of the art. Biochem Med
2015;25(1):73–83
9. Jones GRD. Adventures with Creatinine and eGFR - A
National, International and Personal Story – AACB Roman
Lecture 2014. Clin Biochem Rev 2015;36:75-82

Page 250

eJIFCC2017Vol28No4pp246-250

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

Did creatinine standardization give benefits
to the evaluation of glomerular filtration rate?
Laurence Piéroni1,4, Anne-Sophie Bargnoux1,4, Jean-Paul Cristol1,4,Etienne Cavalier2,4, Pierre Delanaye3

Department of Biochemistry and Hormonology, CHU de Montpellier, PhyMedExp,
University of Montpellier, France
2
Department of Clinical Chemistry, Sart Tilman Hospital and University of Liège, Belgium
3
Department of Nephrology Dialysis Transplantation, Sart Tilman Hospital and University of Liège, Belgium
4
On behalf of the Société Française de Biologie Clinique
1

ARTICLE INFOABSTRACT

Corresponding author:
Jean-Paul Cristol
Department of Biochemistryand Hormonology
CHU de Montpellier
371, Avenue du Doyen Gaston Giraud
34295 Montpellier Cedex 5
FranceEmail: jp-cristol@chu-montpellier.fr

During the last decade, a lot of efforts has been
made to improve the evaluation of renal functions.
Measured Glomerular Filtration Rate (GFR) remains
the only valuable test to confirm or confute the statusof chronic kidney disease (CKD) and is recommended
by Kidney Disease Global Outcomes guidelines whenestimation of GFR is not reliable. However, in routine
clinical practice, serum creatinine remains the oneof the most prescribed biological parameters and isan undeniable factor, alone or in association withother parameters, of the estimation of GFR. Sincemany years, a great improvement in the creatinine
measurements was realized because of the standardization of the methods and fabrication of an international standard with concentration near to physiological ones (SRM967). Standardization according
to Isotopic Dilution Mass Spectrometry dramatically
improves the analytical performances of creatinine
assays resulting in a more accurate estimation of
GFR using creatinine based equations. Indeed, thestandardization of creatinine improves the analytical
performance by reducing the bias and removing theinfluence of the interfering substances.

Key words:
creatinine, standardization,
glomerular filtration rate

Page 251

eJIFCC2017Vol28No4pp251-257

L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye
Did creatinine standardization give benefits to the evaluation of glomerular filtration rate?

However, biological variability of creatinine is
not affected by analytical standardization andremains a limitation to the use of creatinine insome selected populations, having extreme ages
or weights like children, elderly subjects, obese
or malnourished populations. Standardization ofcreatinine assays result in a clear improvementof estimated GFR in general population but alternative methods should be used when creatinine production or metabolism is impaired.


INTRODUCTION
Today, serum creatinine (SCr) is still one of
the most prescribed analyses in medical laboratories to estimate the glomerular filtration
rate (GFR) [1] and it is now recommended tointegrate its value in a predictive equations.
But creatinine is still used in some parts of theworld to evaluate kidney function. Since methods for measuring SCr is potentially prone toseveral interferences, e.g. with bilirubin or
pseudochromogens [2-4], the imprecision of
the SCr measurement has been improved fromthe initial manual Jaffe method with importantinnovations. Earlier in the 1970s, the automatization of the methods began [5-7], followedby the development of kinetic measurements
and by the emergence of enzymatic methods,
almost free from interference by pseudochromogens like proteins [2-4, 8, 9]. Finally, thedevelopment of GC-IDMS or LC-IDMS as reference methods allowed the emergence of IDMS
traceable assays [10].
However, limitations of creatinine as a potential GFR biomarker is not restricted to analytical considerations. First, creatinine levels are
dependent of muscle mass since creatinine is a
product of muscle catabolism of creatine phosphate [11, 12, 13]. Extremely low or extremely
high muscular mass could result in a misinterpretation [14, 15]. Secondly, a tubular secretionof creatinine exists and this secretion could be
responsible for an overestimation of GFR especially during the course of chronic kidney disease [11, 16-18]. Third, Serum creatinine canalso be influenced by diet. Meals rich in proteins such as cooked red meat can increase theserum creatinine. The GFR itself also increases
with such food intakes [11, 13, 19-21]. Fourth,some authors have described extrarenal clearance of serum creatinine, possibly by intestinalbacteria, which could be relevant in advanced
chronic kidney disease (CKD) [22]. Finally, theproduction of creatinine, from muscular creatine, could be influenced negatively in severe
hepatic disease and positively in rhabdomyolysis [11, 23].
These sources of imprecision are “physiologic
limitations” of serum creatinine and one canonly be conscious of them. But the standardization of methods is actually required for reducing analytical errors like bias in the creatinine
measurement. We present here the actions
made during the last decade resulting in standardization of creatinine measurements andtheir possible consequences on GFR estimation.
HOW CAN WE STANDARDIZE CREATININE
MEASUREMENT METHODS?
The concept of the standardization of creatinine measurement was simple. The CreatinineStandardization Program was created by NKDEP’s
Laboratory Working Group in collaboration with
the International Federation of Clinical Chemistry
and Laboratory Medicine (IFCC) and the European
Communities Confederation of Clinical Chemistry(now called the European Federation of Clinical
Chemistry and Laboratory Medicine) to reduce
interlaboratory variation in creatinine assay calibration. The National Institute for Standards
and Technology (NIST) has released a standard
reference material (SRM 967 Creatinine in Frozen
Human Serum) for use in establishing calibrations

Page 252

eJIFCC2017Vol28No4pp251-257

L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye
Did creatinine standardization give benefits to the evaluation of glomerular filtration rate?

for routine creatinine measurement procedures, with demonstrated commutability withnative clinical specimens in routine methods.
These materials were value-assigned with thegas chromatography (GC) -isotope dilution mass
spectrometry (IDMS) and liquid chromatography (LC)-IDMS reference measurement procedures [24]. A concentration of 88,4 µmol/L(1mg/dL) was chosen since this value is comprised in the critical range 1.0-1.5 mg/dL that allows clinical laboratories to verify that method
performances follow recommendations (Total
error in creatinine measurement should not increase the variability in eGFR more than 10% in
eGFR at a serum creatinine concentration of 1.0
mg/dL) [3]. A new SRM 967a was prepared with
two sub-pools, with one having normal levels ofcreatinine (Level 1, 0.8 mg/dL±0.1 mg/dL), andthe other spiked with crystalline creatinine toachieve an elevated level of creatinine (Level 2,
4.0 mg/dL±0.2 mg/dL) to explore a wide rangeof creatinine values.
Since the Creatinine Standardization Program
has requested the manufacturers to standardize their creatinine assays to an IDMS reference
measurement procedure, we can theoretically
expect that the same sample will give the same
result in any laboratory in the world, whatever
the method (Jaffe or enzymatic) and manufacturer, since the calibrators will all be “traceable”
to the higher-order method [25, 26].
But several independent studies have shownthat results obtained with so-called IDMS traceable methods (notably Jaffe assays and somedry enzymatic methods) still provide results thatwere quite far away from the “true value,” asdetermined with a reference method [27, 28].
Importantly, this occurs most of the times when
dealing with lower creatinine values, whereas,
once again, this is the range of values with thelargest impact on eGFR variability. Finally, we canassert that most enzymatic assays on the market in 2017 are IDMS-calibrated [29]. Enzymatic

assays have reached the goal to decrease theinter-assay variability and thus to decrease systematic differences (i.e., bias) between assays[30]. However, the systematic error due to thebias inherent to calibration is only one part ofthe potential error linked to the serum creatinine measurement.
WHY CREATININE STANDARDIZATION
LED TO REDUCING INTERFERENCES
IN CREATININE METHODS?
The first goal to reach when you try to standardize a method is to find a process which allows
you to get a specific method. Two types of methods are used to determine creatinine concentrations: enzymatic and Jaffe’s methods. Both arecolorimetric methods but since the first ones areusing enzymatic reactions, they are more specific than the Jaffe’s ones [27].
In 1886, Jaffe [31] described complex formation between picric acid and creatinine in an
alkaline environment. Since then, several colorimetric methods based on Jaffe’s observation
were commercialized [32]. The total error budget of colorimetric methods was rather due tobias than to imprecision, in particular for low
creatinine concentrations. This bias is due tothe analytical interference by pseudo-chromogens for the Jaffe group [33] or to the calibration used in the dry chemistry method [34]. The
earlier processes to reduce the interference ofpseudo-chromogen effect of proteins [35] on
the reactions based on alkaline picrate were deproteinization or dialysis. Today, however, analyzers use untreated serum or plasma, making
creatinine assays using alkaline picrate reaction
prone to the so-called ‘‘protein error’’ [33]. On
average, this effect produces a positive difference of 27 µmol/L creatinine compared withenzymatic methods [33]. Moreover, before
standardization, each assay was calibrated withspecific material provided by the manufacturers

Page 253

eJIFCC2017Vol28No4pp251-257

L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye
Did creatinine standardization give benefits to the evaluation of glomerular filtration rate?

and particular processes. For example, different Jaffe assays would lead to different serum
creatinine results [3, 25, 34, 36, 37]. Compared
to non-calibrated assays, using IDMS traceable
creatinine (and creatinine-based equations
specifically developed for such standardized assays) leads to a modest but significantly better
performance for eGFR [38].
However, harmonization of creatinine measurement between laboratories is especially important in population studies and on the longitudinal monitoring of renal function in individuals,
with great influence on the establishment ofreference intervals. Ceriotti et al., when trying to identify universally applicable reference
intervals for creatinine via a systematic reviewof the literature, concluded that only data obtained with enzymatic assays had to be considered because of the higher specificity of thisanalytical approach [39]. They explained theirchoice because the subtraction of 18–25 µmol/Lto eliminate protein-related unspecific interference on alkaline picrate assays significantly
improves the correlation of these assays withenzymatic ones. In this situation, the obtainedreference intervals are very similar to those of
the enzymatic methods. However, on individual
samples, especially at the low creatinine concentrations found in children, large differences
can be seen.
Indeed, since the relationship between sCr andeGFR is actually exponential, it implies that
small sCr differences will greatly impact the GFRvalues at low SCr values (corresponding to high
GFR values) but the same difference will haveminimal impact at high SCr values (corresponding to low GFR values). Therefore, if we consider
an analytical error of 17.6 µmol/L in creatinine
measurement for a 60 year-old man presenting acreatinine value of 98.6 µmol/L, this value is notdifferent from 116.2 µmol/L. The corresponding GFR values with the CKD EPI study equation
will be 71 or 58 ml/min/1.73 m2, respectively.

The same example with a serum creatinine of
264 µmol/L and 281.6 µmol/L with the other assay will give CKD-EPI results of 22 and 20 mL/
min/1.73 m2, respectively [3, 40-43]. A relative
low analytical error of 17.6 µmol/L creatinine
can therefore be responsible for a misclassification in the staging of CKD.
Is standardization responsible for the improvement of the imprecision of creatinine assays?
Comparing the analytical imprecision of bothmethods, the coefficient of variation (CV) issystematically better for the enzymatic assays[2, 44]. For low creatinine concentrations presented by children [2], the serum creatinine
concentrations measured with the Jaffe reaction will be higher than with the enzymatic
assay. Therefore, one may prefer enzymatic
assays in specific populations like in children
or in patients with hyperfiltration but also in
specific situations where some Jaffe’s methods
are subject to interferences like bilirubin, ketoacidosis etc.
The gain in imprecision (due to a smaller random error) with the enzymatic assays as compared to Jaffe assays is an intrinsic characteristic of the assay and is totally independent of
the standardization procedure, which only improves the systematic error.
DID STANDARDIZATION
GIVE BENEFIT TO EGFR?
Another source of variability of creatinine is biological variation expressed in an intra-individual
CV. This variation is physiological, independentof the analytical CV and cannot be reduced bystandardization [44].
Indeed, when combining the intra individual CV(5.95%) and analytical CV for Jaffe (5.5%) andenzymatic (2%) methods, in a 60-year old man,this means that for a given GFR, the serum creatinine concentration may vary for a creatinine

Page 254

eJIFCC2017Vol28No4pp251-257

L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye
Did creatinine standardization give benefits to the evaluation of glomerular filtration rate?

concentration of 88.4µmol/L between 80.1 and117 µmol/L if the Jaffe assay is used or between
85.4 and 111.8 µmol/L if the enzymatic assay isused. Using the CKD-EPI equations, this range
of non-different sCr values leads to eGFR values
that may vary between 58 and 92 mL/min/1.73
m2 for Jaffe serum creatinine and between 61
and 84 mL/min/1.73 m2 for the enzymatic assay results. The intrinsic variability of creatinineis thus not so negligible when it is used in theeGFR equation. The relevance of this variation
will be, once again, important in adults withnormal or close to normal serum creatinine values and especially in children.
CONCLUSION
Standardization of creatinine assays is effective
in 2017. This improvement in creatinine measurements has decreased the analytical component of creatinine variability and for assessing
the transferability of creatinine results, a relatively simple recommendation is to use enzymatic assays (to decrease the random error)
and IDMS traceable assays (to decrease the systematic error). Today enzymatic methods haveshown to be effectively calibrated to IDMS [29,44]. However, with an analytical imprecision of
2% (for usual assays), the error due to intra-individual biological variation still remains. Thus,
to overcome this limitation in selected populations (extreme age or body size, muscle diseases including severe denutrition, vegetarian
diet…) recommendation is to measure GFR [1].
REFERENCES
1. KDIGO (2012) Clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney
Int Suppl 2013; 3: 1–150
2. Cobbaert CM, Baadenhuijsen H, Weykamp CW. Prime
time for enzymatic creatinine methods in pediatrics. Clin
Chem 2009; 55:549–558
3. Myers GL, Miller WG, Coresh J et al. Recommendations
for improving serum creatinine measurement: a reportfrom the laboratory working group of the national kidneydisease education program. Clin Chem 2006; 52: 5–18
4. Greenberg N, Roberts WL, Bachmann LM et al. Specificity characteristics of 7 commercial creatinine measurement procedures by enzymatic and Jaffe method principles. Clin Chem 2012; 58: 391–401
5. Arant BS Jr, Edelmann CM Jr, Spitzer A. The congruenceof creatinine and inulin clearances in children: use of theTechnicon AutoAnalyzer. J Pediatr 1972; 81: 559–561
6. Fabiny DL, Ertingshausen G. Automated reaction-rate
method for determination of serum creatinine with theCentrifi- Chem. Clin Chem 1971; 17: 696–700
7. Delanghe J. Standardization of creatinine determination and its consequences for the clinician. Acta Clin Belg
2002; 57: 172–175
8. Fossati P, Prencipe L, Berti G. Enzymatic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement. Clin Chem 1983; 29: 1494–1496
9. McLean MH, Gallwas J, Hendrixson M. Evaluation of an
automated creatininase creatinine procedure. Clin Chem
1973; 19: 623–625
10. Levey AS, Coresh J, Greene T, Marsh J, Stevens LA,
Kusek JW, et al. Expressing the Modification of Diet in
Renal Disease study equation for estimating glomerular
filtration rate with standardized serum creatinine values.
Clin Chem 2007; 53:766–772
11. Perrone RD, Madias NE, Levey AS: Serum creatinine as
an index of renal function: new insights into old concepts.
Clin Chem 1992; 38: 1933–1953
12. Spencer K: Analytical reviews in clinical biochemistry: the estimation of creatinine. Ann Clin Biochem 1986;
23:1–25
13. Heymsfield SB, Arteaga C, McManus C, Smith J, Moffitt S: Measurement of muscle mass in humans: validityof the 24-hour urinary creatinine method. Am J Clin Nutr
1983; 37: 478–494
14. Delanaye P, Cavalier E, Radermecker RP, Paquot N,
Depas G, Chapelle JP, et al: Cystatin C or creatinine fordetection of stage 3 chronic kidney disease in anorexia
nervosa. Nephron Clin Pract 2008; 110:c158–c163
15. Bouquegneau A, Vidal-Petiot E, Vrtovsnik F, CavalierE, Rorive M, Krzesinski JM, et al: Modification of diet inrenal disease versus chronic kidney disease epidemiology collaboration equation to estimate glomerular filtration rate in obese patients. Nephrol Dial Transplant 2013;
28(suppl 4):iv122– iv130
16. Bauer JH, Brooks CS, Burch RN: Clinical appraisal of
creatinine clearance as a measurement of glomerular filtration rate. Am J Kidney Dis 1982; 2: 337-346

Page 255

eJIFCC2017Vol28No4pp251-257

L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye
Did creatinine standardization give benefits to the evaluation of glomerular filtration rate?

17. Shemesh O, Golbetz H, Kriss JP, Myers BD: Limitationsof creatinine as a filtration marker in glomerulopathic patients. Kidney Int 1985; 28: 830-838
18. van Acker BA, Koomen GC, Koopman MG, de Waart
DR, Arisz L: Creatinine clearance during cimetidine administration for measurement of glomerular filtration
rate. Lancet 1992; 340: 1326- 1329
19. Crim MC, Calloway DH, Margen S: Creatine metabolism in men: urinary creatine and creatinine excretions
with creatine feeding. J Nutr 1975; 105: 428–438
20. Preiss DJ, Godber IM, Lamb EJ, Dalton RN, Gunn IR:
The influence of a cooked-meat meal on estimated glomerular filtration rate. Ann Clin Biochem 2007; 44(pt 1):
35–42
21. Mayersohn M, Conrad KA, Achari R: The influence ofa cooked meat meal on creatinine plasma concentrationand creatinine clearance. Br J Clin Pharmacol 1983; 15:
227–230
22. Mitch WE, Walser M: A proposed mechanism for reduced creatinine excretion in severe chronic renal failure.
Nephron 1978; 21: 248–254
23. Papadakis MA, Arieff AI: Unpredictability of clinical evaluation of renal function in cirrhosis. Prospective
study. Am J Med 1987; 82: 945–952
24. Dodder NG, Tai SS, Sniegoski LT, Zhang NF, Welch
MJ. Certification of creatinine in a human serum reference material by GC-MS and LC-MS. Clin Chem 2007; 53:
1694–1699
25. Thienpont LM, Van Landuyt KG, Stockl D, De Leenheer
AP. Candidate reference method for determining serum
creatinine by isocratic HPLC: validation with isotope dilution gas chromatography-mass spectrometry and application for accuracy assessment of routine test kits. Clin
Chem 1995; 41: 995–1003
26. Carobene A, Ferrero C, Ceriotti F, Modenese A, Besozzi M, de Giorgi E, et al: Creatinine measurement proficiency testing: assignment of matrix-adjusted ID GC-MS
target values. Clin Chem 1997; 43: 1342–1347
27. Boutten A, Bargnoux AS, Carlier MC, Delanaye P, Rozet
E, Delatour V, et al. Enzymatic but not compensated Jaffe
methods reach the desirable specifications of NKDEP atnormal levels of creatinine. Results of the French multicentric evaluation. Clin Chim Acta 2013; 419: 132–135

30. Kuster N, Cristol JP, Cavalier E, Bargnoux AS, Halimi
JM, Froissart M, et al: Enzymatic creatinine assays allow
estimation of glomerular filtration rate in stages 1 and 2
chronic kidney disease using CKD-EPI equation. Clin Chim
Acta 2014; 428: 89–95
31. Jaffe M. Ueber den Niederschlag welchen Pikrinsa¨ure
in normalen Harn erzeugt und ueber eine neue Reaction
des Kreatinins. Z Physiol Chem 1886; 10: 391- 400
32. Hanser A-M, Hym B, Michotey O, Gascht D, Marchal A,
Minery M, et al. Comparaison des me´thodes de dosage
de la cre´atinine se´rique. Ann Biol Clin 2001; 59: 737–742
33. Wuyts B, Bernard D, Van den Noortgate N, Van deWalle J, Van Vlem B, De Smet R, et al. Reevaluation of
formulas for predicting creatinine clearance in adults andchildren, using compensated creatinine methods. Clin
Chem 2003; 49: 1011–1014
34. Delanghe JR, Cobbaert CM, Galteau MM, Harmoinen A, Jansen R, Kruse R, et al. Trueness verification of
actual creatinine assays in the European market demonstrates a disappointing variability that needs substantial
improvement. An international study in the frameworkof the EC4 creatinine standardization working group.
Clin Chem Lab Med 2008; 46: 1319–1325
35. Levey AS. Measurement of renal function in chronic
renal disease. Kidney Int 1990; 38: 167–184
36. Delanghe JR, Cobbaert C, Harmoinen A, Jansen R,
Laitinen P, Panteghini M: Focusing on the clinical impact
of standardization of creatinine measurements: a reportby the EFCC working group on creatinine standardization.
Clin Chem Lab Med 2011; 49: 977–982
37. Seronie-Vivien S, Galteau MM, Carlier MC, Hadj-Aissa
A, Hanser AM, Hym B, et al: Impact of standardized calibration on the interassay variation of 14 automated assays for the measurement of creatinine in human serum.
Clin Chem Lab Med 2005; 43: 1227–1233
38. Stevens LA, Manzi J, Levey AS, Chen J, Deysher AE,
Greene T, et al: Impact of creatinine calibration on performance of GFR estimating equations in a pooled individual patient database. Am J Kidney Dis 2007; 50: 21-35
39. Ceriotti F, Boyd JC, Klein G, Henny J, Queraltó J, Kairisto V, et al. Reference intervals for serum creatinine
concentrations: assessment of available data for global
application. Clin Chem 2008;54:559-566

28. Hoste L, Deiteren K, Pottel H, Callewaert N, Martens
F: Routine serum creatinine measurements: how well dowe perform? BMC Nephrol 2015; 16: 21.

40. Delanaye P, Mariat C. The applicability of eGFR equations to different populations. Nat Rev Nephrol 2013; 9:
513–522

29. Pieroni L, Delanaye P, Boutten A, Bargnoux AS, Rozet
E, Delatour V, et al: A multicentric evaluation of IDMStraceable creatinine enzymatic assays. Clin Chim Acta
2011; 412: 2070–2075

41. Delanaye P, Cavalier E, Krzesinski JM, Chapelle JP. Why
the MDRD equation should not be used in patients withnormal renal function (and normal creatinine values)?
Clin Nephrol 2006; 66: 147–148

Page 256

eJIFCC2017Vol28No4pp251-257

L. Piéroni, A.S. Bargnoux, J.P. Cristol, E. Cavalier, P. Delanaye
Did creatinine standardization give benefits to the evaluation of glomerular filtration rate?

42. Delanaye P, Cohen EP. Formula-based estimates of
the GFR: equations variable and uncertain. Nephron Clin
Pract 2008; 110: c48–c53
43. Klee GG, Schryver PG, Saenger AK, Larson TS. Effects ofanalytic variations in creatinine measurements on the classification of renal disease using estimated glomerular filtration rate (eGFR). Clin Chem Lab Med 2007; 45: 737–741

44. Panteghini M: Enzymatic assays for creatinine: time
for action. Scand J Clin Lab Invest Suppl 2008; 241: 84–88
45. Desirable Specifications for imprecision, inaccuracy,
and total allowable error, calculated from data on withinsubject and between-subject biologic variation. Updated and compiled by Dr. Carmen Ricos and colleagues in
2014. Allowable on www.westgard.com

Page 257

eJIFCC2017Vol28No4pp251-257

dariz ti special
n of ur ne
albu Thein measurements
TF-CKDissue:
laboratory diagnostics of chronic kidney diseases

Moving toward standardizationof urine albumin measurements

Jesse C. Seegmiller1, W. Greg Miller2, Lorin M. Bachmann2

1
2

University of Minnesota, Department of Laboratory Medicine and Pathology, Minneapolis, Minnesota, USA
Virginia Commonwealth University, Department of Pathology, Richmond, Virginia, USA

ARTICLE INFOABSTRACT

Corresponding author:
Jesse C. Seegmiller
University of Minnesota
Department of Laboratory Medicine
and Pathology
420 Delaware Street S.E.
Minneapolis, MN 55455
United States of America
Phone: 1-612-625-6198
Fax: 1-612-625-4831
E-mail: jseegmil@umn.edu

Measurement of urine albumin is important for detecting and monitoring kidney disease. At the present time, measurement of urine albumin is not standardized due to the lack of a reference system, which
includes both a reference measurement procedureand certified reference materials. Developing a reference system will provide a means for clinical laboratory measurement procedures to become standardized and will enable successful use of uniform clinicaldecision points. Currently, urine albumin results varyin excess of 40% depending on which commerciallyavailable measurement procedure is utilized for measurement. Clinicians may struggle with classificationof kidney disease in part due to differences in measurements from lack of agreement among laboratory methodologies employed when assessing urine
albumin concentrations. This report focuses on current findings in urine albumin testing, highlights important measurement and reporting considerations,and presents strategies for developing a reference
measurement procedure to enable standardizationof urine albumin measurements.

Key words:
urine albumin, standardization, calibration,reference measurement procedure

Page 258

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

INTRODUCTION
Urine albumin is a diagnostic and prognostic
marker for chronic kidney disease (CKD), diabetes and cardiovascular disease.1,2 When interpreting measurements of urine albumin,
providers must consider the type of urine collection and the methodology used for analyticalmeasurement. The historical standard for measuring the amount of albumin excreted into theurine, known as the urine albumin excretionrate, has been to measure the albumin concentration obtained from a 24-hour urine collection.3 In clinical practice, 24-hour urine collections present problems in terms of specimen
storage and timing accuracy. Thus, assessmentof urine albumin from shorter collection times is
a common clinical practice and presents a moreconvenient collection option. In untimed situations, the urine albumin result should be indexed to urine creatinine concentration and reported as the albumin to creatinine ratio (ACR).
The ACR accounts for hydration and produces
a ratio that has similar diagnostic performance
as a 24-hour urine albumin excretion rate.4-6 A
caveat to these different timing approaches isdifferences in classification of albuminuria depending on timing of collection. Therefore, thecollection method should remain consistentthroughout studies.7 Recommendations are toreport the ACR along with the albumin concentration, preferably collect the first morning
void specimen, and follow-up findings from random urine collections with first morning void
collections.4,8-10
A variety of testing methodologies have been
employed to monitor urine albumin including
turbidimetry 11,12, dipstick 13, radioimmunoassay14,15, immunoturbidimetry16, immunonephelometry 17,18, high performance liquid chromatography 19, liquid chromatography mass
spectrometry20,21, and liquid chromatography
tandem mass spectrometry (LC-MS/MS)22.

Some of these methods are known to have issues with analytical specificity when measuring
urine albumin. One essential attribute for a reference measurement procedure is that it mustbe specific for the measurand it is intended toquantify and not be influenced by matrix effects
or interfering substances that can be present inpatient urine.
This report highlights standardization recommendations for urine albumin measurements
and focuses on methodologies likely suitable foruse as a reference measurement procedure forstandardizing such measurement results.
PREANALYTICAL AND STORAGECONSIDERATIONS FOR URINE ALBUMIN
Several precollection factors have been shownto increase urine albumin excretion such as exercise23, posture24 and fever25. These factors should
be considered when assessing albuminuria forcomprehensive renal workups. Interventions
may not be indicated in patients with the above
conditions unless the albuminuria persists
when the confounding physiological conditions
are no longer present. Nonspecific binding ofalbumin to urine collection containers does notcontribute to measurement error, as binding to
the container has been estimated to be <1% depending on the container hydrophobicity, whichis considered inconsequential.26
A fresh midstream collection for urine albuminmeasurement is preferred.8,27,28 Albumin canremain stable in urine for up to 8 weeks whenstored under refrigerated conditions at 4 oC.29 Forlong term frozen storage of urine albumin samples, a temperature -70 oC or lower is required.
Degradation of albumin in urine causing measurement issues has been reported when stored
at -20 oC over periods of 2 weeks to 3 years.29,30
Therefore, careful attention must be paid to thestorage conditions for urine specimens particularly for investigations using stored samples.

Page 259

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

CURRENT STATE OF URINE
ALBUMIN MEASUREMENTS

However, bias was found to cause a significant
lack of agreement among measurement procedures. The median difference range for routine measurement procedures vs. a comparator LC-MS/MS procedure was approximately
40%. Mean biases ranged from -35% to +34%
for concentrations near 15 mg/L and -15% to+18% for concentrations near 30 mg/L. The results of this study demonstrate that fixed decision thresholds cannot be effectively utilized
due to lack of agreement among routine measurement procedures and therefore standardization is needed.

While the utility of this biomarker is clear, applying disease specific cutoffs for albuminuria
becomes compromised near the decision values due to non-standardized measurement procedures used in clinical laboratories. In a study
that evaluated the state of agreement among
16 quantitative clinical laboratory immunoassay
measurement procedures from in-vitro diagnostics manufacturers, who distribute globally,
results from 332 freshly collected non-frozen
urine albumin samples had total coefficientsof variation (CVs) of 5.2-8.1% and the effects ofsample-specific influences were < 10% for mostmeasurement procedures.31
Table 1

SampleA

The College of American Pathologists offers an
Accuracy Based Urine Survey that uses unalteredpooled frozen human urine as the samples.

Results from the College of American Pathologists- Accuracy Based Urine Survey first mailing in 2017a

Methods

N. Labs

Median,mg/L

Median bias
vs.
LC-MS/MS,%

Siemens Dimension
Vista (IN)

7

16

-1.8

16

18

Abbott Architect c
Systems (IT)

10

13

-20.2

11

13

Beckman AU
Series (IT)

8

13

-20.2

11

14

Roche cobas
c500 Series

9

12

-26.4

10

13

Vitros 5.1
FS/4600/5600

5

15

-8.0

8

16

All methods

58

13

-20.2

8

18

LC-MS/MS

-

16.3

-

-

-

Page 260

eJIFCC2017Vol28No4pp258-267

Low value,
mg/L

High
value,
mg/L

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

B

C

Siemens Dimension
Vista (IN)

7

38

4.1

37

38

Abbott Architect c
Systems (IT)

10

32

-12.3

30

33

Beckman AU Series
(IT)

7

31

-15.1

30

32

Roche cobas
c500 Series

11

32

-12.3

30

34

Vitros 5.1
FS/4600/5600

5

37

1.4

25

38

All methods

59

32

-12.3

25

39

LC-MS/MS

-

36.5

-

-

-

Siemens Dimension
Vista (IN)

7

192

4.1

178

195

Abbott Architect c
Systems (IT)

9

164

-11.1

161

167

Beckman AU
Series (IT)

7

167

-9.4

149

169

Roche cobas
c500 Series

11

155

-15.9

130

173

Vitros 5.1
FS/4600/5600

5

166

-10.0

133

180

All methods

58

164

-11.1

130

195

LC-MS/MS

-

184.4

-

-

-

Data used with permission from the College of American Pathologists(IN) - immunonephelometric, (IT) - immunoturbidimetrica

Page 261

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

Table 1 shows participant results compared to an
LC-MS/MS candidate reference measurement
procedure. Although there were a small numberof participants, the information is representativeand consistent with the previously mentioned
larger study based on individual patient urine
samples.31 The median bias vs. the comparative
method was larger at lower concentrations ofurine albumin with the all methods bias -20% at
16 mg/L, -12% at 36 mg/L, and -11% at 184 mg/L.
The joint committee of the Laboratory Working
Group of the National Kidney Disease Education
Program and the International Federation ofClinical Chemistry and Laboratory Medicine
Working Group for Standardization of Albuminin Urine recommended desirable and optimal
bias goals of ≤13% and ≤7%, respectively, vs. areference measurement procedure.32
These survey results suggest that some measurement procedures can meet these bias goals,
but many do not. The survey also reported ACR
values. Reference measurement procedure results were not available for urine creatinine but
comparison of mean results among different
methods in the survey had differences of 17%,
8.8% and 14% at mean concentrations of 55
mg/dL, 69 mg/dL and 89 mg/dL (4.8 mmol/L,
6.1 mmol/L and 7.9 mmol/L), respectively.
When both creatinine and albumin were used
to calculate the ACR, the differences between
the lowest and highest ACR values for all methods combined were 76% at 15 mg/g, 49% at 60
mg/g, and 65% at 237 mg/g. These differences
will cause misclassification of risk of kidney disease at the commonly used albuminuria decision values of 30 and 300 mg/g creatinine (3.4
and 34 mg/mmol creatinine).
A reference system is in place for urine creatinine
and perhaps needs to be more stringently implemented. However, a reference system does not
yet exist for urine albumin and is the focus of thisreport.

METHODS FOR MEASURING
URINE ALBUMIN
To improve the analytical selectivity in themeasurement of urine albumin, liquid chromatography mass spectrometry (LC-MS) methods
were utilized.20,21 A comparison study of one
LC-MS method to an immunoturbidimetricmethod found the comparability between themethodologies greatly improved when bothmethods employed the same calibrators withthe same calibrator value assignments. Mean
bias improved from -37.8% to 2.2% using thesame calibrators on both platforms.33 A potential shortcoming of the LC-MS urine albumin
method was the lower limit of quantitation of
10-20 mg/L, which is above the level expectedin specimens with normal albumin concentrations.20,33 Other possibilities that could introduce error with this methodology are thepresence of urine albumin fragments containing the N-terminal fragment used in the analysis, which could falsely elevate albumin levels
or modification to the N-terminal portion usedin analysis that would change the mass, which
could falsely lower albumin levels.
In an effort to improve the lower limit of quantitation for urine albumin, a LC-MS/MS method was developed.22 This method employedproteolysis of urinary proteins to produce peptides of albumin as well as peptides from otherproteins present in urine. Large variations inpH (4.5-8) and specific gravity are expected in
the urine of patients with or without a kidney
abnormality.34 pH variations could adversely
affect the trypsin proteolysis process, which
is a critical preanalytic step that occurs priorto LC-MS/MS measurement. Therefore, buffering conditions and dilutions were employedthat provide an optimal environment for trypsin proteolysis. Peptides known to be uniqueto albumin were analyzed and quantitated torepresent the quantity of intact albumin. One

Page 262

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurementsof the key components of this method was theincorporation of an internal standard that consisted of a recombinant form of human serum
albumin isotopically labeled with 15N.
The internal standard served dual purpose:
1. to normalize for any differences in theproteolytic processing among specimens;
2. to provide normalization for LC-MS/MS
analysis.
Several peptides unique to human serum albumin were quantitated and referenced to acalibration curve. The lower limit of quantitation for the LC-MS/MS measurement procedure was found to be 3.13 mg/L.22 Method
comparison studies have been performed examining commercially available immunoassay
platforms to the LC-MS/MS method.31,35 The
LC-MS/MS measurement procedure was used
to perform the comparison study of 16 commercially available measurement procedures
previously described.
Potential challenges for a LC-MS/MS method
include the possibility of albumin fragments inthe urine, post-translational modifications of
the unique peptides monitored, or factors that
inhibit albumin proteolysis. Further investigation of this technique compared urine albumin
concentrations before and after ultrafiltration
using a 10 kDa molecular weight cutoff filter
where differences in the results were smalland suggested minimal signal contribution
from fragments of albumin.36 With the above
cautions appropriately addressed in the measurement procedure details, the LC-MS/MS
method is a good candidate reference measurement procedure for urine albumin. Thismethod provides the necessary sensitivity toassess urine albumin concentrations <5 mg/L.
The ability to quantitate the albumin moleculewith a high degree of analytical specificity byusing proteotypic peptides of albumin that are
not known to be subject to modification anddo not appear in other human proteins, provides support for use of the LC-MS/MS method
as a reference measurement procedure. To ensure high quality results, the LC-MS/MS measurement procedure requires an isotopicallyenriched form of albumin as an internal standard. Procedures for making labeled albumin
have been described.21
A HIGHER ORDER REFERENCE SYSTEMFOR CALIBRATION TRACEABILITY
A higher order reference system is needed toenable all measurement procedures to implement common calibration traceability to achieveequivalent results for urine albumin irrespectiveof the measurement procedure used. A reference system for urine albumin that follows theInternational Organization for Standardization
standard 17511 for calibration traceability hierarchy37 includes three main components:
1. A pure human albumin primary referencematerial.
2. A reference measurement procedure.
3. A human urine matrix based secondaryreference material.
The National Institute for Standards andTechnology (NIST) in the USA is qualifying a recombinant human albumin certified primaryreference material expected to be released in2018 as SRM 2925. SRM 2925 will be a highly
purified solid substance intended to be used
to prepare calibrators for a mass spectrometry
based reference measurement procedure. SRM
2925 is not intended to be used to prepare calibrators for immunoassays. NIST is also preparing an albumin in frozen human urine certified
reference material designated SRM 3666 thatwill include four concentrations intended to be
used to establish the metrological traceability ofcalibration for clinical laboratory measurement
procedures, including immunoassays.

Page 263

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

SRM 3666 will be value assigned using a NIST
reference measurement procedure that is currently in development. The commutability of
NIST SRM 3666 will be validated to ensure it issuitable for use as a calibrator for manufacturer’s selected measurement procedures as well
as for clinical laboratory measurement procedures. It is not known at this time when either
the reference measurement procedure or the
SRM 3666 will be available from NIST.
Since SRM 2925 pure albumin will be available soon, development of suitable reference measurement procedures will provide the
Figure 1

essential components of a reference system toallow standardized calibration traceability forcommercially available clinical laboratory urine
albumin immunoassay procedures.
A reference measurement procedure intended
for use in a calibration traceability hierarchy forclinical laboratory measurement proceduresmust have performance characteristics to ensure acceptable uncertainty in values assigned
to patient samples used as calibrators in the
traceability hierarchy, as described below. In
addition, a reference measurement proceduremust be operational in at least two sites toMetrologic traceability hierarchy for calibrationof urine albumin measurement procedures

Page 264

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

validate equivalent performance to qualify forlisting by the Joint Committee for Traceability inLaboratory Medicine.
Metrologic traceability of calibration is described in the International Organization forStandardization 17511 standard. 37 Figure 1
shows how the reference system components
being developed for urine albumin fit into thetraceability hierarchy. NIST SRM 2925 is a puresubstance primary reference material that isused with a gravimetric reference measurement
procedure to prepare calibrators for an LC-MS/
MS reference measurement procedure. The
LC-MS/MS reference measurement procedure
is used to assign values to a panel of patient’s
urine samples that are used as calibrators for amanufacturer’s selected measurement procedure that is used to assign values to the manufacturer’s working, or master lot, calibrator.
In the case of urine albumin, there will be several concentrations of working calibrator used
to calibrate the manufacturer’s standing immunoassay measurement procedure. The working
calibrators can be prepared as dilutions of a single master lot of working calibrator or as a set ofconcentrations of working calibrators, with eachvalue assigned by the selected measurement
procedure. The manufacturer’s standing measurement procedure is then used to value assign
sequential lots or batches of the manufacturer’s
product calibrator that is used to calibrate theclinical laboratory measurement procedures. In
many cases, the manufacturer’s selected andstanding measurement procedures will be thesame as the clinical laboratory measurement
procedure but operated with a more stringent
protocol for items such as maintenance, calibration, replicate measurements, multiple reagentlots and/or instruments to reduce the uncertainty of the value assignment steps. Thus, metrologic traceability is established from patientresults to the pure substance primary referencematerial.

When NIST SRM 3666, albumin in frozen human urine, becomes available it can replace thepanel of patient urine samples to simplify thetraceability process. In addition, SRM 3666 canbe used by clinical laboratories to verify calibration of their immunoassay measurement procedures for urine albumin.
CONCLUSIONSThe need for standardization of urine albuminmeasurements is clear. Standardization of thismeasurand will assist in applying uniform clinical decision points for various diseases and conditions based on urine albumin to creatinine
ratio values. Standardization of urine albuminmeasurements requires development of both acertified primary reference material and a reference measurement procedure. LC-MS/MS measurement of albumin-specific peptides after
proteolytic digestion under carefully controlled
conditions provides a suitable methodology fora reference measurement procedure. Whenavailable, these reference system components
can be used by immunoassay measurement
procedure manufacturers to achieve metrologic
traceability of calibration to a common reference system. Availability of a commutable frozen human urine reference material will also
be useful as a common calibrator for immunoassays. In addition to standardized metrologic
traceability, urine collection and storage conditions influence the suitability of urine albuminmeasurements and therefore preanalytical processing procedures should be standardized.
REFERENCES
1. Mogensen CE. Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes. N
Engl J Med. 1984;310(6):356-360.
2. Brantsma AH, Bakker SJ, Hillege HL, de Zeeuw D, deJong PE, Gansevoort RT. Cardiovascular and renal outcome in subjects with K/DOQI stage 1-3 chronic kidneydisease: the importance of urinary albumin excretion.
Nephrol Dial Transplant. 2008;23(12):3851-3858.

Page 265

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

3. Keane WF, Eknoyan G. Proteinuria, albuminuria, risk,
assessment, detection, elimination (PARADE): a position
paper of the National Kidney Foundation. Am J Kidney
Dis. 1999;33(5):1004-1010.

18. Marre M, Claudel JP, Ciret P, Luis N, Suarez L, Passa
P. Laser immunonephelometry for routine quantificationof urinary albumin excretion. Clin Chem. 1987;33(2 Pt
1):209-213.

4. Kidney Disease: Improving Global Outcomes (KDIGO)CKD Work Group. KDIGO 2012 Clinical Practice Guideline
for the Evaluation and Management of Chronic Kidney
Disease. Kidney International. 2013;Suppl(3):1-150.

19. Comper WD, Osicka TM, Jerums G. High prevalenceof immuno-unreactive intact albumin in urine of diabetic
patients. Am J Kidney Dis. 2003;41(2):336-342.

5. Witte EC, Lambers Heerspink HJ, de Zeeuw D, Bakker
SJ, de Jong PE, Gansevoort R. First Morning Voids Are
More Reliable Than Spot Urine Samples to Assess Microalbuminuria. In: J Am Soc Nephrol. Vol 20.2009:436-443.
6. Lambers Heerspink HJ, Gansevoort RT, Brenner BM,
et al. Comparison of different measures of urinary protein excretion for prediction of renal events. J Am Soc
Nephrol. 2010;21(8):1355-1360.
7. Younes N, Cleary PA, Steffes MW, et al. Comparison ofurinary albumin-creatinine ratio and albumin excretion
rate in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications
study. Clin J Am Soc Nephrol. 2010;5(7):1235-1242.
8. Miller WG, Bruns DE, Hortin GL, et al. Current issues
in measurement and reporting of urinary albumin excretion. Clin Chem. 2009;55(1):24-38.
9. Saydah SH, Pavkov ME, Zhang C, et al. Albuminuria
prevalence in first morning void compared with previous random urine from adults in the National Health andNutrition Examination Survey, 2009-2010. Clin Chem.
2013;59(4):675-683.
10. Hortin GL. Identifying optimal sample types and decision thresholds for the urinary albumin-creatinine ratio.
In: Clin Chem. Vol 59. United States2013:598-600.
11. Folin O, Denis W. The Quantitative Determination ofAlbumin in Urine. J Biol Chem. 1914;18:273-276.
12. Exton WG. A Simple and Rapid Test for Albumin andother Urinary Proteins. JAMA. 1923;80(8):529-530.
13. Free AH, Rupe CO, Metzler I. Studies with a new colorimetric test for proteinuria. Clin Chem. 1957;3(6):716-727.
14. Keen H, Chlouverakis C. An Immunoassay Method
for Urinary Albumin at Low Concentrations. Lancet.
1963;2(7314):913-914.
15. Woo J, Floyd M, Cannon DC, Kahan B. Radioimmunoassay for urinary albumin. Clin Chem. 1978;24(9):1464-1467.

20. Babic N, Larson TS, Grebe SK, Turner ST, Kumar R,
Singh RJ. Application of liquid chromatography-mass
spectrometry technology for early detection of microalbuminuria in patients with kidney disease. Clin Chem.
2006;52(11):2155-2157.
21. Singh R, Crow FW, Babic N, et al. A liquid chromatography-mass spectrometry method for the quantification ofurinary albumin using a novel 15N-isotopically labeled albumin internal standard. Clin Chem. 2007;53(3):540-542.
22. Seegmiller JC, Barnidge DR, Burns BE, Larson TS,
Lieske JC, Kumar R. Quantification of urinary albuminby using protein cleavage and LC-MS/MS. Clin Chem.
2009;55(6):1100-1107.
23. Poortmans J, Dorchy H, Toussaint D. Urinary Excretionof Total Proteins, Albumin, and β2-Microglobulin DuringRest and Exercise in Diabetic Adolescents With and Without Retinopathy. Diabetes Care. 1982.
24. Uehara K, Tominaga N, Shibagaki Y. Adult orthostatic
proteinuria. Clin Kidney J. 2014;7(3):327-328.
25. Hemmingsen L, Skaarup P. Urinary excretion of ten
plasma proteins in patients with febrile diseases. Acta
Med Scand. 1977;201(4):359-364.
26. Robinson MK, Caudill SP, Koch DD, et al. Albumin adsorption onto surfaces of urine collection and analysis
containers. Clin Chim Acta. 2014;431:40-45.
27. CLSI GP16-A3 Urinalysis; Approved Guideline-Third
Addition. In: Institute CaLS, ed. Third ed. Wayne, PA2000.
28. European urinalysis guidelines. Scand J Clin Lab Invest
Suppl. 2000;231:1-86.
29. Osberg I, Chase HP, Garg SK, et al. Effects of storage
time and temperature on measurement of small concentrations of albumin in urine. Clin Chem. 1990;36(8 Pt
1):1428-1430.
30. Sviridov D, Drake SK, Hortin GL. Reactivity of urinary
albumin (microalbumin) assays with fragmented or modified albumin. Clin Chem. 2008;54(1):61-68.

16. Teppo AM. Immunoturbidimetry of albumin and immunoglobulin G in urine. Clin Chem. 1982;28(6):1359-1361.

31. Bachmann LM, Nilsson G, Bruns DE, et al. State of theart for measurement of urine albumin: comparison ofroutine measurement procedures to isotope dilution tandem mass spectrometry. Clin Chem. 2014;60(3):471-480.

17. Ellis D, Buffone GJ. New approach to evaluation ofproteinuric states. Clin Chem. 1977;23(4):666-670.

32. Miller WG, Seegmiller JC, Lieske JC, Narva AS, Bachmann
LM. Standardization of Urine Albumin Measurements:

Page 266

eJIFCC2017Vol28No4pp258-267

Jesse C. Seegmiller, W. Greg Miller, Lorin M. Bachmann
Moving toward standardization of urine albumin measurements

Status and Performance Goals. The Journal of Applied Laboratory Medicine. 2017;2(3):423-429.
33. Shaikh A, Seegmiller JC, Borland TM, et al. Comparison between Immunoturbidimetry, Size-Exclusion Chromatography, and LC-MS to Quantify Urinary Albumin. Clin
Chem. 2008;54(9):1504-1510.
34. Simerville JA, Maxted WC, Pahira JJ. Urinalysis: a comprehensive review. Am Fam Physician. 2005; 71(6):1153-1162.
35. Seegmiller JC, Sviridov D, Larson TS, Borland TM, Hortin
GL, Lieske JC. Comparison of urinary albumin quantification

by immunoturbidimetry, competitive immunoassay, andprotein-cleavage liquid chromatography-tandem mass
spectrometry. Clin Chem. 2009;55(11):1991-1994.
36. Lieske JC, Bondar O, Miller WG, et al. A reference system for urinary albumin: current status. Clin Chem Lab
Med. 2013;51(5):981-989.
37. ISO 17511:2003 - In vitro diagnostic medical devices -Measurement of quantities in biological samples -- Metrological traceability of values assigned to calibrators andcontrol materials. International Organization for Standardization, Geneva, Switzerland 2003.

Page 267

eJIFCC2017Vol28No4pp258-267

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

Cystatin C is indispensablefor evaluation of kidney disease
Anders Grubb

Department of Clinical Chemistry and Pharmacology, Laboratory Medicine, Lund University,Skåne University Hospital, Lund, Sweden

ARTICLE INFOABSTRACT

Corresponding author:
Anders Grubb
Department of Clinical Chemistry
and Pharmacology
Laboratory Medicine
Lund UniversitySkåne University Hospital
SE-22185 Lund
SwedenPhone: +46 702393773
E-mail: anders.grubb@med.lu.se

The present minireview of the place of cystatin C in clinical medicine emphasizes, and discuss the evidence,that cystatin C-based GFR-estimating equations do not
require the use of vague terms like race and sex, thatcystatin C-based GFR-estimating equations are usefulfor both children and adults, including the elderly, thatthe best GFR-estimation requires simultaneous use ofboth cystatin C- and creatinine-based equations, thatcystatin C-based GFR-estimating equations are superior to creatinine-based equations in predicting endstage renal disease, cardiovascular manifestations,
hospitalisation and death, and, finally that cystatin C
is required to diagnose the new syndrome “Shrunken
Pore Syndrome” with its high mortality and morbidity,
even in the absence of reduced GFR. When automatedlaboratory equipment is available, the cost of cystatin
C is comparable to that of enzymatically determinedcreatinine.

Key words:
cystatin C, GFR, morbidity, mortality,
renal, shrunken-pore-syndrome
Acknowledgement
This study was supported by grants
from the Alfred Österlund Foundation,
the Medical Faculty of the Universityof Lund and from Region Skåne.
Conflict of interest
The author declares that there is no conflict ofinterest regarding the publication of this article.

The conclusion is that cystatin C should be used at least
as often as creatinine in clinical medicine.

Page 268

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

INTRODUCTION
The introduction of creatinine as a marker of
GFR started in 1926 with the publication of anarticle by Poul Brandt Rehberg: “Studies on kidney function. The rate of filtration and reabsorption in the human kidney” (1). Since then theuse of creatinine has been a vital element ofclinical medicine. Cystatin C was suggested to bea marker of GFR in 1979 (2) and a few articles
published before 1994 supported its use as a
GFR-marker (3-5). In 1994 an article with the title “Serum cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine forglomerular filtration rate” was published (6),which initiated widespread studies of cystatin
C as a marker of GFR. Today, October 2017, thesearch string in PubMed “Cystatin C AND (renal
OR kidney)” produces more than 3500 titles.
The information obtained in these 3500 investigations allows the conclusion that the low-cost
analysis of cystatin C should be an integral partof the analysis spectrum for optimal evaluationof the kidney status of a patient.
This is because:
A. Cystatin C-based GFR-estimating equations
do not require the use of vague terms likerace and sex
B. Cystatin C-based GFR-estimating equations
are useful for both children and adults,
including the elderly
C. The best GFR-estimation requires simultaneous use of both cystatin C- and creatinine-based equations
D. Cystatin C-based GFR-estimating equations
are superior to creatinine-based equationsin predicting end-stage renal disease, cardiovascular manifestations, hospitalisationand death
E. Cystatin C is required to diagnose the new
syndrome “Shrunken Pore Syndrome” with

its high mortality and morbidity, even in
the absence of reduced GFR.
CYSTATIN C-BASED GFR-ESTIMATINGEQUATIONS DO NOT REQUIRE THE USE
OF VAGUE TERMS LIKE RACE AND SEX
One of the main advantages of cystatin C compared to creatinine as a GFR-marker is that it
is less dependent upon the body compositionof a patient. For example, while muscle massstrongly influences creatinine, it does not, or
only marginally, affect cystatin C (7-11).
Creatinine-based GFR-estimating equations
therefore contain terms aiming at evaluating
the muscle mass of a specific patient. These
terms refer to the race and sex of the patient.
Specific race-factors have been suggested for
Afro-Americans (12), Japanese (13,14), Chinese(15), Koreans (16), and native Americans andHispanics (17). But “race” is a very vague term,
difficult to define and does not consider the
problem that a major part of the world population represents persons with mixed ethnicity.
In contrast, the cystatin C concentration varies
only marginally with ethnicity and no vague
race terms are therefore required in cystatin
C-based GFR-estimating equations (18-20).
The mean muscle mass of females is lower
than that of males and creatinine-based GFRestimating equations therefore requires significant sex-related factors for females (21).
However, the world is less and less sex-dichotomized and the existence of more than two sexes
is now acknowledged in several countries (22).
This ambiguity in applying creatinine-based
GFR-estimating equations does not apply forsome cystatin C-based GFR-estimating equations, since muscle mass only marginally, or not
at all, influences the cystatin C-level and thus
cystatin C-based GFR-estimating equations do
not require factors for sex (20).

Page 269

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

CYSTATIN C-BASED GFR-ESTIMATINGEQUATIONS ARE USEFULFOR BOTH CHILDREN AND ADULTS,
INCLUDING THE ELDERLY
The strong correlation between muscle massand creatinine poses a special problem concerning the use of creatinine-based GFR-estimating
equations in childhood, since the muscle massstrongly increases with age. As a consequence,different equations generally have to be used
for adults and children (23-25). In contrast, since
the muscle mass does not influence cystatin C
significantly many cystatin C-based equations
work for both children and adults (20, 23-25).
One of them is the CAPA-equation which has
been shown to work from 1 - 50 years of age (20,
25 and unpublished observations by Grubb A, etal.). Another problem related to the use of creatinine-based equations is that the muscle massin the elderly is often considerably reduced,
so that it negatively affects the ability of theseequations to demonstrate a reduced GFR in theelderly. In contrast, cystatin C-based equations
are not significantly influenced by muscle massand therefore useful in identifying reduced GFR
also in the elderly with low muscle mass (26).
THE BEST GFR-ESTIMATION REQUIRESSIMULTANEOUS USE OF BOTH CYSTATIN
C- AND CREATININE-BASED EQUATIONS
Although creatinine-based GFR-estimating
equations are inferior in diagnostic performancecompared to cystatin C-based equations for several populations, it has generally been shownthat the best GFR-estimation requires use ofboth cystatin C and creatinine in the equation(27-31). The best estimates of GFR, producedby cystatin C-based equations, eGFRcystatin C, produce values of which 80-85% are within ±30% of
GFR measured by invasive gold-standard methods and similar figures are valid for the corresponding estimates, eGFRcreatinine, obtained bycreatinine-based equations (27-31). Equations
using both cystatin C and creatinine might produce values of which 90-91% are within ±30% of
GFR measured by invasive gold-standard methods (30,32). Still better results are obtained if themean, eGFRmean= (eGFRcystatin C + eGFRcreatinine)/2of the estimates obtained by a cystatin C-and acreatinine-based equation are used, rather thancomplex equations containing both cystain C
and creatinine (32-34). This is due to that combined equations do not perform optimally in a
number of clinical situations, for example, if thepatient has an abnormally low muscle mass oris treated with a high dose of glucocorticoids. A
strategy for GFR estimation based on the automatic use of a combined cystatin C and creatinine-based equation will, in these cases, have aworse diagnostic performance than a strategy
that only uses the cystatin C- or creatinine-based
GFR-estimating equation not influenced by the
specific patient characteristics (33,34). Such a
strategy thus requires that GFR is estimated byboth a cystatin C- and a creatinine-based equation, producing eGFRcystatin C or eGFRcreatinine, andthat the results are compared. If the two equations produce similar estimates, their average isa very reliable estimate of GFR. If the estimates
do not agree and a specific factor known to disturb either the cystatin C- or creatinine-based
estimate is present, only the estimate producedby the equation not disturbed by this factor, isused (33,34). As a matter of fact, since 1994,
when cystatin C-based estimations of GFR wereintroduced in Lund in parallel with creatininebased estimations, we have had 20-30 cases forwhich eGFRcystatin C and eGFRcreatinine agreed, but
disagreed with GFR measured by our invasive
gold-standard procedure (plasma clearance ofiohexol). In all cases, in which relevant information was available, the error was caused bytechnical problems in the execution of the goldstandard procedure. We therefore consider
that, in practice, eGFRmean based upon agreeing

Page 270

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

eGFRcystatin C and eGFRcreatinine is at least as reliable
as GFR measured by invasive gold-standard procedures (33,34). This strategy is described at themultilingual site www.egfr.se (35), which canalso be implemented to calculate absolute GFRfrom relative GFR, which might be required in,e.g., dosing of medicines cleared by the kidneys.
CYSTATIN C – BASED GFR-ESTIMATINGEQUATIONS ARE SUPERIOR TO
CREATININE-BASED EQUATIONS IN
PREDICTING END-STAGE RENAL DISEASE,CARDIOVASCULAR MANIFESTATIONS,HOSPITALISATION AND DEATH
One important reason to estimate GFR in a patient is to decide whether the patient suffersfrom chronic kidney disease or not, and to classify the degree of the chronic kidney disease, if
present. Both eGFRcystatin C and eGFRcreatinine work
well for this purpose. However, another important aspect of the estimation is how well it
predicts the consequences of kidney disease,e.g., end-stage renal disease, cardiovascular
manifestations, hospitalisation and death, since
this knowledge influences decisions concerning the intensity of the treatment modalities. In
this respect, eGFRcystatin C and eGFRcreatinine differ,because the published scientific studies virtually unanimously show that eGFRcystatin C is significantly superior to eGFRcreatinine (36-39).
The cause for the superiority of eGFRcystatin C as arisk marker is unknown, but observational studies have shown that inflammation, old age, malegender, greater weight, and cigarette smoking
correlate with higher cystatin C levels (40). But
statistical correlations in observational studies do not prove causal connections. A studyof elective surgery of patients demonstrated a
postoperative sharp rise in inflammation of thepatients, with large increases in the levels of
CRP of all patients, but with no increase in thecystatin C levels, thus rejecting the hypothesisthat inflammation causes a raise in the production of cystatin C (41). The correlations between
inflammation, old age, male gender, greater
weight, and cigarette smoking and cystatin C
might be due to that all these factors promote
the development of atherosclerosis, also in therenal arteries, thus producing a decrease in GFR
and an increase in cystatin C (41). These correlations therefore speak in favour of cystatin C as a
GFR-marker and not against it.
CYSTATIN C IS REQUIRED TO DIAGNOSE
THE NEW SYNDROME “SHRUNKENPORE SYNDROME” WITH ITS HIGH
MORTALITY AND MORBIDITY, EVEN
IN THE ABSENCE OF REDUCED GFR
The use of eGFRmean and the simultaneous comparison of eGFRcystatin C and eGFRcreatinine, as the best
way to estimate GFR in clinical practice (32-34,42)
identifies a number of patients with significant
differences between eGFRcystatin C and eGFRcreatinine.
Part of these differences can be explained by factors, such as muscle wasting or treatment withlarge doses of glucocorticoids, known to invalidate the GFR estimations based on creatinine orcystatin C (33). But the majority of the patientswith such differences between eGFRcystatin C andeGFRcreatinine, do not display any known such factor and their eGFRmean is, despite the differences
between eGFRcystatin C and eGFRcreatinine, still the best
way to estimate GFR (41).
Most of the patients displaying these differences
has a pattern of eGFRcystatin C and eGFRcreatinine inwhich eGFRcystatin C is lower than eGFRcreatinine (42,43).
When the levels of low-molecular mass proteinsother than cystatin C, e.g., β2-microglobulin,
β-trace protein, and retinol-binding protein, were
determined in patients with eGFRcystatin C ≤ 60% ofeGFRcreatinine, it was observed that the concentration ratios of these proteins to creatinine were,like the cystatin C-creatinine ratio, higher, than inpatients in whom eGFRcystatin C ≈ eGFRcreatinine (43).

Page 271

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

The genes for these proteins are located at different chromosomes and have different regulation elements and the synthesis of these proteins is not generally known to be influenced
by factors affecting the production of cystatin
C (43). This strongly indicates that the production of these proteins and cystatin C is not coregulated and therefore cannot explain theconcordant increases of their plasma levels.
But the concurrent increase can be explained if
the proteins have a common clearance mechanism by glomerular filtration and that this is

reduced by shrinking of the glomerular pores(43). Therefore, the observation that eGFRcystatin≤ 60% of eGFRcreatinine in a patient indicates theCpresence of a new syndrome, tentatively called“Shrunken Pore Syndrome” (43). The explanation that creatinine and other small molecules
do not simultaneously increase in concentration would then be, that their sieving coefficients are still close to unity (i.e., one) despite
the shrunken pores resulting in reduced sieving
coefficients for proteins similar in size to cystatin C (43-45).

Figure 1A Survival after coronary artery bypass surgery
for patients with GFR > 60 mL/min per 1.73 m2
with and without Shrunken Pore Syndrome (SPS)eGFRcystatin C was estimated using the CAPA equation and eGFRcreatinine using the LMrev equation.
The cut-off level for SPS was eGFRcystatin C ≤ 70% of eGFRcreatinine (red broken line) or eGFRcystatin C ≤ 60% of eGFRcreatinine (redunbroken line).
The unbroken blue line indicates the mortality of patients without SPS (0.90<eGFRcystatin c/eGFRcreatinine <1.10).
The numbers below indicate patients with and without SPS, when the cut-off level was 70%.

Page 272

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

It is noteworthy, that a similar mechanism previously has been suggested for the increase inplasma levels of low-molecular mass proteins in
the third trimester of pregnancy (46-48) and forthe development of still higher concentrationsof low-molecular mass proteins in preeclampsia(49,50). This suggests that the (patho-)physiologic changes in late pregnancy and preeclampsia are similar to those occurring in patients with
“Shrunken Pore Syndrome.”

As “Shrunken Pore Syndrome” was identified recently (43), only a few studies of its clinical consequences have been performed. The first investigation showed, that the long-term mortality inpatients undergoing elective coronary artery bypass grafting was much higher in patients suffering from “Shrunken Pore Syndrome” than in patients without the syndrome (51). This was true
both when the preoperative GFR was normal
or reduced (Figure 1A and B). In this study, theFigure 1B Survival after coronary artery bypass surgery
for patients with GFR < 60 mL/min per 1.73 m2
with and without Shrunken Pore Syndrome (SPS)eGFRcystatin C was estimated using the CAPA equation and eGFRcreatinine using the LMrev equation.
The cut-off level for SPS was eGFRcystatin C ≤ 70% of eGFRcreatinine (red broken line) or eGFRcystatin C ≤ 60% of eGFRcreatinine (redunbroken line).
The unbroken blue line indicates the mortality of patients without SPS (0.90<eGFRcystatin c/eGFRcreatinine <1.10).
The numbers below indicate patients with and without SPS, when the cut-off level was 70%.

Page 273

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney diseasecystatin C-based CAPA-equation was used to produce eGFRcystatin C and the creatinine-based LMrevequation to produce eGFRcreatinine, as both theseequations work not only for adults, but also forchildren (20,52,53). Interestingly, an increase inmortality was not only observed when eGFRcystatin≤ 60% of eGFRcreatinine, but also when eGFRcystatin C
C≤ 70% of eGFRcreatinine (Figure 1 A and B). Ongoingstudies demonstrate that the long-term mortality in “Shrunken Pore Syndrome” increases inversely with the eGFRcystatin C/eGFRcreatinine-ratio,
starting at 0.90. Recently published and ongoingstudies in several different types of populations
corroborate, that “Shrunken Pore Syndrome” isassociated with significantly increased mortality
and morbidity (54,55) and indicate that the syndrome also predicts higher risks for developmentof end-stage renal disease, cardiovascular manifestations and for hospitalisation.
CONCLUSION
The use of cystatin C (or eGFRcystatin C) in addition
to creatinine improves the estimation of GFR,
makes it independent of vague terms like raceand sex, and facilitates its use for children andthe elderly. It also allows the identification of a
new syndrome (Shrunken Pore Syndrome) associated with a high morbidity and mortality.
When automated laboratory equipment is available, the cost of cystatin C is comparable to thatof enzymatically determined creatinine. Cystatin
C should therefore be used at least as often as
creatinine in the clinical routine.
REFERENCES
1. Rehberg PB. Studies on kidney function. The rate of filtration and reabsorption in the human kidney. Biochem J
1926;20:447-60.
2. Löfberg H, Grubb A. Quantitation of γ-trace in humanbiological fluids: indications for production in the central
nervous system. Scand J Clin Lab Invest 1979;39:619–26.
3. Grubb A, Simonsen O, Sturfelt G, Truedsson L, Thysell H. Serum concentration of cystatin C, factor D andbeta-2-microglobulin as a measure of glomerular filtration rate. Acta Med Scand 1985;218:499–503.
4. Simonsen O, Grubb A, Thysell H. The blood serum
concentration of cystatin C (gamma-trace) as a measureof the glomerular filtration rate. Scand J Clin Lab Invest
1985;45:97–101.
5. Grubb A, Löfberg H. Human γ-trace. Structure, function
and clinical use of concentration measurements. Scand J
Clin Lab Invest 1985;45(Suppl. 177):7-13.
6. Kyhse-Andersen J, Schmidt C, Nordin G, Andersson B,
Nilsson-Ehle P, Lindström V, Grubb A. Serum cystatin C,
determined by a rapid, automated particle-enhanced
turbidimetric method, is a better marker than serum
creatinine for glomerular filtration rate. Clin Chem
1994;40:1921–6.
7. Vinge E, Lindergård B, Nilsson-Ehle P, Grubb A. Relationships among serum cystatin C, serum creatinine,lean tissue mass and glomerular filtration rate in healthy
adults. Scand J Clin Lab Invest 1999;59:1–6.
8. Seronie-Vivien S, Delanaye P, Pieroni L, Mariat C, Froissart M, Cristol JP. SFBC ‘Biology of renal function and renal
failure’ working group. Cystatin C: current position andfuture prospects. Clin Chem Lab Med 2008;46:1664–86.
9. Chew JSC, Saleem M, Florkowski CM, George PM. Cystatin C – a paradigm of evidence based laboratory medicine. Clin Biochem Rev 2008;29:47–62.
10. Thomassen SA, Johannesen IL, Erlandsen EJ, Abrahamsen J, Randers E. Serum cystatin C as a marker of therenal function in patients with spinal cord injury. Spinal
Cord 2002;40:524–8.
11. Jenkins MA, Brown DJ, Ierino FL, Ratnaike SI. Cystatin
C for estimation of glomerular filtration rate in patientswith spinal cord injury. Ann Clin Biochem 2003;40:364–8.
12. Levey AS, Greene T, Kusek JW, Beck GJ. A simplified
equation to predict glomerular filtration rate from serum
creatinine [Abstract]. J Am Soc Nephrol 2000;11:A0828.
13. Imai E, Horio M, Nitta K, Yamagata K, Iseki K, Tsukamoto Y, Ito S, Makino H, Hishida A, Matsuo S. Modificationof the modification of diet in renal disease (MDRD) study
equation for Japan. Am J Kidney Dis 2007;50:927–37.
14. Matsuo S, Imai E, Horio M, Yasuda Y, Tomita K, Nitta
K, Yamagata K, Tomino Y, Yokoyama H, Hishida A. Revised
equations for estimated GFR from serum creatinine in Japan. Am J Kidney Dis 2009;53:982-92.
15. Ma YC, Zuo L, Chen JH, Luo Q, Yu XQ, Li Y, Xu JS, Huang
SM, Wang LN, Huang W, Wang M, Xu GB, Wang HY. Modified glomerular filtration rate estimating equation for
Chinese patients with chronic kidney disease. J Am Soc
Nephrol 2006;17:2937-44.

Page 274

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

16. Lee CS, Cha RH, Lim YH, Kim H, Song KH, Gu N, Yu
KS, Lim CS, Han JS, Kim S, Kim YS. Ethnic coefficients forglomerular filtration rate estimation by the Modificationof Diet in Renal Disease study equations in the Korean
population. J Korean Med Sci 2010;25:1616-25.
17. Stevens LA, Claybon MA, Schmid CH, Chen J, Horio
M, Imai E, Nelson RG, Van Deventer M, Wang HY, Zuo L,
Zhang YL, Levey AS. Evaluation of the Chronic Kidney Disease Epidemiology Collaboration equation for estimating
the glomerular filtration rate in multiple ethnicities. Kidney Int 2011;79:555-62.
18. Uhlmann EJ, Hock KG, Issitt C, Sneeringer MR, Cervelli
DR, Gorman RT, Scott MG. Reference intervals for plasma
cystatin C in healthy volunteers and renal patients, asmeasured by the Dade Behring BN II system, and correlation with creatinine. Clin Chem 2001;47:2031-3.
19. Horio M, Imai E, Yasuda Y, Watanabe T, Matsuo S. GFRestimating equation based on standardized serum cystatin C. Am J Kidney Dis 2013;61:197-203.
20. Grubb A, Horio M, Hansson LO, Björk J, Nyman U, Flodin M, Larssson A, Bökenkamp A, Yasuda Y, Blufpand H,
Lindström V, Zegers I, Althaus H, Blirup-JensenI S, Itoh Y,
Sjöström P, Nordin G, Christensson A, Klima H, Sunde K,
Hjort-Christensen P, Armbruster D, Ferrrero C: Generation of a new cystatin C-based estimating equation forglomerular filtration rate using seven assays standardized
to the international calibrator. Clin Chem 2014;60:974-86.
21. Levey AS, Coresh J, Greene T, Stevens LA, Zhang YL,
Hendriksen S, Kusek JW, van Lente F. Chronic Kidney Disease Epidemiology Collaboration. Using standardized serum creatinine values in the modification of diet in renal
disease study equation for estimating glomerular filtration rate. Ann Intern Med 2006;145:247–54.
22. Ainsworth C. Sex redefined. The idea of two sexes is
simplistic. Biologists now think there is a wider spectrum
than that. Nature 2015;518:288-91.
23. Bökenkamp A, Domanetzki M, Zinck R, Schumann G,
Byrd D, Brodehl J. Cystatin C - a new marker of glomerular
filtration rate in children independent of age and height.
Pediatrics 1998;101:875-81.
24. Bökenkamp A, Domanetzki M, Zinck R, Schumann G,
Brodehl J. Reference values for cystatin C serum concentrations in children. Pediatr Nephrol 1998;12:125-9.
25. Leion F, Hegbrant J, den Bakker E, Jonsson M, Abrahamson M, Nyman U, Björk J, Lindström V, Larsson A,
Bökenkamp A, Grubb A. Estimating glomerular filtration
rate (GFR) in children. The average between a cystatin
C- and a creatinine-based equation improves estimationof GFR in both children and adults and enables diagnosing Shrunken Pore Syndrome. Scand J Clin Lab Invest
2017;77:338-44.

26. Colantonio LD, Tanner RM, Warnock DG, Gutiérrez
OM, Judd S, Muntner P, Bowling CB. The role of cystatin-C
in the confirmation of reduced glomerular filtration rate
among the oldest old. Arch Med Sci 2016;12:55–67.
27. Bouvet Y, Bouissou F, Coulais Y, Séronie-Vivien S, Tafani M, Decramer S, Chatelut E. GFR is better estimatedby considering both serum cystatin C and creatinine levels. Pediatr Nephrol 2006;21:1299–306.
28. Ma YC, Zuo L, Chen JH, Luo Q, Yu XQ, Li Y, Xu JS, Huang
SM, Wang LN, Huang W, Wang M, Xu GB, Wang HY; Chinese eGFR Investigation Collaboration. Improved GFR estimation by combined creatinine and cystatin C measurements. Kidney Int 2007;72:1535–42.
29. Tidman M, Sjöström P, Jones I. A Comparison of GFRestimating formulae based upon s-cystatin C and s-creatinine and a combination of the two. Nephrol Dial Transplant 2008;23:154–60.
30. Stevens LA, Coresh J, Schmid CH, Feldman HI, Froissart M, Kusek J, Rossert J, Van Lente F, Bruce RD 3rd,
Zhang YL, Greene T, Levey AS. Estimating GFR using cystatin C alone and in combination with serum creatinine:
a pooled analysis of 3,418 individuals with CKD. Am J Kidney Dis 2008;51:395–406.
31. Schwartz GJ, Munoz A, Schneider MF, Mak RH, Kaskel
F, Warady BA, Furth SL. New equations to estimate GFR
in children with CKD. J Am Soc Nephrol 2009;20:629–37.
32. Nyman U, Grubb A, Sterner G, Björk J. Different equations to combine creatinine and cystatin C to predict
GFR. Arithmetic mean of existing equations performs
as well as complex equations. Scand J Clin Lab Invest
2009;69:619–27.
33. Grubb A. Non-invasive estimation of glomerular filtration rate (GFR). The Lund model: simultaneous use of cystatin C- and creatinine-based GFR-prediction equations,clinical data and an internal quality check. Scand J Clin
Lab Invest 2010;70:65–70.
34. Grubb A, Nyman U, Björk J. Improved estimation ofglomerular filtration rate (GFR) by comparison of eGFRcysand eGFRcreatinine. Scand J Clin Lab Invest 2012;72:73–7.
tatin C
35. www.egfr.se (availability tested November 6, 2017).
36. Jernberg T, Lindahl B, James S, Larsson A, Hansson LO,
Wallentin L. Cystatin C: a novel predictor of outcome insuspected or confirmed non-ST-elevation acute coronary
syndrome. Circulation 2004;110:2342-8.
37. Shlipak MG, Sarnak MJ, Katz R, Fried LF, Seliger SL,
Newman AB, Siscovick DS, Stehman-Breen C. Cystatin C
and the risk of death and cardiovascular events among
elderly persons. N Engl J Med 2005;352:2049–60.
38. Peralta CA, Shlipak MG, Judd S, Cushman M, McClellan W, Zakai NA, Safford MM, Zhang X, Muntner P,

Page 275

eJIFCC2017Vol28No4pp268-276

Anders Grubb
Cystatin C is indispensable for evaluation of kidney disease

Warnock D. Detection of chronic kidney disease with creatinine, cystatin C, and urine albumin-to-creatinine ratio
and association with progression to end-stage renal disease and mortality. JAMA 2011;305:1545–52.

rate in pregnant and non-pregnant women. Indicationsof altered filtration process in pregnancy. Scand J Clin Lab
Invest 2002;62:141–7.

39. Shlipak MG, Matsushita K, Ärnlöv J, Inker LA, Katz R,
Polkinghorne KR, Rothenbacher D, Sarnak MJ, Astor BC,
Coresh J, Levey AS, Gansevoort RT; CKD Prognosis Consortium. Cystatin C versus creatinine in determining risk
based on kidney function. N Engl J Med 2013;369:932–43.

48. Kristensen K, Lindström V, Schmidt C, Blirup-Jensen S,
Grubb A, Wide-Swensson D, Strevens H. Temporal changes of the plasma levels of cystatin C, beta-trace protein,beta-2-microglobulin, urate and creatinine during pregnancy indicate continuous alterations in the renal filtration process. Scand J Clin Lab Invest 2007;67:612–8.

40. Knight EL, Verhave JC, Spiegelman D, Hillege HL,de Zeeuw D, Curhan GC, de Jong PE. Factors influencing cystatin C levels other than renal function and theimpact on renal function measurement. Kidney Int
2004;65:1416–21.

49. Strevens H, Wide-Swensson D, Grubb A. Serum
cystatin C is a better marker for preeclampsia than serum creatinine or serum urate. Scand J Clin Lab Invest
2001;61:575–80.

41. Grubb A, Björk J, Nyman U, Pollak J, Bengzon J, Östner
G, Lindström V. Cystatin C, a marker for successful aging
and glomerular filtration rate, is not influenced by inflammation. Scand J Clin Lab Invest 2011;71:145–9.
42. Björk J, Grubb A, Larsson A, Hansson LO, Flodin M,
Sterner G, Lindström V, Nyman U. Accuracy of GFR estimating equations combining standardized cystatin C andcreatinine assays: A cross-sectional study in Sweden. Clin
Chem Lab Med 2015;53:403–14.
43. Grubb A, Lindström V, Jonsson M, Bäck SE, Åhlund T,
Rippe B, Christensson A. Reduction in glomerular poresize is not restricted to pregnant women. Evidence for a
new syndrome: “Shrunken pore syndrome”. Scand J Clin
Lab Invest 2015;75:333–40.
44. Lund U, Rippe A, Venturoli D, Tenstad O, Grubb A,
Rippe B. Glomerular filtration rate dependence of sievingof albumin and some neutral proteins in rat kidneys. Am
J Physiol 2003;284:F1226–34.
45. Norden AG, Lapsley M, Lee PJ, Pusey CD, Scheinman
SJ, Tam FW, Thakker RV, Unwin RJ, Wrong O. Glomerular
protein sieving and implications for renal failure in Fanconi syndrome. Kidney Int 2001;60:1885-92.
46. Grubb A, Lindström V, Kristensen K, Christensson A,
Wide-Swensson D, Strevens H, Schmidt C, Blirup-Jensen
S. Filtration quality: a new measure of renal disease. Clin
Chem Lab Med 2007;45(Suppl.):S273–4.
47. Strevens H, Wide-Swensson D, Torffvit O, Grubb A.
Serum cystatin C for assessment of glomerular filtration

50. Kristensen K, Wide-Swensson D, Schmidt C, BlirupJensen S, Lindström V, Strevens H, Grubb A. Cystatin C,
beta-2-microglobulin and beta-trace protein in pre-eclampsia. Acta Obstet Gynecol Scand 2007;86:921–6.
51. Dardashti A, Nozohoor S, Grubb A, Bjursten H. Shrunken Pore Syndrome is associated with a sharp rise in mortality in patients undergoing elective coronary artery bypass grafting. Scand J Clin Lab Invest 2016;76:74–81.
52. Björk J, Grubb A, Sterner G, Nyman U. Revised equations for estimating glomerular filtration rate based on
the Lund-Malmö study cohort. Scand J Clin Lab Invest
2011;71:232–9.
53. Nyman U, Björk J, Lindström V, Grubb A. The LundMalmö creatinine-based glomerular filtration rate prediction equation for adults also performs well in children.
Scand J Clin Lab Invest 2008;68:568–76.
54. Purde MT, Nock S, Risch L, Medina Escobar P, Grebhardt C, Nydegger UE, Stanga Z, Risch M. The cystatin C/
creatinine ratio, a marker of glomerular filtration quality:
associated factors, reference intervals, and predictionof morbidity and mortality in healthy seniors. Transl Res
2016;169:80–90.
55. Christensson A, Grubb A, Molvin J, Holm H, Gransbo
K, Tasevska-Dinevska G, Bachus E, Jujic A, Magnusson
M. The shrunken pore syndrome is associated with declined right ventricular systolic function in a heart failure
population - the HARVEST study. Scand J Clin Lab Invest.
2016;76:568-74.

Page 276

eJIFCC2017Vol28No4pp268-276

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

Novel filtration markers for GFR estimation
Amy B. Karger1, Lesley A. Inker2, Josef Coresh3, Andrew S. Levey2,
John H. Eckfeldt1

Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA
William B. Schwartz Division of Nephrology, Tufts Medical Center, Department of Medicine,Tufts University School of Medicine, Boston, MA, USA
3
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Welch Center for Prevention,
Epidemiology and Clinical Research, Johns Hopkins Medical Institutions, Baltimore, MD, USA
1
2

ARTICLE INFOABSTRACT

Corresponding author:
Amy B. Karger
Department of Laboratory
Medicine and Pathology
University of Minnesota
420 Delaware St SE MMC 609,
Minneapolis, MN 55455
United States of America
E-mail: karge026@umn.edu

Creatinine-based glomerular filtration rate estimation (eGFRcr) has been improved and refined sincethe 1970s through both the Modification of Diet in
Renal Disease (MDRD) Study equation in 1999 and
the CKD Epidemiology Collaboration (CKD-EPI) equation in 2009, with current clinical practice dependentprimarily on eGFRcr for accurate assessment of GFR.
However, researchers and clinicians have recognizedlimitations of relying on creatinine as the only filtration marker, which can lead to inaccurate GFR estimates in certain populations due to the influence of
non-GFR determinants of serum or plasma creatinine.
Therefore, recent literature has proposed incorporation of multiple serum or plasma filtration markers
into GFR estimation to improve precision and accuracy and decrease the impact of non-GFR determinants
for any individual biomarker. To this end, the CKD-EPI
combined creatinine-cystatin C equation (eGFRcr-cys)
was developed in 2012 and demonstrated superior
accuracy to equations relying on creatinine or cystatin
C alone (eGFRcr or eGFRcys). Now, the focus has broadened to include additional novel filtration markers
to further refine and improve GFR estimation. Beta2-microglobulin (B2M) and beta-trace-protein (BTP)

Key words:
glomerular filtration rate (GFR),
estimated GFR, filtration markers,
beta-trace protein (BTP),
beta-2-microglobulin (B2M),
metabolomics

Page 277

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

are two filtration markers with established assays that have been proposed as candidates for
improving both GFR estimation and risk prediction. GFR estimating equations based on B2M
and BTP have been developed and validated,with the CKD-EPI combined BTP-B2M equation (eGFRBTP-B2M) demonstrating similar performance to eGFRcr and eGFRcys. Additionally, several studies have demonstrated that both B2M
and BTP are associated with outcomes in CKD
patients, including cardiovascular events, ESRD
and mortality. This review will primarily focus
on these two biomarkers, and will highlight efforts to identify additional candidate biomarkers through metabolomics-based approaches.


INTRODUCTION
It is currently estimated that 15% of US adults,
or about 30 million people, have chronic kidneydisease (CKD)1. CKD is defined as the presenceof kidney damage, or estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, for aduration of at least 3 months2. Once diagnosed,
CKD is staged based on cause of disease, levelof GFR and albuminuria, to provide guidancefor disease management and risk stratification2.
GFR is accepted as the best overall measureof kidney function in health and disease andreflects the product of the number of nephrons and the average single nephron GFR3.
Measured glomerular filtration rate (mGFR)
via quantification of urinary or plasma clearance of an exogenous filtration marker remains
the gold standard for assessing GFR in patientswith CKD. However, GFR measurement is burdensome for patients as well as clinical laboratories. Therefore, clinicians instead routinely
use GFR estimates to diagnose and manage
patients with CKD. The 2012 Kidney Disease
Improving Global Outcomes (KDIGO) guidelines
recommend GFR estimation based on serum or

plasma creatinine (eGFRCr) as the first line test,with eGFR based on cystatin C (eGFRcys) or the
combination of the two (eGFRcr-cys) as a confirmatory test, particularly when there is concern
for inaccurate eGFRcr results in individuals impacted by known non-GFR determinants ofcreatinine, such as extremes of muscle mass, ahigh meat-containing diet, or some dietary supplements such as creatine4.
GFR estimating equations were developed as
early as the 1970s, but it was the 4-variable
Modification of Diet in Renal Disease (MDRD)
Study equation developed in 2000 and re-expressed for use with standardized creatinine5that was the first estimating equation to become widely integrated into routine clinical laboratory reports for assessment of kidney function, due to its reliance on creatinine and readily
available demographic metrics (age, gender andrace)6,7. While this MDRD Study equation was
useful for estimating GFR < 60 ml/min/1.73 m2,
it was found to systematically underestimate
GFR at levels > 60 ml/min/1.73 m2.
Therefore, the Chronic Kidney Disease
Epidemiology Collaboration (CKD-EPI) was
formed in 2003 and set forth to improve theaccuracy of GFR estimating equations by development and validation of equations based on
creatinine or cystatin C in a diverse populationthat included participants across the range of
GFR and age3, with and without CKD, diabetes
and transplants. Cystatin C was selected as acomplimentary candidate filtration marker tocreatinine because it is less affected by nonGFR determinants that impact creatinine8, andseveral studies have demonstrated that it is abetter prognostic marker for predicting development of cardiovascular disease and mortality than creatinine9-11.
The CKD-EPI group developed and validated a
new CKD-EPI creatinine-based eGFR equation,
which was found to have lesser bias compared

Page 278

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

to measured GFR at GFR > 60 than the MDRD
Study equation, and therefore its use was recommended as an improvement over the MDRD
Study equation12. This work was followed bypublication of two papers which demonstrated
that estimating equations which relied on bothcreatinine and cystatin C were superior in precision to equations that relied on only one biomarker alone13,14. These studies laid the groundwork for the main hypothesis driving current
efforts to improve GFR estimation – that incorporation of additional biomarkers into estimation of GFR diminishes the impact of non-GFR
determinants for any given biomarker and improves overall equation performance. Basedon this hypothesis, research in the area of GFRestimation has moved from fine-tuning current
creatinine and cystatin C-based equations to
identifying new endogenous filtration markers
that can be incorporated into GFR estimation to
improve precision.
Beta-2-microglobulin (B2M) and beta-trace protein (BTP) have been identified as two endogenous low molecular weight protein filtration
markers with established assays that have thepotential to improve the accuracy of GFR estimations. Additionally, due to technologic advancesin the field of metabolomics, work is currently
in progress to identify and validate the utility ofadditional, novel filtration markers, with subsequent development of validated assays.

and the amount eliminated through glomerular filtration and tubular metabolism. Due to its
ubiquitous presence on the surface of all cells,
B2M elevation is seen with diseases associated with high cell turnover, such as many malignancies. Therefore, B2M is most commonlymeasured along with serum albumin to risk
stratify multiple myeloma patients using theInternational Staging System (ISS)16, with higher levels of B2M associated with higher tumor
burden and more aggressive subtypes, due toincreased shedding of B2M15.
B2M was first suggested as a biomarker for glomerular filtration in the 1980s17,18, however, as
an acute phase reactant that increases in a variety of inflammatory and infectious disorders,
its potential as a candidate for a single-marker
equation was limited19,20. Despite this shortcoming a handful of research groups derived
GFR estimating equations based on B2M alone,
but data supporting the performance and validity of these equations is lacking21-24.

BETA-2-MICROGLOBULIN (B2M)

Elevation of B2M in patients with CKD, especially end stage renal disease (ESRD), has been
traditionally attributed to impaired removal
secondary to decreased glomerular filtration.
However recent literature has put forth the
hypothesis that an additional source of B2Melevation in patients with CKD may be the interference of uremic solutes with the non-covalent binding of B2M to MHC molecules, leading to an increase in shedding of B2M into thecirculation15.

B2M is a 11.8 kD protein which associates withboth classical and non-classical MHC Class I
molecules on the surface of all cells and is critical for antigen presentation15. It is freely filteredby the glomerulus, with more than 99.9% reabsorbed and metabolized in the proximal tubule15. Serum/plasma B2M concentrations are
impacted by the amount generated and shedby nucleated cells, body distribution kinetics,

Due to its established use as a prognostic marker for multiple myeloma, B2M is routinely measured in many clinical laboratories by a variety
of methods – nephelometry, turbidimetry, or
immunoassay25. However, studies have demonstrated that B2M assays are not harmonized or
standardized leading to discordance between
methods25,26. While the WHO 1st International
Standard for B2M was developed in 1985 27,

Page 279

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

and a B2M certified reference value in the serum protein standard ERM-DA470k/IFCC was
assigned in 2015 by the Institute for Reference
Materials and Measurements (IRMM)28, manufacturers have not universally adopted use of
ERM-DA470k/IFCC for calibration of their measurement procedures25.
BETA-TRACE PROTEIN (BTP)
BTP, also known as lipocalin prostaglandin D2
synthase (L-PGDS), is a 23-29 kDa protein. The
variation in size depends on the degree of posttranslational glycosylation29, with the larger isoforms of BTP in serum and urine, and smaller
isoforms with truncated side chains in cerebrospinal fluid (CSF)29. BTP was first noted to beelevated in patients with CKD in 1987, as an incidental finding in a study focused on BTP as amarker for CSF leak30. Its specific potential as afiltration marker was not suggested until 1997,
in a study that observed very high levels of BTP
in patients on hemodialysis31.
Table 1

The first GFR estimating equations based on BTP
were derived in 2007 by White and colleagues,
in a cohort of 163 adult kidney transplant patients with measured GFR. These equations,known as the White equations, performed
comparably to the MDRD Study equation, withevidence of improved performance at higher
GFRs32. The following year, researchers led by
Dr. Uwe Pöge developed 3 additional BTP-based
GFR estimating equations from a cohort of 85
kidney transplant patients validated in a separate cohort of 102 kidney transplant patients33.
The three Pöge equations were compared to
the re-expressed MDRD Study equation andWhite equation 1 (based on BTP and urea). The
Pöge BTP-formula 3 had better accuracy andprecision than White equation 1, and demonstrated a slightly smaller bias and higher 10%
accuracy when compared to the re-expressed
MDRD Study equation33. The generalizability ofthese equations to clinical populations otherthan kidney transplant recipients has not been

GFR estimating equations based on BTP
developed by White32 and Pöge33

Description

Development
population

Equation

White Equation 1(BTP & urea)

N = 163, kidney transplantpatients

eGFR = 112.1 x BTP-0.662 x Urea-0.280 x(0.880 if female)White Equation 2(BTP & Cr)

N = 163, kidney transplantpatients

eGFR = 167.8 x BTP-0.758 x Cr-0.204 x(0.871 if female)

Pöge BTP-formula 1(BTP alone)

N = 85, kidneytransplant patientseGFR = 47.17 x BTP-0.7933

Pöge BTP-formula 2(BTP & Cr)

N = 85, kidneytransplant patientseGFR = 974.31 x BTP-0.2594 x Cr-0.647

Pöge BTP-formula 3(BTP & urea)

N = 85, kidneytransplant patientseGFR = 89.85 x BTP-0.5541 x Urea-0.3018

White and Pöge formulas utilize units of mg/L for BTP, mmol/L for creatinine, and mmol/L for urea.

Page 280

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

established, and these minor differences were
not deemed sufficient enough to recommend
replacement of the MDRD Study equation forroutine clinical practice33. (Table 1)
While GFR estimating equations based on BTP
appear promising, a major hurdle involves thelack of standardization amongst currently available BTP assays29,34. Unlike creatinine, cystatin C
and B2M, there are currently no certified reference materials available for BTP. Additionally,
given the known variation in post-translational
modification which creates a variety of glycoprotein epitopes, immunoassays utilizing different antibodies would be expected to give disparate BTP results.
USING BTP AND B2M
TO IMPROVE GFR ESTIMATION
Given the various shortcomings of relying on BTP
or B2M alone for GFR estimation, the CKD-EPI
investigators evaluated the utility of combining
the markers35. Data was pooled from 3 separate
research studies involving a total of 3,551 subjects with CKD due to a variety of causes, eachwith GFR measured based on urinary clearanceof iothalamate35. Equations were developed using either BTP or B2M concentrations alone andin combination (Table 2).
Table 2The performance of the three equations wascompared to the CKD-EPI creatinine- and cystatin C-based equations based on precision(Table 3). Their analysis demonstrated that the
combined BTP-B2M equation had similar performance to both the creatinine and cystatin
C equations but did not represent an improvement over either equation35. Additionally, thecombined BTP-B2M equation was not as accurate as the combined creatinine-cystatin C
equation35. Lastly, averaging the BTP-B2M equation with the creatinine-cystatin C equation did
not lead to improvement in equation performance35. Limitations of this work included theabsence of participants without CKD and an external validation population. (Table 3)
While the non-GFR determinants of creatinine
were already well-established, it was important
to more fully characterize the non-GFR determinants of cystatin C, BTP and B2M. Preliminary
studies demonstrated evidence for non-GFR
determinants of cystatin C, including inflammation, immunosuppressive therapies, thyroid disease and obesity36-39, but there were few studies
that had evaluated the non-GFR determinantsof BTP and B2M. Therefore, in 2016 the CKDEPI investigators published a cross-sectional
analysis of these same CKD cohorts which characterized the non-GFR determinants for thesethree biomarkers40. Their analysis showed that

CKD-EPI BTP and B2M equations35

Description

Development
population

EquationBTP

N = 2,380, chronic kidneydisease patients

GFR = 55 x BTP-0.695 x 0.998age x
0.899 if female

B2M

N = 2,380, chronic kidneydisease patients

GFR = 133 x B2M-0.852

BTP-B2M

N = 2,380, chronic kidneydisease patients

GFR = 96 x BTP -0.278 x B2M-0.588

Page 281

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

Table 3

Performance of CKD-EPI GFR Estimating Equations(Adapted from Inker et al.35)
Description

Inter-quartile
range (95% CI)

1-P30 (%)(95% CI)

1-P20 (%) (95% CI)

BTP

15.0 (14.1, 15.9)

23.6 (21.3, 26.1)*

43.6 (40.8, 46.5)

B2M

12.9 (12.2, 13.8)

18.4 (16.2, 20.8)*

37.2 (34.6, 40.1)

BTP-B2M

12.1 (11.4, 13.0)

15.5 (13,3, 17.7)*

35.4 (32.5, 38.1)

Creatinine

11.6 (10.9, 12.4)

16.4 (14.2, 18.6)*

34.5 (31.7, 37.3)

Cystatin C

11.4 (10.6, 12.4)

16.9 (14.9, 18.6)*

34.8 (32.1, 37.6)

Creatinine-Cystatin C

9.3 (8.7, 10.1)

11.3 (9.5, 13.2)

25.5 (23.1, 28.0)

Average of Creatinine-Cystatin
C + BTP-B2M

10.2 (9.5, 11.0)

9.6 (8.0, 11.4)

25.0 (22.6, 27.6)

P30 and P20 are the percentage of GFR estimates > 30% and > 20% from measured GFR*P < 0.001 when compared to the creatinine-cystatin C equation

Table 4

Summary of major non-GFR determinants for filtration markers40,41

GFR biomarker

Non-GFR determinant profile

Creatinine

Male sex, black race, elevated urine creatinine, age

Cystatin C

Male sex1, smoking, body mass index (BMI) and C-reactive protein (CRP)

BTP

Male sex1, urine protein excretion, non-black race, body mass index (BMI)

B2M

Urine protein excretion, smoking and C-reactive protein (CRP)

The association between male sex and creatinine was stronger than the associations between
male sex and BTP or cystatin C

1creatinine was more strongly associated withmale sex, black race and elevated urine creatinine than BTP, B2M or cystatin C. In addition,
each filtration marker exhibited unique profilesof non-GFR determinants (Table 4).

In 2017, non-GFR determinants of these filtration markers were further characterized in 2
community-based, predominantly elderly cohorts (Table 4)41. Again, creatinine was found to
more strongly associate with age and sex than

Page 282

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

cystatin C, BTP or B2M. Additionally, both cystatin C and B2M had significant associations
with CRP, confirming prior studies demonstrating a relationship between inflammation and inflammatory diseases and these biomarkers15,42.
Not all associations were duplicated between
the two studies, and therefore more researchis needed. Both studies did provide evidence
that each filtration marker has unique non-GFR
determinant profiles, providing a foundationof support for the hypothesis that combining
multiple markers with differing non-GFR determinants for GFR estimation has the potential tominimize bias and imprecision, thereby improving accuracy. Additionally, eGFRcys, eGFRBTP andeGFRB2M were less influenced by race than eGFRcr, thus introducing the possibility of developing a multiple marker estimating equation without creatinine which would eliminate the needfor race specification.

health, but could also be due to confounding by
non-GFR determinants of cystatin C 46. These results raised the question of whether B2M and
BTP have prognostic value beyond creatinine oreGFRcr alone10. The first study proposing B2M asa prognostic marker was published in 2008, anddemonstrated that B2M was an independent
predictor of overall mortality in a communitybased elderly population47. BTP was first proposed as a prognostic marker in a 2010 study
which found that it was a strong predictor for
future CKD progression48.

PROGNOSTIC VALUE OF BTP AND B2M

The study found that, similar to cystatin C,
B2M is a stronger predictive marker than eGFRcr for outcomes such as cardiovascular disease, kidney failure and mortality49. BTP levels
also predicted these outcomes more strongly
than eGFRcr, although not to the degree ofcystatin C and B2M levels49. This study was followed up by a similar analysis performed on
6,445 subjects from the Third National Healthand Nutrition Examination Survey (NHANESIII), a general population cohort spanning therange of adulthood, ages 20 and older50. This
study also demonstrated that BTP and B2M
were stronger prognostic markers than eGFRcr, for all-cause mortality, cardiovascular disease, and coronary heart disease mortality50.
Additionally, incorporating 4 markers – creatinine, cystatin C, B2M and BTP – into a risk prediction model led to moderate improvementin 10-year risk prediction compared to eGFRcr,
when adjusted for mortality and cardiovascular risk factors50.

Like cystatin C, BTP and B2M are promising biomarkers in CKD not only due to their potential
role in improving GFR estimation, but also dueto their role as prognostic indicators. Patientswith CKD have a significantly increased risk for
cardiovascular disease, hospitalization and mortality compared to the general population, andtherefore there is interest in predicting theseoutcomes43-45.
In 2005, cystatin C was found to be a stronger predictor of mortality and cardiovascular outcomesthan creatinine and eGFRcr10. Additionally there isa marked discordance in mortality prediction between eGFRcr and eGFRcys at higher eGFRs, withhigher eGFRcr associated with increased mortality while higher eGFRcys is associated with decreased mortality46. This discordance is thought
to be due to non-GFR determinants of creatinine
such as muscle wasting that would confound its
association with outcomes in individuals in poor

Based on the promise of these initial studies,in 2012 researchers took a more comprehensive look at BTP and B2M as prognostic markers, by examining their association with risks
for mortality, cardiovascular disease and kidney failure in a large group of subjects fromthe Atherosclerosis Risk in Communities (ARIC)
study (n = 9,988), a middle-aged general population cohort49.

Page 283

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

While these studies supported the utility of B2M
and BTP as prognostic markers in the general
population, they did not examine their utility inclinically relevant sub-populations, such as diabetics or patients with chronic kidney disease,or in racial groups other than Whites or AfricanAmericans. Therefore, their role as risk predictors in a type 2 diabetic Pima Indian cohort was
examined in 201551.

based on the four filtration markers (creatinine,cystatin C, BTP and B2M) alone and in combination with each other, through analysis of thethree cohorts described above (ARIC, NHANES
III, Pima) combined with three CKD study populations–Chronic Renal Insufficiency Cohort(CRIC), Modification of Diet in Renal Disease(MDRD) study and African American Study ofKidney Disease (AASK)54.

This study found that both BTP and B2M wereassociated with ESRD, with BTP having the stronger association51. Interestingly in this study only
B2M, and not BTP, was associated with mortality,
after adjustment for other mortality risk factors
and kidney function markers51. Therefore, B2M
may be a more useful prognostic marker than
BTP in this subpopulation of Pima Indian diabetics. To further address the potential role of BTP
and B2M in clinically significant subpopulations,a cohort of CKD patients was examined to specifically look at B2M and BTP’s role in predicting cardiovascular events, ESRD and mortality52.
This study demonstrated that both B2M and
BTP were independently associated with ESRD
and all-cause mortality, and B2M was associatedwith risk for cardiovascular events in these patients with mild or moderate CKD52. Additionally,
a 4-marker composite score generated fromeGFRcr, eGFRcys, B2M and BTP levels was independently associated with all three outcomes– ESRD, all-cause mortality and cardiovascular
events52. Of note, this analysis showed that BTP
and B2M are associated with ESRD, and B2M andthe 4-marker composite score were significantly
associated with all-cause mortality and cardiovascular events even after adjustment for mGFR,
indicating that non-GFR determinants contributeto risk prediction52. These findings support priorstudies that have shown that B2M or BTP haveprognostic value beyond measured GFR51,53.

Consistent with the data supporting association of B2M and BTP with risk outcomes, thisstudy found that eGFRB2M and eGFRBTP modestly improved prediction of ESRD and mortality
over eGFRCr54. Additionally, this meta-analysis
demonstrated that higher eGFRB2M and eGFRBTP
are associated with lower mortality, similar toeGFRcys54, consistent with the hypothesis that
increased mortality associated with higher
eGFRcr reflects confounding by non-GFR determinants of creatinine such as muscle wastingin patients in poor health. Additionally, eGFR
based on the average of the estimated GFRs
from all 4 biomarkers provided the best overall
performance for risk prediction, albeit only amodest improvement over eGFRCr54.

Lastly, a recent individual patient meta-analysis
from the CKD Biomarkers Consortium study
also examined the association between eGFRThis study and others together demonstrate thatcombining multiple filtration markers provides
the best overall performance for predicting risk
outcomes.
LOOKING TO THE FUTURE—KIDNEY METABOLOMICS
Advances over the last decade in mass spectrometry and associated chromatography methods have led to an explosion of metabolomics
studies aimed at discovering novel biomarkers
for various diseases55.
In 2010, the first targeted metabolomics studies in CKD patients identified novel uremic toxins, but the studies were too limited in size andpower to draw firm conclusions about the identified metabolites56-58.

Page 284

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

In 2012, the first large-scale targeted metabolomics study in subjects spanning the range of
GFR was performed using 3,011 samples fromthe KORA F4 study for metabolite discovery, and984 samples from the TwinsUK study for metabolite validation59. A total of 22 metabolites and516 metabolite ratios were identified as having
a significant association with eGFRcr, with acylcarnitines having the strongest association59.

metabolites (C-mannosyltryptophan, pseudouridine, and O-sulfo-L-tyrosine) were significantly
associated with development of low eGFRcr65.
Studies comparing metabolites to measured
GFR have been reported and could yield moreaccurate estimates of GFR whose generalizability and robustness will need to be tested.

This cross-sectional analysis was soon followed
by a targeted longitudinal metabolomics study in2013, aimed at determining whether the same ordifferent metabolites and metabolite ratios wereassociated with development of eGFRcr decline
over time independent of baseline eGFR60. The
study examined associations between 140 metabolites and 19,460 ratios with the incidence of
decreased eGFRcr and eGFRcr decline over a 7 year
period in 1,104 subjects from the KORA study60.
This longitudinal analysis demonstrated that theacylcarnitines overall did not significantly associate with eGFRcr decline over time. Rather, thestudy identified one metabolite and two ratiosthat had a significant association with change ineGFRcr over time – spermidine, the kynurenineto-tryptophan ratio, and the phosphatidylcholine diacyl C42:5-to-phosphatidylcholine acylalkyl C36:0 ratio – all of which were supported bysmaller, prior studies61-64.

While there have been marked improvements inthe accuracy of GFR estimation using serum- orplasma-based biomarkers over the last 20 years
with refinement of equations based on creatinine and cystatin C, inaccuracy of estimated GFRremains a challenge due to the impact of non-GFR
determinants of these biomarkers. B2M and BTP
hold promise as candidate endogenous filtration
markers that have the potential to improve the accuracy of both GFR estimation and risk prediction.

In 2016, the first large-scale non-targeted metabolomics study was published, with metabolite discovery performed on samples from 1735
Kora study subjects, and validated in 1164 samples from the TwinsUK study65. A non-targeted
approach has the advantage of identifying previously unrecognized CKD-associated metabolites. Of the 493 small molecules quantified
in the study, 54 metabolites had a validated
significant association with eGFRcr, with 6 metabolites demonstrating a significant pairwise
correlation: C-mannosyltryptophan, pseudouridine, N-acetylalanine, erythronate, myo-inositol and N-acetylcarnosine65. Additionally, three

CONCLUSION

Additionally, cystatin C, B2M and BTP are less affected by race than creatinine, and therefore provide the potential opportunity to estimate GFR
without the need for race specification. Kidney
metabolomics research is in the early phases ofmetabolite discovery and validation, with workon the horizon to assess the clinical feasibility ofusing additional, new biomarkers for improved
GFR estimation and risk prediction. Thus, the focus is shifting to the concept of estimating GFR
with a panel of several serum or plasma biomarkers, to minimize the impact of each individual
biomarker’s non-GFR determinants.
Additionally, multiple studies on BTP and B2M
as prognostic markers support the idea that risk
prediction also improves when multiple markers are combined. Therefore, novel biomarkers
identified via metabolomics profiling in chronic
kidney disease patients will likely be combinedwith biomarkers such as creatinine, cystatin
C, B2M and BTP, for future incorporation into
multi-biomarker estimating equations for GFR
and multi-biomarker risk prediction models.

Page 285

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

REFERENCES
1. Centers for Disease Control and Prevention National
Chronic Kidney Disease Fact Sheet. 2017; https://www.
cdc.gov/diabetes/pubs/pdf/kidney factsheet.pdf.
2. Levey AS, Coresh J. Chronic kidney disease. Lancet.
2012;379(9811):165-180.
3. Levey AS, Inker LA, Coresh J. GFR estimation:from physiology to public health. Am J Kidney Dis.
2014;63(5):820-834.
4. Stevens PE, Levin A, Members KDIGOCKDGDWG.
Evaluation and management of chronic kidney disease:synopsis of the kidney disease: improving global outcomes 2012 clinical practice guideline. Ann Intern Med.
2013;158(11):825-830.
5. Levey AS, Coresh J, Greene T, et al. Using standardized
serum creatinine values in the modification of diet in renal disease study equation for estimating glomerular filtration rate. Ann Intern Med. 2006;145(4):247-254.
6. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth
D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group.
Annals of internal medicine. 1999;130(6):461-470.
7. Levey AS GT, Kusek J, Beck G. A simplified equation topredict glomerular filtration rate from serum creatinine.
In. [abstract A0828]. Vol 11:155A. J Am Soc Nephrol2000.
8. Galteau MM, Guyon M, Gueguen R, Siest G. Determination of serum cystatin C: biological variation and reference values. Clin Chem Lab Med. 2001;39(9):850-857.
9. Menon V, Shlipak MG, Wang X, et al. Cystatin C as a risk
factor for outcomes in chronic kidney disease. Ann Intern
Med. 2007;147(1):19-27.
10. Shlipak MG, Sarnak MJ, Katz R, et al. Cystatin C andthe risk of death and cardiovascular events among elderly
persons. N Engl J Med. 2005;352(20):2049-2060.
11. Madero M, Sarnak MJ. Association of cystatin C
with adverse outcomes. Curr Opin Nephrol Hypertens.
2009;18(3):258-263.
12. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern
Med. 2009;150(9):604-612.
13. Stevens LA, Coresh J, Schmid CH, et al. Estimating GFR
using serum cystatin C alone and in combination withserum creatinine: a pooled analysis of 3,418 individuals
with CKD. Am J Kidney Dis. 2008;51(3):395-406.
14. Inker LA, Schmid CH, Tighiouart H, et al. Estimating
glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med. 2012;367(1):20-29.

15. Argyropoulos CP, Chen SS, Ng YH, et al. Rediscovering
Beta-2 Microglobulin As a Biomarker across the Spectrumof Kidney Diseases. Front Med (Lausanne). 2017;4:73.
16. Greipp PR, San Miguel J, Durie BG, et al. International staging system for multiple myeloma. J Clin Oncol.
2005;23(15):3412-3420.
17. Leroy D, Mauriat F, Dechaux M, Chopin N, Broyer M,
Sachs C. [Beta 2 microglobulin. Index of glomerular filtration in children]. Arch Fr Pediatr. 1984;41(1):43-47.
18. Nolte S, Pringsheim W, Künzer W. [Beta 2 microglobulin in the serum as a parameter of glomerular kidney
function in the first days of life]. Monatsschr Kinderheilkd. 1986;134(10):725-728.
19. Filler G, Priem F, Lepage N, et al. Beta-trace protein,cystatin C, beta(2)-microglobulin, and creatinine compared for detecting impaired glomerular filtration rates inchildren. Clinical chemistry. 2002;48(5):729-736.
20. Miyata T, Jadoul M, Kurokawa K, Van Ypersele de Strihou C. Beta-2 microglobulin in renal disease. J Am Soc
Nephrol. 1998;9(9):1723-1735.
21. Bianchi C, Donadio C, Tramonti G, Consani C, Lorusso
P, Rossi G. Reappraisal of serum beta2-microglobulin as
marker of GFR. Renal failure. 2001;23(3-4):419-429.
22. Donadio C. Serum and urinary markers of early impairment of GFR in chronic kidney disease patients: diagnostic accuracy of urinary β-trace protein. Am J Physiol
Renal Physiol. 2010;299(6):F1407-1423.
23. Trollfors B, Norrby R. Estimation of glomerular filtration rate by serum creatinine and serum beta 2-microglobulin. Nephron. 1981;28(4):196-199.
24. Ikezumi Y, Uemura O, Nagai T, et al. Beta-2 microglobulin-based equation for estimating glomerular filtration rates in Japanese children and adolescents. Clin Exp
Nephrol. 2015;19(3):450-457.
25. Fedele PL, Choy KW, Doery JC, Grigoriadis G, Shortt
J, Lu ZX. Inter-laboratory discordance of beta-2 microglobulin results: impact on the validity of the international staging system for multiple myeloma. Br J Haematol.
2014;166(6):951-953.
26. Tichý M, Maisnar V, Palicka V, et al. International
Staging System required standardization of biochemical laboratory testing in multiple myeloma. Neoplasma.
2006;53(6):492-494.
27. WHO Technical Report Series No. 745. In:1987:21.
28. Auclair G, Zegers I, Munoz A, et al. The certificationof the mass concentration of beta-2-microglobulin in human serum: ERM-DA470k/IFCC. In. European Report EUR
26972 EN. Luxembourg, European Union2015.

Page 286

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

29. White CA, Ghazan-Shahi S, Adams MA. β-Trace protein: a marker of GFR and other biological pathways. Am
J Kidney Dis. 2015;65(1):131-146.
30. Felgenhauer K, Schädlich HJ, Nekic M. Beta traceprotein as marker for cerebrospinal fluid fistula. Klin
Wochenschr. 1987;65(16):764-768.
31. Hoffmann A, Nimtz M, Conradt HS. Molecular characterization of beta-trace protein in human serum andurine: a potential diagnostic marker for renal diseases.
Glycobiology. 1997;7(4):499-506.
32. White CA, Akbari A, Doucette S, et al. A novel equation to estimate glomerular filtration rate using betatrace protein. Clinical chemistry. 2007;53(11):1965-1968.
33. Pöge U, Gerhardt T, Stoffel-Wagner B, et al. Beta-traceprotein-based equations for calculation of GFR in renaltransplant recipients. Am J Transplant. 2008;8(3):608-615.
34. White CA, Akbari A, Eckfeldt JH, et al. β-Trace Protein
Assays: A Comparison Between Nephelometric and ELISA
Methodologies. Am J Kidney Dis. 2017;69(6):866-868.
35. Inker LA, Tighiouart H, Coresh J, et al. GFR Estimation
Using β-Trace Protein and β2-Microglobulin in CKD. Am J
Kidney Dis. 2016;67(1):40-48.
36. Rule AD, Bergstralh EJ, Slezak JM, Bergert J, Larson TS. Glomerular filtration rate estimated by cystatin
C among different clinical presentations. Kidney Int.
2006;69(2):399-405.
37. Risch L, Herklotz R, Blumberg A, Huber AR. Effects of
glucocorticoid immunosuppression on serum cystatin C
concentrations in renal transplant patients. Clin Chem.
2001;47(11):2055-2059.

cardiovascular events, and hospitalization. The New England journal of medicine. 2004;351(13):1296-1305.
44. Matsushita K, van der Velde M, Astor BC, et al. Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality ingeneral population cohorts: a collaborative meta-analysis. Lancet. 2010;375(9731):2073-2081.
45. Bash LD, Astor BC, Coresh J. Risk of incident ESRD: acomprehensive look at cardiovascular risk factors and 17
years of follow-up in the Atherosclerosis Risk in Communities (ARIC) Study. Am J Kidney Dis. 2010;55(1):31-41.
46. Shlipak MG, Matsushita K, Ärnlöv J, et al. Cystatin C
versus creatinine in determining risk based on kidney
function. N Engl J Med. 2013;369(10):932-943.
47. Shinkai S, Chaves PH, Fujiwara Y, et al. Beta2microglobulin for risk stratification of total mortality in the elderly population: comparison with cystatin
C and C-reactive protein. Archives of internal medicine.
2008;168(2):200-206.
48. Spanaus KS, Kollerits B, Ritz E, et al. Serum creatinine,cystatin C, and beta-trace protein in diagnostic staging
and predicting progression of primary nondiabetic chronic kidney disease. Clin Chem. 2010;56(5):740-749.
49. Astor BC, Shafi T, Hoogeveen RC, et al. Novel markers
of kidney function as predictors of ESRD, cardiovasculardisease, and mortality in the general population. Am J
Kidney Dis. 2012;59(5):653-662.
50. Foster MC, Inker LA, Levey AS, et al. Novel filtration
markers as predictors of all-cause and cardiovascularmortality in US adults. Am J Kidney Dis. 2013;62(1):42-51.

38. Knight EL, Verhave JC, Spiegelman D, et al. Factors influencing serum cystatin C levels other than renal function and the impact on renal function measurement. Kidney Int. 2004;65(4):1416-1421.

51. Foster MC, Inker LA, Hsu CY, et al. Filtration markers as predictors of ESRD and mortality in Southwestern
American Indians with type 2 diabetes. Am J Kidney Dis.
2015;66(1):75-83.

39. Manetti L, Pardini E, Genovesi M, et al. Thyroid function differently affects serum cystatin C and creatinine
concentrations. J Endocrinol Invest. 2005;28(4):346-349.

52. Foster MC, Coresh J, Hsu CY, et al. Serum β-Trace Protein and β2-Microglobulin as Predictors of ESRD, Mortality, and Cardiovascular Disease in Adults With CKD in the
Chronic Renal Insufficiency Cohort (CRIC) Study. Am J Kidney Dis. 2016;68(1):68-76.

40. Liu X, Foster MC, Tighiouart H, et al. Non-GFR Determinants of Low-Molecular-Weight Serum Protein Filtration Markers in CKD. Am J Kidney Dis. 2016;68(6):892-900.
41. Foster MC, Levey AS, Inker LA, et al. Non-GFR Determinants of Low-Molecular-Weight Serum Protein Filtration Markers in the Elderly: AGES-Kidney and MESA-Kidney. Am J Kidney Dis. 2017.
42. Shlipak MG, Katz R, Cushman M, et al. Cystatin-C andinflammatory markers in the ambulatory elderly. Am J
Med. 2005;118(12):1416.
43. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu
CY. Chronic kidney disease and the risks of death,

53. Tangri N, Inker LA, Tighiouart H, et al. Filtration markers may have prognostic value independent of glomerular filtration rate. J Am Soc Nephrol. 2012;23(2):351-359.
54. Inker LA, Coresh J, Sang Y, et al. Filtration Markers as Predictors of ESRD and Mortality: Individual Participant Data Meta-Analysis. Clin J Am Soc Nephrol.
2017;12(1):69-78.
55. Gowda GA, Djukovic D. Overview of mass spectrometry-based metabolomics: opportunities and challenges.
Methods Mol Biol. 2014;1198:3-12.

Page 287

eJIFCC2017Vol28No4pp277-288

Amy B. Karger, Lesley A. Inker, Josef Coresh, Andrew S. Levey, John H. Eckfeldt
Novel filtration markers for GFR estimation

56. Kim CD, Kim EY, Yoo H, et al. Metabonomic analysisof serum metabolites in kidney transplant recipients withcyclosporine A- or tacrolimus-based immunosuppression. Transplantation. 2010;90(7):748-756.
57. Rhee EP, Souza A, Farrell L, et al. Metabolite profiling identifies markers of uremia. J Am Soc Nephrol.
2010;21(6):1041-1051.
58. Toyohara T, Akiyama Y, Suzuki T, et al. Metabolomic
profiling of uremic solutes in CKD patients. Hypertens
Res. 2010;33(9):944-952.
59. Goek ON, Doring A, Gieger C, et al. Serum metabolite
concentrations and decreased GFR in the general population. Am J Kidney Dis. 2012;60(2):197-206.
60. Goek ON, Prehn C, Sekula P, et al. Metabolites associate with kidney function decline and incident chronic
kidney disease in the general population. Nephrol Dial
Transplant. 2013;28(8):2131-2138.

61. Saito A, Takagi T, Chung TG, Ohta K. Serum levels ofpolyamines in patients with chronic renal failure. Kidney
Int Suppl. 1983;16:S234-237.
62. Schefold JC, Zeden JP, Fotopoulou C, et al. Increased
indoleamine 2,3-dioxygenase (IDO) activity and elevatedserum levels of tryptophan catabolites in patients withchronic kidney disease: a possible link between chronic
inflammation and uraemic symptoms. Nephrol Dial
Transplant. 2009;24(6):1901-1908.
63. Wang Y, Liu H, McKenzie G, et al. Kynurenine is an
endothelium-derived relaxing factor produced during inflammation. Nat Med. 2010;16(3):279-285.
64. Peralta CA, Katz R, Shlipak M, et al. Kidney function
decline in the elderly: impact of lipoprotein-associatedphospholipase A(2). Am J Nephrol. 2011;34(6):512-518.
65. Sekula P, Goek ON, Quaye L, et al. A MetabolomeWide Association Study of Kidney Function and Disease in the General Population. J Am Soc Nephrol.
2016;27(4):1175-1188.

Page 288

eJIFCC2017Vol28No4pp277-288

special issue:kidney
The laboratory
diagnostics of chronic kidney diseases
sTF-CKDof chronic
diseaseA pathway to national guidelines for laboratory
diagnostics of chronic kidney disease – examples
from diverse European countries

Vanja Radišić Biljak1, Kristin Moberg Aakre2, Dogan Yucel3, Anne-Sophie
Bargnoux4, Jean-Paul Cristol4, Laurence Piéroni4

Department of Medical Biochemistry and Laboratory Medicine, Merkur University Hospital, Zagreb, Croatia,
on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM)
2
Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway
3
Department of Medical Biochemistry, Ankara Training and Research Hospital, University of Health Sciences,
Altindag, Ankara and Turkish Biochemical Society
4
Laboratoire de Biochimie, CHRU de Montpellier, PhyMedExp, University of Montpellier, France,
on behalf of the Société Française de Biologie Clinique (SFBC)
1ARTICLE INFOABSTRACT

Corresponding author:
Vanja Radišić Biljak
Department of Medical Biochemistry
and Laboratory Medicine
Merkur University Hospital
Zajčeva 19
10000-Zagreb
Croatia
Phone: +385917967867
E-mail: vanja.radisic@gmail.com

The principal benefit of guidelines is to improve
the quality of care received by patients. In the 2012
Clinical Practice Guideline for the Evaluation andManagement of Chronic Kidney Disease (KDIGO) was
released and it is designed to provide information andassist decision making. This review gives a brief overview of a various national CKD guidelines that rely on
the newly released KDIGO guidelines. All of the included countries (France, Turkey, Norway and Croatia)
are non-English speaking countries and they differ in
population and socio economic aspects. Examples
shown in this review may provide valuable experiencefor countries that are in process of creating their national CKD guidelines.

Key words:
chronic kidney disease (CKD),
national guidelines,
laboratory medicine, KDIGO

Page 289

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

INTRODUCTION
For patients (and almost everyone else in healthcare), the greatest benefit that could be achieved
by guidelines is to improve health outcomes (1).
In the 2012 Clinical Practice Guideline for the
Evaluation and Management of Chronic Kidney
Disease (2) was released by the The Kidney
Disease: Improving Global Outcomes (KDIGO).
This guideline serves to update the 2002
KDOQI Clinical Practice Guidelines for Chronic
Kidney Disease: Evaluation, Classification, andStratification following a decade of focused research and clinical practice in CKD. Although it
is designed to provide information and assist
decision making, it is not intended to define astandard of care, and should not be construed
as one, nor should it be interpreted as prescribing an exclusive course of management (2).
As a comparison to worldwide recognized British(3), Australian (4, 5) and American CKD guidelines(2), this article gives a brief overview of various
national CKD guidelines from diverse European
countries: France, Turkey, Norway and Croatia.
All of the included countries are non-English
speaking countries and they differ in population and socio economic aspects. Through theseexamples variations in practice will be shown
when the needs of individual patients, available
resources, and limitations unique to a specificcountry, were taken into account.
FRENCH GUIDELINES FOR ESTIMATIONOF GLOMERULAR FILTRATION RATE (EGFR)– PAST, PRESENT AND THE FUTURE
I) Analytical evaluation and improvementof creatinine measurement: involvementof the scientific societies
As early as 2002, the “Société Française deBiologie Clinique” (SFBC) recognized the glomerular filtration rate (GFR) estimation as a major
health problem (6) and created the “Creatinine”
Working Group. This laboratory working group

initiated a multicentric study to evaluate inter-assay variation and accuracy of 17 creatinine assaysof which 14 were commercially available automated assays (4 enzyme assays, 1 compensatedJaffe assay, and 9 non-compensated Jaffe assays)(7). Using 30 frozen human samples, they demonstrated that a very high inter-assay variation
persisted since the median inter-assay coefficientof variation (CV) was 14.2% for 20 low samples(45–150 µM) and 7.7% for 10 high samples (250–350 mM). In addition, the inaccuracy, assessedwith three certified reference materials, appears
to be relatively high, especially for the lowest
concentration with biases ranging from –2.9% to+57.5% for the low level (68.7 µM) (8).
In 2008 the newly formed working Group «Bio­
logie des fonctions rénales et de l’insuffisancerénale» involving Nephrologists and medical
Biologists was supported by both the SFBC andthe “Sociéte de Néphrologie”. This group decided to perform a new study after the publication of the “Laboratory Working Group of the
National Kidney Disease Education Program”
recommendations (NKDEP) for in-vitro diagnostic (IVD) manufacturers (9), highlighting the needfor developing methods for creatinine measurement that are reproducible and traceable toisotope dilution mass spectrometry (IDMS). Ourevaluation involved 25 clinical laboratories, 12
enzymatic and 4 compensated Jaffe creatinine
automated assays. Creatinine was measured inserum pools ranging from 35.9±0.9 μmol/L to
174.5±3.1 μmol/L (IDMS determination). This
study demonstrates substantial improvementsin the calibration, traceability and precision of
the enzymatic methods, reaching the total analytical error of 8% for the majority of enzymatic
methods (10). Moreover, most of these assays
allowed accurate creatinine measurements forcreatinine levels lower than 40 μmol/L. By contrast, this requirement was never obtained forthe compensated Jaffe methods at the criticallevel of 74.4±1.4 μmol/L (11).

Page 290

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney diseaseII) Time to recommendations:a step by step improvement
Based on the international recommendations
and our own French studies, we were able topublish some French recommendations. The
first recommendation (12), published in 2002
by the «Agence National d’Accréditation etd’Eva­luation en Santé» (ANAES, former nameof Haute Autorité de Santé, HAS) recommended that laboratory analysts should provide anestimation of GFR value using the CockcroftGault formula for every request for serum creatinine, but no analytical guidelines for creatinine measurement were suggested.
However, as soon as the Kidney Disease Outcome
Quality Initiative (K/DOQI) classification was proposed (6) following a position statement from
Kidney Disease: Improving Global Outcomes(KDIGO) (13), an update of the French position statement about estimation of GFR andproteinuria has been developed by the «Société
de Néphrologie» in 2009.
For renal function measurement, it is recommended to estimate GFR from serum creatinine
using IDMS traceable simplified modification ofdiet in renal disease (MDRD) equation.
These recommendations were published in
the French journal of the Société Française deNéphrologie: Néphrologie Therapeutique (14).
This guidelines was further supported by
the report of «Agence Française de Sécurité
Sanitaire des Produits de Santé (AFSSAPS)» in
2010 recommend an IDMS traceable creatinine assay. They advised the use of enzymatic
assays in specific populations like in pediatricpatients or in specific situations where Jaffe
assays are known to be subject of interferences (15).
Through their collaborative works, the SFBC
working group published its own recommendations in the French journal of the SFBC: “LesAnnales de Biologie Clinique”, highlighting theuse of IDMS-traceable creatinine assay and theuse of CKD-EPI equation (16). Finally, the «Haute
Autorité de Santé», driven by the French Ministryof Health, meet an expert panel involving clinicalbiologists, nephrologists, and geriatricians.
These guidelines, available on the Web site of
the HAS in 2011-2012, recommend an IDMS
traceable enzymatic creatinine assay in all clinical situations because of better analytical specificity, sensitivity and performances of enzymatic
assays compared to Jaffe assays (17).
Further to the publication of KDIGO 2012clinical practice (2), the HAS recommend that
French clinical laboratories report eGFR in
adults using the 2009 CKD-EPI creatinine equation which gives the best performance in termsof accuracy. Pending the full adoption of the
CKD-EPI equation by health professionals, the175 MDRD formula may be used in the meantime but Cockroft and Gault formula should beomitted. It should be noted that the ethnic adjustment factor into the equation does not apply in France due to the non-validation of thiscorrection in non-American black people andto the French law for preventing any ethnic
discrimination.
Since it has been demonstrated that mild tomoderate CKD is associated with adverse clinical outcomes, the KDIGO working group decided not to combine stage 1–2 CKD.
A precise eGFR above 60 mL/min/1.73 m² is thus
valuable. Results of the SFBC study support theuse of CKD-EPI equation rather than MDRD andfound that accurate enzymatic assays allow estimation of eGFR until 90 mL/min/1.73 m² with
MDRD and 120 mL/min/1.73 m² with CKD-EPI
equation.
In all cases, compensated Jaffe creatinine assays
lead to important errors in eGFR and should be
avoided (18).

Page 291

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

III) Results and limitations after morethan 10 years of scientific communication
Data from the French biochemistry external
quality assessment scheme «ProBioQual» (Centre
lyonnais d’études pour laPROmotion de la
BIOlogie et du contrôle de QUALité, LYON FRANCE) underline the emergence of standardized Jaffe assays in 2008, and a gradual
implementation of enzymatic assays in Frenchlaboratories since the creation of these guidelines (Figures 1 and 2).
In 2017, about 65% of French laboratories
use enzymatic creatinine assays. In addition,
all laboratories give the creatinine result withan estimation of GFR. Some of them add thestaging of CKD according to the KDIGO fromstage I to V. As a limitation, these recommendations are clearly designed for adult population, no French recommendations are edited
for children.
Figure 1

Reliable estimates of high eGFR are importantfor drug dosing, nevertheless, the HAS draws attention to the difficulties of drug dosage adjustments. For example, there are currently conflicting recommendations between the current CKD
guidelines (19) and the «Groupe Français d’Etude
sur l’Hémostase et la Thrombose (GEHT)» regarding the use of direct oral anticoagulants for theprevention and treatment of thromboembolic
disease in patients with reduced renal function.
Clinicians are reluctant to use the MDRD or CKDEPI formula reported by the lab since the estimation of renal function with the Cockcroft andGault formula is used for the pharmacokinetics
studies and the development of drug dosing
guidelines. In addition, the values obtained with
the Cockcroft formula in patients >75 years are
systematically lower than the values obtainedwith the MDRD formula. This should allow a safeuse of prescription of direct oral anticoagulants.

Creatinine assays before French recommendations

Page 292

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

Figure 2

Creatinine assays after French recommendationsIV) Lessons and perspectives
The strong involvement of the French scientificsocieties, mainly the “Société de néphrologie”
and the SFBC leading to the constitution of joint
working group associating clinicians and medical
biologists, allows the creation of the French recommendations for GFR estimation. In addition,these collaborative groups allow the initiation
and realization of multicentric studies. Similarly
to the creatinine study, the SFBC group conducted a multicentric evaluation of automatedcystatin C assays before (2008) and after (2015)
standardization using the certified referencematerial ERM-DA 471. In the latter study, we
showed that bias remains the major componentof the combined uncertainty because of possible

problems associated with the implementation of
traceability (20). Although some manufacturers
have clearly improved their calibration protocols
relative to ERM-DA471, most of them failed tomeet the criteria for acceptable cystatin C measurements. As a result, no recommendations are
currently available in France for cystatin C.
To date, the SFBC and Société de Néphrologie
have initiated two working groups, one about thebiomarkers of Chronic Kidney Disease – Bone andMineral disorders and one about urinary markers
of renal dysfunctions. These groups produced reviews for the journal of the SFBC (“Les Annales
de Biologie Clinique”) and organized multicentricevaluations of albuminuria and urinary calcium
and phosphate determinations.

Page 293

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

INCREASING AWARENESSABOUT CHRONIC KIDNEY DISEASEAND IMPLEMENTATION OF A NEW
PRACTICE GUIDELINE IN TURKEY
CKD is an important and growing public healthproblem in Turkey like in all over the World. For
the year 2015, general incidence and generalprevalence of end stage renal disease in Turkey
were 147.3 and 935.4 per million population.
But the prevalence of CKD is very high, 15.7%
in Turkey. Currently, there are about 100 000
patients undergoing hemodialysis or peritoneal
dialysis in our country. Hence, the awarenesslevel should be increased. Currently, awarenesslevel is <10% in the World and not more than
2% in Turkey.
Although the laboratory examinations are sinequo non for screening, diagnosis, evaluation,
staging and monitoring of CKD, these examinations are mostly analysed by using different analytical methods and techniques and therefore
different results can be obtained and reportedwith different units. For this reason, a uniform
and standard approach is required for laboratory practice. From this point of view, the Turkish
Biochemical Society (TBS) planned a strategy
through implementation of a series of steps.
These steps are presented as follows.
The working groupTBS organized a working group on CKD (WGCKD) in 2014. The WG-CKD was essentially consisted of laboratory specialists from different
level hospital laboratories and the representatives of main diagnostic companies.
The survey
The WG-CKD at first coordinated a questionnaire for Turkish laboratories. The survey included questions addressing the assessmentof awareness about the CKD and especially on
creatinine and urinary albumin measurements

such as instrument use, creatinine and urine
albumin methods and their traceability, calibration and control procedures, external quality assessment scheme, reporting of eGFR,
reporting of creatinine and albumin results.
There were similar questions also relating toserum cystatin C and urine total protein in thesurvey.
About 100 specialists from different hospitallaboratories, a total of 94 labs, participated in
the survey. The major analytical systems and reagents for creatinine were of Roche Diagnostics(29.4%), Abbott Diagnostics (28.24%), BeckmanCoulter Inc. (27.06%), Siemens Healthcare(5.88%), and Mindray (1.18%). More than 90%of the laboratories were using the Jaffe method
and only 8% were using the enzymatic creatinine method. The methods were traceable toSRM 967 (50.79%), SRM 914 (33.33%) and SRM
909 (11.11%), essentially. Creatinine results
were mostly reported with conventional units(mg/dL, 95.18%).
Reference ranges recommended by diagnostic
companies were used (about 80%) and age and/or sex-related reference ranges were reported (89.73%) by a majority of the labs. Only
49.30% and 18.31% of the laboratories werereporting eGFR for adult and pediatric population, respectively. Mostly CKD-EPI formula wasused (44.74%) for eGFR, and cystatin C use
was only 10.53%). Cystatin C was measured
by nephelometric and turbidemetric methods
and only 5.2% of the labs were participate inan EQAS. Urine albumin was measured by turbidimetric (86.00%) and nephelometric (12%)
methods and all specimen types, 24-h urine,
random, first morning and second morning,were accepted. The majority of the laboratories (88.37%) did not use decision limits forurine albumin.

Page 294

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

The guideline
The WG-CKD decided to prepare a short guideline based on KDIGO 2012 clinical practiceguideline for the evaluation and management of
CKD for laboratory specialists. The guideline wascompleted and published in 2015 and included
the following key recommendations (21).
1. Creatinine assays should be traceable to areference material which creatinine concentration assigned by GC-IDMS technique.
2. When reporting the creatinine result, eGFR
should also be reported in adult (>18 years)
population. A warning expression should be
included in the report form if eGFR result is<60 mL/min/1.73 m2.
3. eGFR values should be expressed quantitatively up to 90 mL/min/1.73 m2 by CKD-EPI
equation. Above 90 mL/min/1.73 m2, eGFRvalues can be expressed quantitatively or>90 mL/min/1.73 m2.
4. eGFR equations of the adult population
should not be used for pediatric population.
Different equations utilizing also patientheight should be used. The enzymatic creatinine assay should be preferred. eGFR basedon cystatin C can be used for confirmation in
the pediatric population.
5. Cystatin C measurements, at least wheneGFR based on creatinine is not reliable andfor confirmation should be encouraged.
6. Proteinuria or albuminuria values should bereported in proportion to creatinine.
Implementation of the guideline
The guideline was was accepted by the Ministryof Health and it was circulated by Department
of Elderly Health and Disables, Public HealthInstitution of Turkey, under the Turkey ’s
Prevention and Control Program of Kidney
Diseases (2014 – 2017) in December 2015. The
guideline was also announced from the websiteof the Department of Laboratory Services,
Ministry of Health (http://dosyamerkez.saglik.
gov.tr/Eklenti/2621,kbh1pdf.pdf?).
Currently, the guideline is implemented by all
public and private medical laboratories at alllevels, primary, secondary, and tertiary healthinstitutions across Turkey. In this connection, eGFR is reported with CKD-EPI formula
through serum creatinine in adult population
and with Schwartz formula in the pediatricpopulation; proteinuria and albuminuria arealso interpreted and reported according to
the guideline.
We hope, the implementation of the guidelineby all medical laboratories, will have importantconsequences on standardisation and harmonisation of laboratory tests relating to CKD
and of course on patient safety.
NORWEGIAN RECOMMENDATIONSFOR DIAGNOSING CKDIn Norway, two recommendations regarding diagnosing CKD have been published within thelast years. One is the recommendation fromthe Norwegian Society of Medical Biochemistry(NSMB) regarding estimation of GFR based on
creatinine measurements (22), and the other isthe Diabetes Guideline (23) from the Norwegian
Directorate of Health that includes a chapter on
diagnosis and follow-up on diabetes nephropathy. Amongst other things this guideline describes how urinary albumin testing should beundertaken.
NSMBs recommendations for estimating GFR
The recommendation was worked through by
a working group consisting of five specialists inlaboratory medicine and one laboratory technician, and was published in 2016. The groupgot feedback from local nephrologists during
their work.

Page 295

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

The main messages in the recommendations•

Creatinine should be measured using an enzymatic assay•

eGFR should be calculated using the CKDEPI formula•eGFR results should be multiplied by 1.15 if
the patient is Afro-American•

Renal disease should be classified according
to the guideline from KDIGO (2)Implementation of the recommendation
In 2017 most Norwegian laboratories use an
enzymatic assay. The last numbers from theNorwegian EQA scheme (NOKLUS) showsthat 57 laboratories use enzymatic assays andonly two uses the Jaffe method. This is an improvement from before the recommendations
were produced, when 8 laboratories used the
Jaffe method. NSMB has not yet evaluated if
Norwegian laboratories have changed formulafor eGFR calculations from the MDRD formula to
the CKD-EPI formula, but oral communications
with the main laboratories in Norway indicatethat this change has been undertaken.
The guideline for diagnosing
and follow up of Diabetes Nephropathy
This guideline is part of the official Norwegian
Diabetes Guideline. It was produced by a working
group established by the Norwegian Directorate
of Health and gives recommendations regarding the diagnosis and follow-up of renal diseasein diabetes patients. The group members wereendocrinologists, nephrologists, general practitioners and a specialist in laboratory medicine.
Recommendations related to diagnosing renal
disease focused on eGFR and measurement ofurinary albumin. The group recommended thatthese tests were conducted on a yearly basis
and more often if positive results or progressive
disease were detected.

The laboratory specialist was also a member ofthe eGFR working group described above, so
recommendations related to eGFR were harmonized between the two groups and are identically to those described above.
Some information was available regarding
followed up and diagnosis of albuminuria in
diabetes patients in Norway when the work
started. This task is primarily done in primarycare, and > 95% of general practitioners screen
diabetes patients for albuminuria (24). Generalpractitioners commonly use high quality quantitative point of care instruments that measure
albumin/creatinine ratio in morning or spot
samples (25).
The main recommendations related
to urine albumin measurements•

Urine albumin should be measured as albumin/creatinine ratio.

•

A morning sample or a random spot sample
should be used.

•

Two positive samples are necessary to diagnose albuminuria. The second sample
should be taken within 3 months from thefirst sample.

•

Albuminuria should be classified as recommended by KDIGO (2).

•Physical activity, acute inflammatory response and urinary tract infection may leadto false positive results and should therefore be avoided during testing.

•

Biological variation is high and reference change values of 100-200% may be
expected.

Implementation of the guideline
The implementation of this guideline has not
yet been evaluated by the Norwegian healthcare authorities.

Page 296

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

ADVANTAGES AND OBSTACLES
IN CREATING NATIONAL CHRONICKIDNEY DISEASE LABORATORYRECOMMENDATIONS IN CROATIA
In 2013, the Joint Croatian Working Group(JCWG) for laboratory diagnostic of CKD on
the behalf of Croatian society of medical biochemistry and laboratory medicine (CSMBLM)
and Croatian chamber of medical biochemists(CCMB) conducted a survey across Croatian
medical-biochemistry laboratories to assess
the current practice in this area of laboratory
medicine. The results from the survey were
published in the presented article in the firstissue of national Biochemia Medica Journal in2015 (26).
The results of the survey showed that there isa large heterogeneity among Croatian laboratories regarding measuring methods, reporting units and reference intervals (cut-off values), both for creatinine and urine albumin
or protein. The two key prerequisites for CKD
screening, automatic reporting of eGFR and albuminuria or proteinuria assessment, are not
implemented nationwide. There is a need forharmonization in laboratory diagnostics of CKD
in Croatia (26). There is still a substantial number of laboratories that use the non-standardized uncompensated Jaffe method, almost one
quarter of all Croatian medical-biochemistrylabs. Only about 11% of laboratories use enzymatic method. The rest of laboratories measure creatinine with compensated Jaffe method traceable to IDMS method and Standard
Reference Material 967.
The majority of laboratories that participatedin the survey generally do not report resultsfor eGFR (75%). Among laboratories that report eGFR, there is a statistically signifcant difference in distribution by type of institution (P< 0.001), with the lowest number of laboratories from primary health care institutions.

The most prevalent equation for calculating
eGFR, at the time point when the survey was
conducted, was MDRD equation for standardized creatinine, which was in accordance withthe recommendations of Croatian Chamber atthat time. However, there were some answers
indicating using the MDRD equation for standardized creatinine with the results of serum
creatinine measured with non-standardized uncompensated Jaffé method, and reporting of results for eGFR calculated with MDRD equation
as an exact number regardless of eGFR value.
Majority of laboratories that participated in thesurvey do not measure urine albumin or protein (75%), predominantly in primary healthcare laboratories. There is a large heterogeneity
among type of sample recommended for measuring urine albumin or protein and reporting
units, consequently. The results indicate thatassessment of albuminuria and proteinuria in alarge number of laboratories is still performedin 24-hour urine samples.
The most important issue that occured is thefact that laboratories still use non-standardized
methods for creatinine results and do not report eGFR values. Also, the majority of laboratories do not measure urine albumin, especiallyin primary care health setting. These facts set
the background for the process of standardization and harmonization in this area of laboratory medicine which resulted in issuing firstnational recommendations for laboratory diagnostics of chronic kidney disease in Croatia (27).
These national recommendations, based on therelevant 2012 KDIGO Guideline, represent thefirst step in accomplishing the goal of standardization and harmonization in this area of laboratory medicine. The recommendations were published on English language, however Croatiantranslation was printed in a form of a booklet
and distributed to every medical-biochemistrylaboratory in Croatia (Figure 3).

Page 297

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

Figure 3

The main page of a Croatian national recommendations for laboratory
diagnostics of chronic kidney disease (booklet)

The national recommendations are mainly
based on the KDIGO 2012 guidelines, however,novel literature findings are also incorporated.
Considering the results obtained via conductedsurvey, our main goal was to provide recommendations that can be easily applied in every
medical biochemistry laboratory in Croatia. We,
as a WG and authors of recommendations, decided to start at the basic laboratory tests usedin laboratory diagnostics of CKD: creatinine,eGFR, urine albumin-to-creatinine ratio (ACR)

and urine protein-to-creatinine ratio (PCR). The
text of the national recommendations is organized to identify critical points in four major
laboratory tests used in basic laboratory diagnostics of CKD. The draft of the recommendations was sent to numerous national and international experts for their comments. The
manuscript was also made available for public
consultation. All comments were carefully considered and incorporated into the final versionof the recommendations.

Page 298

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

It is rather difficult to give unique and uniform
recommendations, regarding a large heterogeneity amongst methods and populations. Our
intention was to point out to some weak points
in pre-, post- and analytical phase, as well assome basic pediatric considerations, but every
laboratory must set their own specifications
for method performance and handling thespecimens, according to their possibilities andconditions.
The main messages in the recommendations
are as follows:•

Creatinine should be measured using an enzymatic assay•

eGFR should be calculated using the CKDEPI formula•

Urine albumin should be measured as albumin/creatinine ratio.

•

A morning sample or a random spot sample
should be used.

1. Creatinine assays should be traceable to areference material which creatinine concentration assigned by GC-IDMS technique.
2. When reporting the creatinine result, eGFR
should also be reported in adult (>18 years)
population. A warning expression should be
included in the report form if eGFR result is<60 mL/min/1.73 m2.
3. eGFR values should be expressed quantitatively up to 90 mL/min/1.73 m2 by CKD-EPI
equation. Above 90 mL/min/1.73 m2, eGFRvalues can be expressed quantitatively or>90 mL/min/1.73 m2.
4. eGFR equations of the adult population
should not be used for pediatric population.
Different equations utilizing also patientheight should be used. The enzymatic creatinine assay should be preferred. eGFR basedon cystatin C can be used for confirmation in
the pediatric population.

So, our final goal for 2017, as a Joint Working
Group, will be a complete implementation ofnational guidelines.
Every member of the WG participates in theimplementation process. We inted to provide
relevant information to every medical biochemist in our geografically diverse country.
To facilitate implementation of national guidelines the members of a national WG gave a series of lectures entitled: „The role of laboratory
testing in detection and classificaton of chronic
kidney disease: national recommendations”.
To assess the national recommendations implementation process, our subsequent actions
include repeating a slightly modified survey
by the end of 2017. The biggest challenge remains introduction of albuminuria measurement in primary health care laboratories. Thisis a regulatory issue that requires the involment of the State and our health care system
to finance the introduction of the new tests inprimary health care labs that represent about
70% of medical-biochemistry labs in Croatia.
This problem is already presented twice toresponsible regulatory bodies, however noagreement was made so far.
Future plans also include cooperation with
Croatian Society for Nephrology, Dialysis andTransplantation of Croatian Medical Association– initial contact has alreadly been establishedand there is good will to continue with this project in the future.
CONCLUSION
Guidelines call attention to increasing awareness
to CKD and implementation of a new guideline
for medical laboratories (Turkey), clinical services,and preventive interventions (France, Norway).
As seen in Croatian example, services that were
not previously offered to patients may be available as a response to newly released guidelines.

Page 299

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

Explicit guidelines improve clinical practice;
however clinical guidelines will achieve the fullpotential only if appropriate strategies are selected at each stage of the implementation (28).
Examples shown in this review may provide valuable experience for countries that are in processof creating their national CKD guidelines.
REFERENCES
1. Woolf SH, Grol R, Hutchinson A, Eccles M, Grimshaw
J. Potential benefits, limitations, and harms of clinicalguidelines. BMJ 1999;318:527-530
2. Kidney Disease: Improving Global Outcomes (KDIGO)CKD Work Group. KDIGO 2012 clinical practice guideline
for the evaluation and management of chronic kidneydisease. Kidney Inter Suppl 2013;3:1-150
3. National Institute for Health and Care Excelence (NICE):
Chronic kidney disease in adults: assessment and management. Available at: https://www.nice.org.uk/guidance/cg182 Accessed September 26th 2017
4. Johnson DW, Jones GRD, Mathew TH, Ludlow MJ,
Doogue MP, Jose MD, Langham RG, Lawton PD, McTaggart SJ, Peake MJ, Polkinghorne K, Usherwood T. Australasian Creatinine Consensus Working Group. Chronic
kidney disease and automatic reporting of estimated glomerular filtration rate - new developments and revised
recommendations. Med J Aust 2012;197(4):222-223
5. Johnson DW, Jones GRD, Mathew TH, Ludlow MJ,
Chadban SJ, Usherwood T, Polkinghorne K, Colagiuri S,
Jerums G, MacIsaac R, Martin H. Australasian Proteinuria
Consensus Working Group. Chronic kidney disease andmeasurement of albuminuria or proteinuria: a position
statement. Med J Aust 2012;197(4):224-225
6. National Kidney Foundation. K/DOQI clinical practiceguidelines for chronic kidney disease: evaluation, classification, and stratification. Kidney Disease Outcome Quality Initiative. Am J Kidney Dis 2002;39:S1–246
7. Séronie-Vivien S, Galteau MM, Carlier MC, Hadj-Aissa
A, Hanser AM, Hym B, Marchal A, Michotey O, PouteilNoble C, Sternberg M, Perret-Liaudet A; SFBC “Créatinine” de la Section “Assurance qualité”. [Improving theinterlaboratory variation for creatinine serum assay]. Ann
Biol Clin (Paris). 2004 Mar-Apr;62(2):165-75. In French.
8. Séronie-Vivien S, Galteau MM, Carlier MC, Hadj-Aissa
A, Hanser AM, Hym B, Marchal A, Michotey O, PouteilNoble C, Sternberg M, Perret-Liaudet A; Creatinine Working Group of the Société Française de Biologie Clinique.
Impact of standardized calibration on the inter-assay
variation of 14 automated assays for the measurementof creatinine in human serum. Clin Chem Lab Med.
2005;43(11):1227-33
9. Myers GL, Miller WG, Coresh J, Fleming J, Greenberg N,
Greene T et al. Recommendations for improving serum
creatinine measurement: a report from the laboratory
working group of the national kidney disease education
program. Clin Chem 2006; 52:5-18
10. Piéroni L, Delanaye P, Boutten A, Bargnoux AS, Rozet
E, Delatour V, Carlier MC, Hanser AM, Cavalier E, Froissart
M, Cristol JP; Société Française de Biologie Clinique. A
multicentric evaluation of IDMS-traceable creatinine enzymatic assays. Clin Chim Acta. 2011;412(23-24):2070-5
11. Boutten A, Bargnoux AS, Carlier MC, Delanaye P, Rozet
E, Delatour V, Cavalier E, Hanser AM, Froissart M, Cristol
JP, Piéroni L; Société Française de Biologie Clinique. Enzymatic but not compensated Jaffe methods reach the desirable specifications of NKDEP at normal levels of creatinine. Results of the French multicentric evaluation. Clin
Chim Acta. 2013;419:132-5
12. Agence National d’Accréditation et d’Evaluation en
Santé (ANAES). Diagnostic de l’insuffisance rénale chezl’adulte: recommandation pour la pratique clinique 2002.
In French.
13. Levey AS, Eckardt KU, Tsukamoto Y, Levin A, Coresh
J, Rossert J et al. Definition and classification of chronic
kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int
2005;67:2089-100
14. Groupe de travail de la Société de Néphrologie. [Evaluation of glomerular filtration rate and proteinuria forthe diagnosis of chronic kidney disease]. Nephrol Ther.
2009;5(4):302-5. In French.
15. Agence Française de Sécurité Sanitaire des Produitsde Santé (AFSSAPS). Rapport du Contrôle de marché desdispositifs médicaux de diagnostic in vitro de dosage dela créatinine, état des lieux, notices et tracabilité 2010.
In French.
16. Bargnoux AS, Boutten A, Cambillau M, Carlier MC,
Cavalier E, Cristol JP, Hanser AM, Piéroni L, Delanaye P,
Frimat L, Froissart M; Working group of the French Society of Clinical Chemistry and the French Society of Nephrology. [Recommendations for the selection and alignment techniques for the determination of creatinine].
Ann Biol Clin (Paris). 2011;69(1):9-16. In French.
17. Haute Autorité de Santé (HAS). Évaluation du débit defiltration glomérulaire, et du dosage de la créatininémie
dans le diagnostic de la maladie rénale chronique chezl’adulte. 2011. In French.
18. Kuster N, Cristol JP, Cavalier E, Bargnoux AS, Halimi
JM, Froissart M, Piéroni L, Delanaye P; Société Française
de Biologie Clinique (SFBC). Enzymatic creatinine assays

Page 300

eJIFCC2017Vol28No4pp289-301

V. Radišić Biljak, K. Moberg Aakre, D. Yucel, A.S. Bargnoux, J.P. Cristol, L. Piéroni
A pathway to national guidelines for laboratory diagnostics of chronic kidney disease

allow estimation of glomerular filtration rate in stages 1
and 2 chronic kidney disease using CKD-EPI equation. Clin
Chim Acta. 2014;428:89-95
19. Stevens LA, Nolin TD, Richardson MM, Feldman HI,
Lewis JB, Rodby R, Townsend R, Okparavero A, Zhang YL,
Schmid CH, Levey AS; Chronic Kidney Disease Epidemiology Collaboration. Comparison of drug dosing recommendations based on measured GFR and kidney function
estimating equations. Am J Kidney Dis. 2009;54(1):33-42
20. Bargnoux AS, Piéroni L, Cristol JP, Kuster N, Delanaye
P, Carlier MC, Fellahi S, Boutten A, Lombard C, GonzálezAntuña A, Delatour V, Cavalier E; Société Française de Biologie Clinique (SFBC). Multicenter Evaluation of Cystatin
C Measurement after Assay Standardization. Clin Chem.
2017;63(4):833-841
21. Abusoglu S, Aydin I, Bakar F, Bekdemir T, Gulbahar O,
et al. A short guideline on chronic kidney disease for medical laboratory practice. Turk J Biochem 2016;41:291-301
22. NSMBs anbefaling for kreatininbasert estimering
av GFR. Available at: http://legeforeningen.no/Fagmed/
Norsk-forening-for-medisinsk-biokjemi/norsk-selskapfor-medisinsk-biokjemi/Nyheter/NSMBs-anbefaling-forkreatininbasert-estimering-av-GFR/ Accessed September
26th 201723. Nasjonal faglig retningslinje for diabetes. Available
at: https://helsedirektoratet.no/retningslinjer/diabetes
Accessed September 26th 201724. Aakre KM, Thue G, Subramaniam-Haavik S, Cooper J,
Bukve T, Morris HA, Müller M, Lovrencic MV, Plum I, Kallion K, Aab A, Kutt M, Gillery P, Schneider N, Horvath AR,
Onody R, Oosterhuis W, Ricos C, Perich C, Nordin G, Sandberg S. Diagnosing microalbuminuria and consequences
for the drug treatment of patients with type 2 diabetes:
an international survey in primary care. Diabetes Res Clin
Pract 2010;89:103-9
25. Aakre KM, Thue G, Subramaniam-Haavik S, Bukve T,
Morris H, Müller M, Lovrencic MV, Plum I, Kallion K, AabA, Kutt M, Gillery P, Schneider N, Horvath AR, Onody R,
Oosterhuis W, Ricos C, Perich C, Nordin G, Sandberg S.
Postanalytical External Quality Assessment of Urine Albumin in Primary Health Care: An International Survey. Clin
Chem 2008;54:1630-6
26. Radišić Biljak V, Honović L, Matica J, Knežević B, ŠimićVojak S. Laboratory diagnostics of chronic kidney disease
in Croatia: state of the art. Biochem Med 2015, 25:73-83
27. Radišić Biljak V, Honović L, Matica J, Krešić B, Šimić
Vojak S. The role of laboratory testing in detection andclassification of chronic kidney disease: national recommendations. Biochem Med 2017;27(1):153-76
28. Grimshaw JM, Russell IT. Effect of clinical guidelines
on medical practice: a systematic review of rigorous evaluations. The Lancet 1993;342:1317-1322

Page 301

eJIFCC2017Vol28No4pp289-301

TF-CKD special issue: The laboratory diagnostics of chronic kidney diseases

A summary of worldwide national activities
in Chronic Kidney Disease (CKD) testing

Roberto Ruiz-Arenas1, Rosa Sierra-Amor1, David Seccombe2,
Stella Raymondo3, Maria Stella Graziani4, Mauro Panteghini4,
Tewogbade A. Adedeji5, Shanthi Naidu Kamatham6, Vanja Radišić Biljak7
Alianza Mexicana para Prevenir las Enfermedades Crónicas, A.C. (AMPEC)CEQAL Inc., Vancouver, British Columbia, Canada
3
Asociación Bioquímica Uruguaya (ABU)
4
Italian Society of Clinical Chemistry and Clinical Molecular Biology (SIBioC)
5
Association of Clinical Chemists of Nigeria (ACCN)
6
Association of Medical Biochemists of India (AMBI)
7
Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM)
1
2

ARTICLE INFOABSTRACT

Corresponding author:
Vanja Radišić Biljak
Department of Medical Biochemistry
and Laboratory Medicine
Merkur University Hospital
Zajčeva 19
10000-Zagreb
Croatia
Phone: +385917967867
E-mail: vanja.radisic@gmail.com

Chronic kidney disease (CKD) is a major public healthissue worldwide and is associated with adverse healthoutcomes, especially in low- and middle-income
countries. In a cash limited healthcare system, guidelines that improve the efficiency of health care free up
resources needed for other healthcare services. This
short review presents some examples from national
acitivities in CKD testing, including countries throughout the globe: Mexico in North America, Uruguay inSouth America, Italy in Europe, Nigeria in Africa andIndia in Asia. Considering the fact that treatment of
CKD is cost-effective and improves outcomes, this observation argue in favor of including CKD in nationalguidelines and noncommunicable chronic disease(NCD) programs. This diverse example of national activities fullfil the very first step in achieving this goal.

Key words:
chronic kidney disease (CKD),
national activities, laboratory medicine,
KDIGO

Page 302

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

INTRODUCTION
Chronic kidney disease (CKD) is a major public health issue worldwide and is associatedwith adverse health outcomes, especially in
low- and middle-income countries (1-4).
Considering the fact that CKD is associatedwith high health-care costs, CKD is readily
identifiable, treatment of CKD is cost-effective
and improves outcomes (5), many countries
are developing or refining national strategies
for CKD (6). Howevever, despite two decadesof widely accepted CKD clinical practice guidelines, such as the Kidney Disease Outcomes
Quality Initiative (KDOQI) and recently The
Kidney Disease: Improving Global Outcomes(KDIGO) 2012 Clinical Practice Guideline for
the Evaluation and Management of Chronic
Kidney Disease (7) and continuing medical
education for physicians, recent reports frommany developed countries indicate that CKD
care remains suboptimal (8).
In a cash limited healthcare system, guidelinesthat improve the efficiency of health care free
up recources needed for other healthcare services (9). Perhaps the intial step to create a
guideline is to explore the current status of CKD
testing in a national enviroment. Therefore, theaim of this article was to present a summaryof worldwide national activities in CKD testingin various countries without already developed
national CKD guidelines.

in the Mexican population will provide opportunities for slowing and in some cases preventing
the natural progression of this disease to endstage and the need for dialysis.
The early recognition of CKD may be achieved
through an estimation of glomerular filtration
rate (eGFR) – a well-recognized index of renal
function. The calculation of eGFR for a given
patient is based upon their age, gender andserum Creatinine (Cr) test result. The accurate
measurement of creatinine is therefore an essential requirement for the accurate assessment of renal function. Significant differences
exist between clinical laboratories (CL) for themeasurement of creatinine. Standardizing themeasurement and reporting of this analyte by
CLs is an essential pre-requisite for the accurate
diagnosis and management of CKD. In this shortreport, we examined the accuracy of creatinine
measurements from some CLs in Mexico.
CLs nationwide were invited to participate, anda questionnaire was distributed. The CLs thatvoluntarily accepted to participate received 3
sets of human serum samples (3 samples/set)with differing concentrations of creatinine provided by CEQAL. The creatinine reference values in these samples had been assigned by the
ID/MS reference method for the measurementof creatinine (12).

Creatinine standardization
Mexican Pilot Study
to determine accuracy and trueness

Each CLs recorded the measurement of Cr ineach set of samples (one sample set analyzed oneach of three separate days), the methodology,
and the manufacturer´s information were also
provided. Intra and inter-run coefficient of variation (CV) as well as total error percentage (TE %)were calculated and used for comparison.

In Mexico, a national end-stage renal disease
registry has not been developed. However, datafrom single state registries (10) and from the
US Renal Data System (11) indicate that some
Mexican states have an unusually high incidence
and prevalence of CKD. Early recognition of CKD

A total of 17 CLs, 5 from public and 12 from private
sector participated voluntarily. The mean CV% was
4.56 (1 to 18.04 %) and mean TE% was 16.6 (3.9
to 47.9%). When grouped, public CLs had a mean
CV% of 3.93 and a mean TE% of 19.0, and private
CLs of 4.82% and 15.7%, respectively.

NORTH AMERICA

Page 303

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Figure 1

Precision and accuracy of 17 Mexican clinical laboratories
measuring creatinine using different methods and instruments

When compared individually to international
standards, 4 CLs had a “minimum acceptableperformance” (≤11.4 TE %), 3 a “desirable performance” (≤7.6 TE %), and 10 an “undesirable
performance” (between 13.41 and 47.93 TE %).
None had an “optimum performance” (≤3.8
TE %) (see Figure 1).
Most of the participating laboratories wereoperating Jaffe methods (see Figure 2). The bimodal nature of the Jaffe results may reflect differences between Jaffe and Jaffe compensatedmethods. This level of detail was not captured
in this study. Some of the observed between
method differences may have been due in partto the non-specificity of the Jaffe method. The
between method precision data show that better precision can be achieved with a closed
system (dry chemistry) as opposed to an open
system (Jaffe). These data serve to highlight themethodological differences (in addition to calibration issues) that can exist between laboratories and the significant impact that they canhave on reported test results.
The performance data from this accuracy based
assessment pilot study serve to highlight the
inaccuracy and variability of creatinine test results from a small sampling of Mexican laboratories. A desirable and/or minimum level of
performance was achieved by 41% of the laboratories whereas an unacceptable level of performance was recorded for 59% of the participating laboratories. The methods used werealkaline picrate (n=1), colorimetric (n=1), dry
chemistry (n=4) and Jaffe (n=11). These data
demonstrate that there is an urgent need for anationwide creatinine standardization programin Mexico that is directed towards improving
the accuracy and reporting of creatinine test

Page 304

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Figure 2

Precision and trueness data
sorted according to creatinine method used

results throughout the country. Standardized
measurements of creatinine in Mexico will identify opportunities for preventing CKD while atthe same time making sure that “most at risk”
Mexicans are accurately identified, diagnosed
and managed appropriately.
Recommendations for improving serum Cr measurement (13) as well as IFCC/WASPaLM TF-CKD
survey results guided us to initiate this pilot study.

medical services, allowing them to be available
with equity for the entire population.
Our country has a Renal Health Program (PSR),
dependent on the FNR and the Ministry of
Public Health (MSP), executed by the Honorary
Committee on Renal Health (CHSR). The main
purpose of PSR is to prevent CKD and improve
the quality of life of the patients who carry it,
seeking their integration into the SIS.

Interaction between laboratories and clinicians has taken place in a unique way throughthe External Evaluation Program conducted by
the Committee for Standardization and Quality
Control (CECC).

In view of the enormous economic losses thecountry was suffering, the CHSR contacted the
CECC, looking for our help , with the hypothesisthat the diagnosis of CKD could be improved as
the quality of the creatinine result improved.
Creatinine is the main tool for diagnosis of CKD
throughout the glomerular filtration rate calculation. It was through the CECC that they could
integrate all the laboratories in the country to
standardize creatinine.

The Integrated Health System (SIS) in Uruguay has
a National Fund of Resources (FNR), whose mission is to provide financing for highly specialized

This Project was declared of National Interest by
the Government, and CECC obtained a grant fromthe National Innovation Research Agency (ANII).

SOUTH AMERICA
Standardization of creatinine in Uruguay
CKD in Uruguay has a prevalence of 7%.

Page 305

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Given the need to have a Reference Laboratory
to obtain target values of creatinine withtraceability to reference methods and advice
for the design of standardization procedures,
the CECC incorporated into the national project the Reference Laboratory of the Argentine
Biochemical Foundation (LARESBIC), which
was formalized by an agreement that was
signed in 2010.
The standardization was done using a reference
method calibrated with certified materials by
NIST and DGKL, with traceable values to theprimary method of Isotopic Dilution and Mass
Spectrometry.
The preparation, packaging and distribution in
dry ice of the material used (native serum), wasin charge of the CECC. An aliquot of vials wassent to LARESBIC Argentina under the same
conditions, for the assignment of reference values (creatininase/sarcosine oxidase).
Four surveys were madeLevel I: 98 Laboratories. 3 creatinine levels/3
samples/3days Date: March 2008
Level II: 73 Laboratories. 3 creatinine levels/3
samples/3days Date: November 2008
Level III: 101 Laboratories. 5 creatinine levels/2
samples/3days Date: September 2009

the laboratories can incorporate the corrections
that arise from the standardization, for which it
will be necessary to lower their intra-laboratorial CVs through the Internal and External Control
and using homogenous systems with traceability to the primary reference method.
EUROPESTATUS OF LABORATORY TESTS FORCHRONIC KIDNEY DISEASE IN ITALY
1. Educational activities
by the Italian Society of Clinical Biochemistry
and Laboratory Medicine (SIBioC)
After the issuing of the 2012 Kidney Disease
Improving Global Outcomes Initiative (KDIGO)
guideline (7), which included specific recommendations for the clinical laboratory, SIBioC
has worked to diffuse the guideline content
among the professional community in Italy,
with the aim to help laboratories to implement
the recommendations in their daily practice.
This was obtained through the publication of
a number of papers in Biochimica Clinica, the
SIBioC official journal, and the efforts of SIBioC
Working Groups dealing with renal disease, thatorganized training courses using both traditional meeting and e-learning approaches.

Level IV: Laboratories. 5 creatinine levels/2
samples/3days Date: March 2011

2. Cooperation between SIBioC
and clinical organisations

Imprecision, Systematic Error, Total Error andRegression Parameters were obtained.

In 2009, a joint SIBioC-Italian Society on
Nephrology recommendation was released (14)and, more recently, the Italian Minister of Healthhas issued a national guideline on identificationand prevention of CKD in adults, with the proactive contribution of laboratory experts (15).

Eleven years after the start of these activities,
and with four surveys conducted and continuous monitoring through our EQAs, the evolution of the analyte is considered to have beenfavorable in statistically significant terms.
The analytical quality parameters have improved since the first distribution, but in thelast two the situation has been maintained. This
positive impact will deepen in the measure that

3. Laboratory tests
a. Serum creatinine
Two national evaluations have been recently published on the status of creatinine measurement

Page 306

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testingin Italy, both using commutable control materials
with target values assigned by a higher-order reference procedure (16,17). Data seem to indicatethat the standardization efforts are still having effects lower than expected. In 2014, only 41% oflaboratories showed optimal performance [i.e., a
Table 1

total error (TE)<4.5%], while 16.6% were unable
to reach even the minimum quality goal (i.e.,
TE<13.3%). It should be noted that enzymatic
methods, although strongly promoted by SIBioC(18,19), are used in a minority of laboratories(∼25%).

Main results of the 2015 national survey on the urine albuminmeasurement, compared with the results obtainedin a similar survey performed in 2007 (Italy)

2007

2015

24-h collection

43%

16%

First morning void

9%

59%

Second morning void-

6%

Random30%

19%Type of sample

Partecipation to an EQAS

Yes

28%

44%

No

72%

56%

mg/mmol creatinine or mg/g creatinine

15%

52%

µg/min9%5%mg/24 h

33%

9%

mg/L

29%

26%

Measurement unit

Page 307

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testingb. eGFRAFRICA

Data on the use of equations to derive the GFR
are still sparse and heterogeneous (17).

NATIONAL ACTIVITIES IN CKD TESTING:
THE NIGERIAN CURRENT SITUATION

In 2013, employed equations were MDRD(69%), CKD-EPI (15%), Cockcroft-Gault (7%) and
Schwartz (1%).

Prevalence

More importantly, ∼25% of laboratories did notoffer any eGFR option.
c. Urine albumin
In 2007 and 2015, SIBioC did two surveys tocheck the use of this parameter. As reported in
the Table 1, results were encouraging in showing the improvement of adherence to the KDIGO
recommendations.
EQAS show some variability among different
commercial measuring systems, even if the whole
performance is not so bad.
In 2014, 3.6% of laboratories were unable to fulfil
the minimum quality level, while 88.6% showed
good performance (defined as a TE<11%) (17).
4. Final remarks
SIBioC is dedicating many efforts to the standardisation of laboratory procedures for CKD
diagnostics, through the action of its Working
Groups and the publication of recommendations.
The situation is, however, far from optimal withlarge room for improvement, as indicated by
EQAS and surveys results. In particular, somegoals, indicated by Graziani et al. (17) with thecorresponding indicators, have been identified
for being pursued in a relatively short time.
It is also worthy to note that many of these objectives (availability of eGFR and use of CKD-EPI
equation, report of albuminuria in the recommended terminology and unit, use of the recommended sample for urine albumin) can beachieved at no additional costs under the directcontrol of the clinical laboratory.

Various studies with different results possibly
because mild-moderate cases excluded; metaanalysis for Saharan Africa gives prevalence as
13.9% (20). Prevalence of CKD in a Nigerian family practice population: 250 consecutive, newly
registered patients during 2005-2006; 45% had
increased urine albumin on first testing, persistent in 12.4%; 20% had low e-GFR on first testing, persistent in 10% (21).
Causes of CKD in Nigeria
Hypertension 30%; chronic glomerulonephritis
28%; diabetes 3%; obstructive uropathy 5%;others 3.9%; unknown 30% (Arogundade et al2005); 37% CKD in a population of diabetic subjects (22); 38% CKD in HIV positive population in
outpatient clinic (23); 24% CKD (e-GFR MDRD)
in a HIV/AIDS population in tertiary referral unit
(24,25).
The enormity of CKD in Nigeria
The Situation in a Teaching Hospital in SouthEast Nigeria showed CKD accounts for 8-10% of
hospital admissions; death from end-stage renal
disease constituted 22% of medical deaths (26).
Current/existing practices
Since information on activities in CKD testing at
national level on how nephrologists/laboratories investigate patients can mainly be provided by national surveys, most of which is questionnaire-based with voluntary participation.
A questionnaire was sent to the membershipof Association of Clinical Chemists of Nigeria(ACCN) in different hospitals across Nigeria(representing a complex mixture of private andpublic clinical laboratories) to seek information

Page 308

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testingon the current practices. The information obtained was then collated, analysed, and summarized for this submission.
Not surprisingly, there is evidence of differences in practice across laboratories in different
parts of Nigeria. These differences are seen in
all aspects of CKD testing including methods
being used, commercial assay kits, commercial standards, commercial controls (SRMs or
CRMs), and even some slight variations exist
in creatinine reference interval values acrosslaboratories in different states.

Creatinine assay
Most institutions use commercial enzymatic (kinetic) assay kits.
Use of automation in creatinine assay
This is also widely distributed across thecountry, though less widely than the Roche
Reflotron Point-of-Care machine. Automation
systems in current use in different parts of thecountry include: ARCHITECT C4000, ABBOTTLABORATORIES, USA, Miura 200, ISE, Italy and
TC-Matrix, Teco Diagnostics, USA etc.

Chronic Kidney Disease testingin Nigeria (Summarized in Table 2)

Use of Point-of-Care Testing (POCT)
in creatinine assay

1. Serum creatinine: in routine use, mostlycommercial enzymatic and kinetic assay kits

Roche Reflotron machine, a point of care clinical chemistry equipment that measures creatinine with the result being made available within
5 minutes is widely distributed and used acrossthe country (available in 196 health institutions
across the country). Also, point of care testing
equipment for measuring albumin to creatinine
ratio is being used in some centers.

2. eGFR (Cockcroft-Gault): still being used bysome (though very few), based on somevalidation studies done in some populationgroups3. eGFR (creatinine-based, MDRD):
widely in routine use
4. eGFR (creatinine-based CKD-EPI):
widely in routine use

External quality assurance of creatinine(and other analytes) assays

5. Albumin/creatinine ratio: in routine use
7. Urinalysis (dipstick): mainly in routine
screening/medical tests

A few public/governmental institutions partake in EQA (e.g UKNEQAS, with satisfactory
results); several of the modern private institutions/laboratories are partakers with satisfactory results.

8. Conventional 24-hour urine profile:still being used in some institutions

Routine reporting of eGFR/ standardisation

6. Urine protein: in routine use

9. eGFR (Cystatin C based CKD-EPI):
currently for research use only, not yet
routine10. eGFR (Creatinine with cystatin C-based
CKD-EPI): currently for research use only,
not yet routine
11. Kidney injury molecule 1 (KIM 1): currently
for research use only, not yet routine

In Nigeria, some clinical chemists and clinicians
have worked on use of eGFR to determine/evaluate renal function, both in the normal or apparently healthy population and in various diseases - “the studies cited under the introduction
above”. A cut-off value of ≥60ml/min/1.73m2
for the apparently healthy (non-CKD subjects)
population using the 4-variable MDRD equation has been established in some regions/Page 309

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Table 2

Summary of CKD testing in Nigeria
Currently
for research
onlyRemarksNO

IDMS Traceable;
Commercial enzymatic
assay kits; Commercial
standards and controls(SRMs and CRMs),
e.g. Randox; EQA
results satisfactory ininstitutions that engage

YES

NO

Still being used by some(very few) based on
validation studies donein some populationgroupseGFR(creatinine-based,
MDRD)

Automation,
Enzymatic, Kinetic
Spectrophotometry,
POCT (Roche
Reflotron)

YES

NO

Mostly 4-variable MDRD;IDMS Traceable

eGFR(creatinine-based
CKD-EPI)

Automation,
Enzymatic, Kinetic
Spectrophotometry,
POCT (Roche
Reflotron)

YES

NO

Mostly IDMS Traceable

YES

Mostly commercial
ELISA kits with controls
and calibrators

YES

Mostly commercial
ELISA kits with controls
and calibrators

YES

Mostly commercial
ELISA kits with controls
and calibrators

CKD
testing approach

Current assay
methods/practiceSerum creatinine

Automation,
Enzymatic, Kinetic
Spectrophotometry,
POCT (Roche
Reflotron)*Jaffe End point still
done in some healthinstitutions

eGFR(creatinine-based,
Cockcroft-Gault)

Automation,
Enzymatic, Kinetic
Spectrophotometry,
POCT (Roche
Reflotron)eGFR(Cystatin C based
CKD-EPIeGFR(Creatinine withcystatin C-based
CKD-EPI)
Kidney injury
molecule 1(KIM 1)ELISA assays
Creatinine(Automation,
Enzymatic, Kinetic
spectrophotometry);
Cystatin C: ELISA
ELISACurrently
in routine
use

YES

NO

NO

NO

Page 310

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Urine Albumin/creatinine ratio

Albumin:
Automation, ELISA,POCT equipment

YES

NO

Mostly commercial
ELISA kits with controls
and calibrators

24 hourUrine protein

Spectrophotometric

YES

NO

In cases that require 24
hour quantification (e. gNephrotic syndrome)NO

Mostlyat Point-of-Careand in routine
screening/medical tests

Urinalysis (dipstick
with/without
microalbuminuria
template)

StripsYES

zones of the country. Available data from different studies done in Nigerian population groups
is consistent with the cut-off value of <60mL/
min/1.73m2 for CKD, using the 4-varaible MDRD
equation. Sanusi et al. in Ile-Ife, South- western
Nigeria (27, 28), Adebisi in Ilorin, North-central
Nigeria (29), and Agaba et al. in Jos, Northeastern Nigeria (30) have proved that the MDRD
equation is reliable alternative to measurementof endogenous creatinine clearance (Crcl) in theestimation of GFR in Nigerians. Currently, only a
few clinical chemistry laboratories report eGFR.
However, in many/most institutions, the nephrologists commonly use the creatinine result
from the laboratories for eGFR (mostly MDRD
and CKD-EPIcr equations).
CKD task force in Nigeria

to IDMS, even though there is no Laboratory/
Nephrology Working Group put in place yet.
Conclusion and way forward
Need for ACCN to have a CKD Task Force whowill work towards standardization of assays ofcreatinine and other markers of CKD across thecountry, and also collaborate with the nephrologists (Nigerian Association of Nephrology) in
the following areas: agreement to recommend
the routine reporting of e-GFR; agreement on
clinical practice guidelines; joint initiative topromote the benefits of testing for CKD; preparation of educational support materials for laboratory personnel, family doctors and patients,
renal physicians (31).

ACCN will constitute a task force.

ASIA

Collaboration with nephrologists

SUMMARY OF NATIONAL ACTIVITIESIN CKD TESTING IN INDIAThe Nigerian Association of Nephrology (NAN)
developed Guidelines for the Detection andManagement of Chronic Kidney Disease (CKD),
2nd May, 2011, though without involving theclinical chemists.
However, there is a level of collaboration being
practised by nephrologists and clinical chemists in some centres/institutions in reportingeGFR, especially in the aspect of creatinine assay method, standardisation and traceability

In the large ethnicity of India, uniformity in testing is not available for CKD, and many a time notaffordable. There is no one statutory control forthe laboratory diagnosis in correlation with clinical diagnosis for various reasons at present.
The testing can be categorized into the following types of laboratories generally available inline with scope of services, Basic composite/
Medium/Advanced laboratories.

Page 311

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Awareness and protocol of testing is available,
but actual performance is seen in some teaching, corporate and centres of excellence hospitals which are not in the reach of the population
specially in outreach places and lower socio economic strata. Hence most of the understanding
and publications are from these laboratories
which may not reflect the accurate incidence.
International guidelines are followed in the best
possible way available. Most of the publications
are from the advanced laboratories.
The IDMS traceability creatinine is used in majority of the Medium and Advanced Laboratories(32). Though MDRD equation is used quite commonly, the appropriateness of the same in linewith creatinine traceability is many a time questionable. There is variation in use of the formulaof MDRD among the laboratories
In the higher centres, creatinine is reportedwith eGFR and vice versa. Protein creatinine
ratio is done in all 3 categories of laboratories
and albumin creatinine ratio in the medium
and advanced laboratories. Standardization of
the eGFR in institutes have been done initially by comparison with isotope renogram andfound to be superior to CG formula (33). The
Schwartz formula for pediatric population iswell accepted clinically, CKD-EPI children has
not yet taken off (34).
Serum Cystatin usage is minimal as a regular
tool of evaluation mainly due to cost and eGFR
using the same, is sporadic in publication.
Both medium and large laboratories have goodresults of proficiency testing. Laboratories areunder a national accreditation programme(though not yet mandatory). With different ethnicities to arrive at national reference interval,
would take time. Though study evaluations aredone in different areas of the country, these
may be considered cross-sectional due to thediversity of individuals living in different areas.

The distinct features to be addressedin the country under statutory guidance
1. Uniformity of use in creatinine assay
2. Biological reference interval of the serum
creatinine, protein creatinine ratio and albumin creatinine ratio, sex and gender wise
3. Selection of MDRD and CKD EPI equation
uniformity (35)
4. Awareness of general medical practitioners in use of the equation only with standardized IDMS creatinine assay
5. To always correlate with regional higher
centres on knowhow of assays, reference
intervals with national and internationalguidelines
6. A regional state wise registry to know theCKD burden in the country (36)
7. To work with instrument and reagent
vendors to help in this achievement on aGovernment statutory.
CONCLUSION
This short review presents some examples fromworldwide national acitivities in CKD testing.
Considering the fact that already mentioned
treatment of CKD is cost-effective and improves
outcomes, this observation argue in favor of including CKD in national guidelines and noncommunicable chronic disease (NCD) programs (5).
The described activities fullfil the very first stepin achieving this goal.
Acknowledgement
Cueto-Manzano A1, Garcia-Garcia G2, CortesSanabria L1, Martinez-Ramirez HR1, MercadoSalgado G3, Simeon A. Adebisi4, Tunji Soriyan5,
Ibrahim O. R5, Olatunbosun Olawale6, Idris

Page 312

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

Mohammed7, Chinelo Onyenekwu8, Ify D.
Nnakenyi9, Alaje A.10, Ayinbuomwan E11
Hospital de Especialidades, CMNO, IMSS,
México2
Hospital Civil de Gudalajara, SSA, México
3
Alianza Mexicana para Prevenir las
Enfermedades Crónicas, A.C. (AMPEC), México
4
Department of Chemical Pathology, Benue
State University, Makurdi, Nigeria
5
Department of Chemical Pathology, Lagos
University Teaching Hospital, Lagos, Nigeria
6
Department of Chemical Pathology, Olabisi
Onabanjo University Teaching Hospital,
Sagamu, Ogun State, Nigeria
7
Department of Chemical pathology, Aminu
Kano Teaching Tospital, Kano State, Nigeria
8
Department of Chemical Pathology, Babcock
University Teaching Hospital, Lishan-Remo,
Ogun State, Nigeria
9
Department of Chemical Pathology Universityof Nigeria Teaching Hospital, Enugun, Enugun
State Nigeria
10
Department of Chemical Pathology, Obafemi
Awolowo University/Obafemi Awolowo
University Teaching Hospital, Ile-Ife, Osun
State, Nigeria
11
Department of Chemical Pathology Universityof Benin Teaching Hospital, Benin, Edo State
Nigeria.
1

REFERENCES
1. Bello AK, Levin A, Manns BJ, Feehally J, Drueke T, Faruque L et al. Effective CKD care in European countries:
challenges and opportunities for health policy. Am J Kidney Dis 2015;65(1):15-25

4. Black C, van der Veer SN. Unlocking the Valueof Variation in CKD Prevalence. J Am Soc Nephrol
2016;27(7):1874-77)
5. Tonelli M, Agarwal S, Cass A, Garcia Garcia G, Jha V,
Naicker S, Wang H, Yang CW, O’Donoghue D. How to
advocate for the inclusion of chronic kidney disease ina national noncommunicable chronic disease program.
Kidney Int 2014;85(6):1269-74
6. Radišić Biljak V, Moberg Aakre K, Yucel D, Bargnoux A-S,
Cristol J-P, Piéroni L. A pathway to national guidelines forlaboratory diagnostics of chronic kidney disease – examples from diverse European countries. eJIFCC, in press
7. Kidney Disease: Improving Global Outcomes (KDIGO)CKD Work Group KDIGO 2012 clinical practice guideline
for the evaluation and management of chronic kidneydisease. Kidney Int Suppl 2013;3:1-150
8. Al Shamsi S, Al Dhanhani A, Sheek-Hussein MM, Bakoush O. Provision of care for chronic kidney disease by
non-nephrologists in a developing nation: a national survey. BMJ Open 2016;6(8): e010832
9. Woolf SH, Grol R, Hutchinson A, Eccles M, Grimshaw
J. Potential benefits, limitations, and harms of clinicalguidelines. BMJ 1999;318:527-530
10. Da Silvera-Martínez R, Jiménez-Gutiérrez R. Optimización de la creatinina al estimar la tasa de filtración glomerular en el laboratorio. Rev Med Inst Mex Seguro Soc
2011;49(5):481-486
11. http://www.usrds.org, Accessed 20th July 2017
12. Komenda P, Beaulieu M, Seccombe D, Levin A. Regional implementation of creatinine measurement standardization. J Am Soc Nephrol. 2008;19:164–169
13. Myers GL1, Miller WG, Coresh J, Fleming J, Greenberg
N, Greene T, Hostetter T, Levey AS, Panteghini M, Welch
M, Eckfeldt JH. Recommendations for improving serum
creatinine measurement: a report from the Laboratory
Working Group of the National Kidney Disease Education
Program. Clin Chem 2006;52(1):5-18
14. Zoccali C, Cappelletti P, Plebani M. Valutazione
di laboratorio della funzionalità renale. Biochim Clin
2009;33:144-5

2. Radhakrishnan J, Remuzzi G, Saran R, Williams DE,
Rios-Burrows N, Powe N et al. Taming the chronic kidneydisease epidemic: a global view of surveillance efforts.
Kidney Int 2014; 86(2):246-250

15. Istituto Superiore di Sanità, Sistema nazionale per leLinee Guida. Linea Guida 23. Identificazione, prevenzione
e gestione della malattia renale cronica nell’adulto. www.
snlg-iss.it

3. Couser WG, Remuzzi G, Mendis S, Tonelli M. The contribution of chronic kidney disease to the global burden of major noncommunicable diseases. Kidney Int
2011;80(12):1258-1270

16. Carobene A, Ceriotti F, Infusino I, Frusciante E, Panteghini M. Evaluation of the impact of standardization
process on the quality of serum creatinine determinationin Italian laboratories. Clin Clin Acta 2014;427:100-6

Page 313

eJIFCC2017Vol28No4pp302-314

R. Ruiz-Arenas et al.
A summary of worldwide national activities in Chronic Kidney Disease (CKD) testing

17. Graziani MS, Secchiero S, Terreni A, Caldini A, Panteghini M. La diagnostica di laboratorio della malattia
renale cronica in Italia: armonizzare è d’obbligo. Biochim
Clin 2015;39:617-26
18. Panteghini M. Enzymatic assays for creatinine: time
for action. Biochim Clin 2008;32:203-8
19. Ceriotti F. Determinazione della creatinina: per i laboratori è tempo di agire. Biochim Clin 2010;34:9-10
20. Stanifer JW, Jing B, Tolan S, Helmke N, Mukerjee R,
Naicker S, Patel U. The epidemiology of chronic kidneydisease in sub-Saharan Africa: a systematic review andmeta-analysis. Lancet Glob Health. 2014;2(3):e174-81
21. Afolabi MO, Abioye-Kuteyi EA, Arogundade FA, Bello
IS. Prevalence of CKD in a Nigerian family practice population. SA Fam Pract 2009; 51:131-137
22. Adebisi SA. Routine reporting of estimated glomerular filtration rate (eGFR) in African Laboratories and theneed for its increased utilisation in Clinical Practice. The
Nigerian Postgraduate Medical Journal. 2013;20(1):57-62
23. Enem CP, Arogundade F, Sanusi A, et al. Renal disease
in HIV-seropositive patients in Nigeria: an assessment ofprevalence, clinical features and risk factors. Nephrol Dial
Transplant. 2008;23(2):741–746
24. Adedeji TA, Adebisi SA, Akande AA, Adedeji NO, Ajose
AO, Idowu AA, Fawale MB, Olanrewaju TO, Okunola O,
Akinsola A. Sustained Improvement in Glomerular Filtration Rate after Four Weeks on Highly Active Antiretroviral
Therapy. Journal of Therapy and Management in HIV Infection. 2014;2:50-57
25. Adedeji TA, Adedeji NO, Adebisi SA, Idowu AA, Fawale
MB, Jimoh KA. Prevalence and Pattern of Chronic Kidney
Disease in Antiretroviral-Naive Patients with HIV/AIDS.
Journal of the International Association of Providers of
AIDS Care. 2015;14(5):434-440
26. Ifeoma I. Ulasi and Chinwuba K. Ijoma. The Enormityof Chronic Kidney Disease in Nigeria: The Situation in a

Teaching Hospital in South-East Nigeria. J Trop Med 2010;
Article ID 501957, 6 pages
27. Sanusi AA, Akinsola A, Ajayi AA. Creatinine clearance
estimation from serum creatinine values: evaluation andcomparison of five prediction formulae in Nigerian patients. Afr J Med Med Sci 2000; 29:7–11
28. Sanusi AA, Arogundade FA, Akintomide AO, Akinsola
A. Utility of predicted creatinine clearance using MDRD
formula compared with other predictive formulas in Nigerian patients. Saudi J Kidney Dis Transpl. 2009;20(1):86-90
29. Adebisi SA, Adekunle BA and Etu AK. Creatinine
Clearance: alternative approach to traditional 24- hoururine collection in normal individuals. Afr J Med Med Sci
2001;30:27-30
30. Agaba EI, Wigwe CM, Agaba PA, Tzamaloukas AH. Performance of the Cockcroft-Gault and MDRD equationsin adult Nigerians with chronic kidney disease. Int Urol
Nephrol 2009; 41: 635–642
31. Graham Beastall. Chronic Kidney Disease: Steps to improve testing for CKD. Sixth Scientific Conference of the
Association of Clinical Chemists of Nigeria (ACCN), October 12-14, 2016.
32. Bhowmik D, Agrawai A, Panda S. Assessing the prevalence of chronic kidney disease in the community: Estimating glomerular filtration rate is the Achilles heel. Indian J Nephrol 2014;24(6):411-12
33. Chakravarthi R, Hussaini S, Makesh Prasad K, Naidu
S, Harikrishana, Shekkar, Laxmi. Estimation of GFR in helathy Indian population. Indian J Nephrol 2007;17:105
34. Sethi SK. Estimating accurate glomerular filtration
rate in children. Indian Pediatrics 2014;51:263-264
35. Mulay AV, Gokhale SM. Comparison of serum creatinine-based estimating equations with gates protocol for
predicting glomerular filtration rate in Indian population.
Indian J Nephrol 2017;27(2):124-128
36. Rao M, Pereira BJG. Chronic kidney disease in India –a hidden epidemic. Indian J Med Res 2007;126:6-9

Page 314

eJIFCC2017Vol28No4pp302-314

Evaluation of the correlation coefficientof polyethylene glycol treated and direct prolactin
results and comparability with different assay
system results
Shyamali Pal

R B Diagnostic Private Limited

INFO

ABSTRACT

Corresponding author:
Shyamali Pal
Technical Director & Consultant Biochemist
R B Diagnostic Private Limited
P- 857, Block-A, Lake Town
Kolkata-700089
India
Phone: +91-9874415344
Email: shy23_pal@yahoo.co.in

The presence of Macro prolactin is a significant causeof elevated prolactin resulting in misdiagnosis in all
automated systems. Poly ethylene glycol (PEG) pretreatment is the preventive process but such processincludes the probability of loss of a fraction of bioactive prolactin.

Key words:
macro prolactin, bioactive prolactin,
correlation coefficient, regression coefficient

Surprisingly, PEG treated EQAS & IQAS samples in
Cobas e 411 are found out to be correlating with direct results of at least 3 immunoassay systems andtreated and untreated Cobas e 411 results are comparable by a correlation coefficient. Comparison of
EQAS, IQAS and patient samples were done to findout the trueness of such correlation factor. Studywith patient’s results have established the correlation coefficient is valid for very small concentrationof prolactin also.
Materials and methods
EQAS, IQAS and 150 patient samples were treatedwith PEG and prolactin results of treated and untreated samples obtained from Roche Cobas e 411.
25 patient’s results (treated) were compared withdirect results in Advia Centaur, Architect I & Access2
systems.
Page 315

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

Statistical calculations
Correlation coefficient was obtained from trendline of the treated and untreated results. Twotailed p-value obtained from regression coeffi­
cient(r) and sample size.
Results and discussion
The correlation coefficient is in the range(0.761-0.771). Reverse correlation range is(1.289-1.301). r value of two sets of calculated
results were 0.995. Two tailed p- value is zero
approving dismissal of null hypothesis.
Conclusion•

The z-score of EQAS does not always assure
authenticity of results•

PEG precipitation is correlated by the factor
0.761 even in very small concentrations

non reactive immunoglobulin G molecule witha molecular mass of approximately 150-170kDa
causing a prolonged clearance rate. Though
M-PRL is non reactive but it interferes with prolactin immunoassay and causes false elevationof prolactin (1, 2, 3).
The probable reasons for elevation may be:•

The assay antibodies are probably having
affinity to different epitopes on PRL withwhich they react. The elevation of result isdependent on the availability of such epitopes on the M-PRL complex (4)•The coupling of same pair of antibodies todifferent solid phase and signal generating
system (5, 6)•

Incubation time has also been shown to be
directly related to the reactivity with M-PRL(6). It was observed that Roche Elecsys
System showed maximum elevated results(5, 7)

Abbreviations
GFC: gel filtration chromatography
PEG: polyethylene glycol
EQAS: external quality assurance system
M-PRL: macro prolactin
PRL: prolactin
ECLIA: electro-chemiluminescence
immunoassay
CLIA: clinical laboratory improvement
amendments
IQAS: internal quality assurance system
r: regression coefficient

INTRODUCTION
The presence of Macro prolactin (M-PRL) is aknown cause of misdiagnosis, unnecessary investigation and inappropriate treatment. M-PRL
in human blood consists of monomeric bioactive
prolactin (PRL) of molecular mass 23kDa and a

A study was done to examine the frequency of
Macroprolactinemia in clinical practice and theability of immunoassay systems to distinguishbetween M-PRL and PRL using 300 hyperprolactinemic serum samples. Overall, 71 results
dropped to within the normal range following
treatment of serum samples with PEG, indicating that 24% of hyperprolactinemia are approximately misdiagnosed due to interference by
M-PRL. Ten out of these samples where elevation of results was due to interference of M-PRL
were tested at 18 clinical laboratories. Two setsof PRL measurements of these serum samples
were obtained from each of the nine most commonly used immunoassay systems. Across thenine assay systems, differences in the PRL estimates ranged from 2.3- to 7.8-fold. Elecsys users reported the highest PRL levels. Somewhat
lower values were reported for DELFIA systems
followed by Immuno-1, AxSYM, and Architectassay system.

Page 316

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

The Immulite 2000 assay generated PRL levels
equivalent to approximately 50% of those reported by the high-reading methods. The lowest PRL levels were reported by Access, ACS:
180, and Centaur systems (4, 8).
Two system of separation of M-PRL from bioactive PRL became popular, PEG precipitation, and
Gel Filtration Chromatography (GFC)(9,10). A
HPLC method has been developed using Agilent
Zorbax GF-250 Column, tris buffer with saline at
pH 7.2 which was found to have equal efficiency of GFC but still not very popular(11). GFC istime consuming and expensive, so not suitable
for regular clinical laboratory performances.
Therefore, PEG precipitation became common
method of precipitation of M-PRL. Karolina etal (12) assessed elevation effect of M-PRL in
27 patients among which 19 with functional
hyperprolactinemia and 8 with prolactinoma
between PEG precipitation and ultracentrifugation. A high diagnostic agreement (95.9%)
and positive correlation coefficient (r=0.506,
p<0.001) was found out between two precipitation method. Both precipitation methods
showed equal efficacy in functional hyperprolactinemia, and PEG precipitation was bettermethod in prolactinoma.
Kit inserts of different systems (13, 14, 15, 16)
mentioned that the PRL results may get affected due to the presence of M-PRL and PEG
precipitation has been suggested where elevated result is obtained. It was also stated that afraction (approximately 14%) of active prolactin
may get co precipitated during PEG pre- treatment. The dilution effect also to be taken into
consideration. No instruction on cutoff value
above which PEG pretreatment to be done wasmentioned in the inserts. Hence, a correlation
study was felt to be necessary to get a guideline
regarding such cutoff value.
In the current study, the author treated all samples
twice, direct assay and after PEG precipitation. A

reverse correlation of both results was done andregression coefficient (r) was calculated. The reverse correlation was necessary to substantiate
the authenticity of correlation coefficient. The
reason of finding out such correlation was to findout:•Should PEG precipitation be done for all
samples irrespective of normal/or elevatedresults? (i.e., whether there should be alower cutoff?)•

Does the fraction of PRL being co precipitated with M-PRL affect patients’ clinicalstatus?

•Though M-PRL is still being mentioned as interfering molecule in all inserts but PEG pretreated results of Cobas e 411 are in agreement with untreated results of Access-2
system. Hence, the underlying problem of
elevated prolactin results is still a matter ofconcern in Cobas e 411.

•

Whether the correlation coefficients arewithin an acceptable uncertainty both in direct and reverse direction?

MATERIALS AND METHODS
Materials
A total of 150 patient samples were collected at
random with direct prolactin results from 0.25300 ng/ml. As the basic aim of the study was tocheck the dilution effect during PEG treatment
and transferability of the expected values soclinical case history has not been considered tomake the study a blind trial. Prolactin has beenmeasured twice, direct measurement & after
PEG precipitation in EQAS samples (BIORAD,
Cycle 13), BIORAD immunoassay control levels
1, 2 & 3, lot 40330 and above mentioned patient samples.
Total 25 number samples were selected andtested in 4 different systems Roche Cobas e 411,
Abbott Architect I, Advia Centaur and Access 2.

Page 317

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

The laboratory performed tests in Cobas e 411& Access 2 and outsourced in accredited laboratories having Advia Centaur & Abbott Architect
I systems. It was informed not to treat the
samples with PEG. In Access 2 and Cobas e 411
these 25 samples were measured twice ie, direct estimation and after PEG precipitation.

using Lumiphos* 530 as Chemiluminescent substrate (14).

Methods

Advia Centaur
Chemiluminescence Immuno assay (CLIA) (16).

Cobas e 411
Electro Chemiluminescence Immunoassay(ECLIA) (13, 17).
Access 2
Chemiluminescence Immunoassay (CLIA) (14).
The Access Prolactin assay is a simultaneous
one-step immunoenzymatic (“sandwich”) assay
Table 1

Abbott, Architect i
Prolactin assay is a two-step immunoassay using
Chemiluminescent Micro particle Immunoassay(CMIA) technology with flexible assay protocols,
referred to as Chemiflex (15).

Precipitation using PEG
25% solution of poly ethylene glycol 6000(PEG) is
prepared using deionized water. It is stable for 7
days. Sample and PEG solution was mixed in equalvolume. Mixed for 10 seconds and centrifuged for
5-30 minutes between 1500-10000g (13).

Comparison of EQAS values, BIORAD, Cycle-13
Peer mean of comparedsystems(ng/mL)

PEG
treatedPEG
DirectLab
zresults
X
resultresults*
score(Cobas-e 1.289 X0.771(ng/mL)
Cobas
Advia
Access Archi
411)(ng/mL) (ng/mL)
e 411 Centaur**2**
tect**(ng/mL)

Sample
no.

N#

1

165

13.3

-0.67

13.9

9.01

9.9

10.6

10.6

10.57

10.25

2

217

19

-0.71

19.9

12.3

13.3

14.7

15.1

15.14

14.65

3

232

33.75

-0.56

35

22.2

23.3

25.7

26.6

26.63

26.02

4

236

47.16

-1.48

52.7

33.2

35.4

39.8

40.2

40.1

36.36

5

235

19.9

-0.25

20.2

11.9

13.4

14.3

15.4

15.37

15.34

6

243

54.63

0.40

53.3

33.6

35

39.3

40.34

40.56

42.12

7

238

14.96

0.75

14.2

8.84

9.91

10.5

11

10.8

11.53

Page 318

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

8

232

19.84

-0.41

20.4

11.8

13.2

14.7

16.2

15.52

15.3

9

241

36.24

0.16

35.9

22.1

23.2

26.5

26.9

27.32

28

10

232

54.11

-0.03

54.2

34

34.6

40.6

41.85

41.24

41.71

11

246

14.56

0.01

14.6

8.91

9.67

10.5

11.55

11.11

11.45

12

225

37.03

0.43

36.1

22.1

23

26.6

28.2

27.47

28.55

r***

-

-

-

-

0.998

0.997

0.999

-

-

0.9995p-value: <0.0001*The lab has enrolled for Roche Cobas e 411 only.
**Peer mean obtained from BIORAD, monthly EQAS assessment sheet.
# Participant laboratories of Roche Cobas e 411.

Fi Whenu e compared
1 Correof EQASr***withation
PEG pretreatedresults
Figure 1

Correlation of EQAS results of Cycle 13

Correlation of EQAS results45
y = 0.771x
R² = 0.987

40
35
30
25
20
15
10
5
0

0

10

20

30

Page 319

eJIFCC2017Vol28No4pp315-327

40

50

60

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

Figure 2

Reverse correlation of EQAS results Cycle 13

Reverse correlation of EQAS
60
y = 1.289x
R² = 0.987

50
40
30
20
10
0

0

5

10

15

20

25

30

35

40

45

Calculations

Results and discussion

PEG treated results were multiplied by 2(1+1ratio). EQAS results of direct and PEG treated values were compared twice, putting PEG resultson Y-axis and direct result on X-axis and reversing the axis.

The z-scores of EQAS results (Immunoassay,
BIORAD, Cycle 13) showed no outlying score in
the complete cycle but remarkable discrepancy was observed with the peer mean of otherimmunoassay systems.

Correlation factors obtained from trend lines(Table 1, Figures 1 & 2). The factors were 0.761& 1.306.

The laboratory observed recurrent complaintsof elevated prolactin results from the patientsthough EQAS and IQAS results were appropriate to the peer mean values.

In a similar manner correlation factors obtained
for Trilevel Immunoassay controls. Correlation
factors were 0.762 & 1.307 (Table 2, Figures 3& 4).
Such correlation was checked from 150 patient’s
data ranging from (0.298-355) ng/ml. Factors
were 0.761 & 1.306 (Table 4, Figures 5 & 6)

The patients obtained results mainly from
Abbott Architect, Advia Centaur systems andlaboratory started comparing results with
Access 2 system.
The difference in results from Cobas e 411 andcomparability of results of other systems were

Page 320

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

Table 2

Correlation of IQC value (Lot 40330, BIORAD, immunoassay trilevel)

ControlWithoutPEG(ng/mL )After
pretreatment
with PEG(ng/mL )Access-2(ng/mL )Advia
Centaur(ng/mL )Architect-i(ng/mL )

L1

9.05

6.86

6.54

5.75

7.37

L2

27.3

21.1

17.9

15.6

21.5

L3

55.8

42.4

39.2

37.7

49.4

p-value: <0.01

Figure 3

Correlation of BIORAD trilevel immunoassay control (Lot 40330)

Correlation of IQAS
4540

y = 0.7623x
R² = 0.9998

35
30
25
20
15
10
5
0

0

10

20

30

IO 321A
Page

eJIFCC2017Vol28No4pp315-327

40

50

60

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

Figure 4

Reverse correlation of BIORAD trilevel immunoassay control (Lot 40330)

Reverse correlation of IQAS
60

y = 1.307x
R² = 0.999

50
40
30
20
10
0

0

1
Table
3

Sl.no.

5

10

15

20

25

30

35

40

45

Comparison of PEG treated PRL values of Roche Cobas e 411
with direct results of other Immunoassay systems
Sample: Patient sample chosen at random of concentration range
6ng/mL - 365ng/mL
Roche Cobas e 411(ng/mL)
Directresult

PEG treatedresult

1

8.5

6.3

2

10.2

3
4

AbbottArchitect(direct)(ng/mL)

Access 2 Beckman(ng/mL)

Advia
Centaur(direct)(ng/mL)

Directresult

PEG treatedresult

6.8

7.1

6.9

6.0

7.6

7.9

7.5

7.1

8.5

9.8

8

7.2

7.3

7.7

7.8

10.1

8

7.9

8.3

8.8

9.1

Page 322

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

5

18.8

14.8

15.6

14.3

13.8

15.8

6

20.1

16.6

14.8

15.9

14.8

17.1

7

28

20.4

21.1

19.2

20.5

18.5

8

29

21.1

22.5

18.5

19

22.5

9

30.5

24

26.8

23.8

24

23.6

10

41

30

31.1

28.8

27.5

32.7

11

38.8

32

29.4

31.5

30.8

29

12

52

39.4

41.6

40.8

41.5

42.5

13

58.9

46.2

43.5

48.3

49

45.1

14

66.2

50.5

55

49.1

50.2

47

15

66.8

50.6

53.8

48.5

47.9

53

16

73

56

55

55.5

54.7

51

17

75

57

60.1

61.2

58.9

62.8

18

87

65

66

69

71.2

59

19

108

84.8

80

75

73.2

78

20

110

86

78

92

90

90

21

168

124

118

132

124

125

22

139

103

112

119

115

114

23

302

235

228

215

209

230

24

103

78

82

75

70

81

Page 323

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

25

471

355

341

362

355

358

r*

-

-

0.99880.99670.997

0.9985p-value: <0.001
r* -- Each series of instrument results compared with Cobas e 411 after PEG treatment results

Table 4

Correlation of prolactin direct & PEG treated results

Total
no. ofpatients

Rangeof analysis(ng/mL)

Correlation
factorof PEG
to direct
results

Correlation
factorof direct
to PEG
results

Regr. ofdirect result&
PEGX1.306

Regr. ofDi rectx0.761& PEG results

150

0.298-355

0.761

1.306

0.995

0.995

Figure 5Comparison of data with PEG on Y-axis

Comparison of data with PEG on Y-axis

results after PEG pretreatment

250
y = 0.761x
R² = 0.991

200
150
100
50
0

0

50

100

150
direct estimation results

Page 324

eJIFCC2017Vol28No4pp315-327

200

250

300

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

Figure 6

Comparison of data with PEG on X-axis (reverse comparison)

Comparison of data with PEG on X-axis
300
y = 1.301x
R² = 0.991

direct estimation results

250
200
150
100
50
0

0

50

100

150

200

250

results after PEG pretreatment

observed. Hence, the laboratory started comparing peer mean of EQAS and IQAS results in all
chemiluminescent immunoassay systems. EQAS
samples pretreated with PEG and compared theresults with direct results. The correlation coefficients obtained from the slope twice.
i. Plotting PEG results on Y axis and direct
results on X-axis:
The correlation coefficient was 0.761 for
EQAS results of Cycle 16 (Table 1, Fig. 1)
ii. Plotting PEG results on X axis and direct
results on Y-axis:
The correlation coefficient was 1.289 for
EQAS results of Cycle 16 (Table 1, Fig. 2) i.e.
reverse comparison.
The two step crosschecking was done and correlation coefficients were evaluated to confirm

validity of the same within a limit of acceptable
uncertainty.
It was also observed that PEG treated Cobas e
411 results are in accordance with Immunoassay
systems Abbott Architect, Access 2 & Advia
Centaur, r being 0.998, 0.997 & 0.999. The reason may be the EQAS samples either pretreatedwith PEG or the methods in other systems weremodified so that involvement of M-PRL is notaffecting the patient’s results like Cobas e 411.
The insert of Lot 40330, BIORAD reflected similar consistency in results of above mentioned
Systems and elevated prolactin results in Cobas
e 411. So Trilevel immunoassay results wereobtained and evaluated following the sameprocedure.

Page 325

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin results

The correlation coefficients were found out tobe 0.762 and 1.307 with R2 0.999 (Table 2, Fig.
3, Fig. 4). The correlation coefficients of EQAS
and IQAS both in direct and reverse direction
are within the limit of acceptable uncertainty.

CONCLUSION•

Correlation coefficient 0.761 is validated.

•

When results are correlated in a wide range
it can be concluded that PEG treatment to
be done irrespective of concentration ofprolactin within normal reference interval
or above the biological reference interval.
Within normal reference range result does
not exclude the presence of M-PRL.

•As the PEG treated results are in correlation
with other systems hence chance of inappropriate diagnosis is less. At least it may
be concluded the precipitation of PRL is sosmall that it will not affect the results andinterpretation.

•

Possibility of systematic bias for the elevatedresults in Cobas e 411 cannot be excluded as
it is not expected that presence M-PRL will
be measurable by a correlation coefficient.

•

Current peer mean results and range of
IQAS and comparison data of EQAS, BIORAD
is affirmative of the fact that the results of
PRL in Cobas e 411 is yet elevated than other systems and needs modification/amendment of method. As peer mean of EQAS and
IQAS are worldwide data hence the problem is a universal one and needs immediate
resolution.

Now, to exclude PEG precipitation effect 25
samples were treated in the laboratory in Cobas
e 411 and Access 2. In both systems direct andPEG treated sample results were recorded. The
Advia Centaur & Abbott Architect results obtained from accredited laboratories and it was
instructed to send direct results only (Table 3).
No remarkable deviation in results were noted
between direct and after PEG treatment resultsin Access 2. The r value of Abbott Architectcompared to PEG treated results of Cobas e
411 was 0.9988. The same for Access direct
was 0.9967, for Access-PEG 0.997 and Advia
Centaur was 0.9985. Though more than 25
samples could not be compared considering financial viability the range of prolactin results
sent for comparison was extended from 6.0365.0 ng/mL (Table 3).
The previous and current PRL insert of Roche
Diagnostics were compared. No amendment
in the procedure was observed (17). Though
the inserts mentioned PEG treatment but nocutoff was instructed. To assess the validityof the laboratory defined correlation constant
150 samples were tested directly and after
PEG precipitation. Correlation constants obtained are 0.761 & 1.301 with R2 0.991 (Table 4,
Figures 5, 6). Hence, the correlation constant isalmost same for IQAS & human sample (0.761& 0.762). Slightly different for EQAS samples(0.771) but such deviation is negligible and wellwithin acceptable range. The EQAS cycle is acurrent one and difference in peers values confirm that Cobas e 411 values are still elevatedand such elevation is measurable by a correlation coefficient.

REFERENCES
1. Beda-Maluga K, Pisarek H, Komorowski J, Pawlikowski
M, Swietostawaski J, Winczyk K. The detection of macroprolactin by precipitation and ultra filtration methods.
Endocrynol Pol.62 (6); p.529-36. 2011.
2. Jurrige V. A possible cause of hyperprolactinemia, misdiagnosis and mistreatment. Pathology in Practice. Endoc
rinology.26(7);p.26-30,2006.
3. Vaisya R, Gupta R, Arora S. Macroprolactin- A frequentcause of misdiagnosed hyperprolactinemia in clinicalpractice. J.Reprod. Infertil.11(3); p.161-7,2010.
4. Smith T P, Suliman A M, Fahie- Wilson M N, McKenne T J. Cross variability in the detection of prolactin
in sera containing big-big prolactin(macroprolactin) by

Page 326

eJIFCC2017Vol28No4pp315-327

Shyamali Pal
Evaluation of correlation coefficient of polyethylene glycol treated and direct prolactin resultscommercial immunoassays. J Clin Endocrinol Metab.87;
p.5410-5, 2001.
5. Fahie Wilson M N, Brunsden P, Surrey J, Everitt A. Macroprolactin and the Roche Elecsys Prolactin Assay: Characteristic of the reaction and detection by precipitation
with poly ethylene glycol. Clin Chem. 46:p.1993-5,2000.
6. Hekim C, Alfyhan H, Leinonen J T, Stenman V H. Effectof incubation time on recognition of various forms of prolactin in serum by DELFIA assay. Clin Chem. 48, p.2253-6,
2002.
7. Schneider W, Marcovitz S, Al-Shammari S, Yago S,
Chavalier S. Reactivity of Macroprolactin in common automated systems. Clin Biochem. 34,p.469-73,2001.
8. Leslie H, Courtney C H, Bell P M, Hadden D R,McCance D
R, Ellis P K,Sheridan B, Atkinson A B. Laboratory and clinical experience in 55 patients with macroprolactinemia
identified by a simple polyethylene glycol precipitation
method. J Clin Endocrinol Metab. 86, p.2743-6, 2001.
9. Fahie-Wilson M N, John R, Ellis A R. Macroprolactin:
high molecular mass forms of circulating prolactin. Ann
Clin Biochem. 42,p.175-92,2005.

10. Gibney J, Smith T P, McKenna T J. The impact on clinical practice of routine screening for macroprolactin. J Clin
Endocrinol Metab.90(7), p.3927-32,2005.
11. Bell D A, Hoad K, Leong L, Bakar J A, Sheehan P, Vasikaran S D. A high pressure liquid Chromatography method for separation of prolactin forms. Ann Clin Biochem.
49. p.285-8, 2012.
12. Karolina B M, Pisarek H, Romanowska I, Komorowski
J, Swietoslawski J, Winczyk K. Ultrafiltration – an alternative method to poly ethylene glycol precipitation for macroprolactin detection. Arch Med Sci. 11(5),1001-7,2015.
13. Kit insert for Prolactin: Roche Diagnostics, p.1-5, 2010.
14. Kit insert for Prolactin: Access 2, Beckman Coulter,
p.1-4,2010.
15. Kit insert for Prolactin: Abbott Diagnostics, p.1-6,2014.
16. Kit insert for Prolactin: Advia Centaur, p.1-5,2013.
17. Kit insert for Prolactin: Roche Diagnostics, p.1-4, 2016.

Page 327

eJIFCC2017Vol28No4pp315-327

Lab test findings in the elderly
Barna Vásárhelyi1, Lóránd A. Debreczeni2
1
2

Department of Laboratory Medicine, Faculty of Medicine of Semmelweis University, Budapest, Hungary
Unit of Laboratory Medicine, “Szent Imre” Teaching Hospital, Budapest, Hungary (retired)

INFO

LET TER TO THE EDITOR

Corresponding authors:
Barna Vásárhelyi
E-mail: vasarhelyi.barna@med.semmelweis-univ.hu

A specific issue of the eJIFCC, with several papers[i.e. 1-6], focuses on the harmonization of laboratory information provided by different services withdifferent clinical settings. Hopefully, these publications will also consider the fact that the reference
interval of several analytes is different in elderly patients compared to the younger age groups.

Lóránd A. Debreczeni
E-mail: loranddebreczeni@gmail.com
Key words:
reference intervals of analytes in elderly,risk of chronic morbidities

A common accompanying phenomenon in the elderly (above 75 years of age) is the increased riskof chronic morbidities. This is associated with anincreased requirement for health care activities
that include the patients’ assessment and monitoring. Blood sampling and laboratory testing are
an inherent part of this process.
When a clinician considers the results of laboratory
tests (as ’good’ or ’bad’), the reference range usedby the laboratory is the cornerstone and the basisof further evaluations. In the majority of laboratories healthy reference range is declared according
to test descriptions and literature; just a minority
perform a systematic analysis among individuals of
a population considered as healthy. However, this iswhere the problem lies: in general, blood donors orother adults aged 20-40 years are enrolled to assess
Page 328

eJIFCC2017Vol28No4pp328-332

Barna Vásárhelyi, Lóránd A. Debreczeni
Letter: Lab test findings in the elderly

healthy reference range. And very rarely elder(and completely healthy) subjects are asked toparticipate. This is not an issue until the analyte
to be tested is not affected by the age itself.
This factor, however, has a direct or indirect effect on a number of laboratory tests, as it ishighlighted in a recent review [7]. In addition
to age and comorbidities (e.g. diabetes mellitus, rheumatism, osteoporosis, etc.) elder patients also have various disadvantages such as
obesity, low socioeconomic status, disabilities
and poor (unhealthy) diet. These all may havean impact on laboratory results. (see Table 1)
Therefore, it is a common scenario that thelaboratory reports of elderly patients may present a number of flags indicating a deviationfrom ’normal reference range’ [8, 9]. That isoften alarming for the patient (and his or her
physician). A resultant consequence (provided that the doctor is not fully aware, whether aslight abnormality is acceptable for the patient)
Table 1is the prescription of novel laboratory tests thatgenerate novel questions.
The direction of anticipated abnormalities(compared to the reference range obtained inyounger subjects) is summarized in Table 2.
SOME ANALYTESWITH PARTICULAR IMPORTANCE
Decreased hemoglobin/hematocrit
In the elderly, the impairment of the intestinal
absorbance of iron and vitamin B12 may leadto a decrease in hemoglobin and erythrocyte
synthesis. Occult blood loss is also common.
There is an increased tendency for hemolysis.
Therefore, it is recommended to decrease thelower level of reference range of hemoglobin(e.g. 115 g/l and 110 g/l for males and females,
respectively). However, it is challenging to differentiate real anemia from the effect of aging.
In the majority of patients the cause of anemia

Factors having an effect on the result of lab tests in the elderly

Physiological changes
gonadal hormones’ levels are decreasing, bone loss is increasing; renal function is impaired;
blood fat levels are increased.
Life style modification
inactivity and associated alteration of body compartments; muscular mass is decreased;
less supply of vitamin D due to less exposure to sun.
Dietary factors
problems with digestion and absorption, dental problems lead to insufficient intake of nutrient
rich food – vitamin and mineral trace deficiency. Further risks are alcoholosim and obesity.
In US patients (according to CDC) aged 50 – 74 years up to 40 per cent of cancers are linked toobesity. Increased body weight is a risk factor for at least 13 cancers (i.e. esophageal, thyroid,
breast, gall-bladder, gastric, liver, pancreas, renal, ovarian, uterine and colorectal cancers). [10]Medicinal therapy(due to co-morbidities) may also cause abnormality in some lab test results.
Advertisement
e.g. dietary supplements.
Page 329

eJIFCC2017Vol28No4pp328-332

Barna Vásárhelyi, Lóránd A. Debreczeni
Letter: Lab test findings in the elderly

Table 2

Some analytes with altered results in the elderly
Increasingalkaline phosphatase, antinuclear antibody, fibrinogen,
FSH, LH, SHBG, gamma glutamil transferase, gastrin, uric acid,interleukin-6, insulin, cholesterol, parathormone (PTH),
prostate specific antigen (PSA), rheuma factor, copper, triglycerol, ESR
Decreasing

aldosterone, vitamin B12, dihydroepiandrosterone (DHEA), vitamin D,
ferritin, phosphate, HDL-cholesterol, IGF-1, interleukin-1, calcium (total),creatinine clearence, creatine kinase, magnesium, growth hormone,
estradiol, free testosterone, T3, ironis a chronic disease e.g. occult blood loss or renal failure. Anemia is of particular importance aselderly patients with anemia are at higher risk
for circulatory and oxygenation problems hallmarked by fatigue, dyspnea, paresthesia (thatare often attributed to elder age and, therefore,
is not treated.)
The area of gas exchanging alveolar surface isalso decreased, leading to a decrease in arterial oxygen tension by 4 mmHg per decade; thisprocess results in latent hypoxia. Hypoxia is often associated with cognitive problems (thatare further aggravated by the side effects ofmedicines used commonly).
Increase in blood glucose levels
Serum glucose levels increase proportionally toage, while glucose tolerance is decreasing. The
reference range of fasting glucose is wider in theelderly (3.9 – 6.7 mmol/l). However, blood glucose levels are often low due to decreased bodyweight and dietary problems. Simultaneously,
serum insulin levels also increase indicating insulin resistance; this is responsible for impaired
glucose tolerance observed in up to 25% of patients above 75 years. Therefore, postprandial

blood glucose levels are often higher when performing an oral glucose tolerance test (upper
limit = 5.5 mmol/l + [age in years /18].
Increased erythrocyte
sedimentation rate (ESR)The ESR is increasing proportionally with age(in general by 0.22 mm/h per year above 20
years of age), but its exact cause is not known.
Therefore, the upper limit of reference range
in the eldery is 40 mm/h and 45 mm/h inmales and females, respectively. (The contribution of the common occurrence of systemic
inflammation in the elderly to high ESR is not
fully clear. One should remember not to use
ESR as a basis of diagnosis of inflammation in
the elderly.)
Decrease of iron levels and stores
Serum iron levels decrease in the elderly, probably due to impaired production of gastric juice.
Simultaneously, iron stores decrease also. The
other common cause of low iron levels and systemic iron deficiency is chronic blood loss; therefore, malignancy should be searched for.

Page 330

eJIFCC2017Vol28No4pp328-332

Barna Vásárhelyi, Lóránd A. Debreczeni
Letter: Lab test findings in the elderly

Increase of total cholesterol
and triglyceride levels
Total cholesterol levels increase by up to
1 mmol/L in 60 years of age. No further elevation is anticipated thereafter; rather, in very
old subjects the level of this analyte decreases.
Triglyceride levels increase by 30 and 50 per
cent in males and females, respectively, between 30 and 80 years of age. HDL-levels increase somewhat in aged men, while decreasein aged women.
Decreased renal function
In general, aged people take several medicines
simultaneously. The metabolites are partly excreted via the kidney. Therefore, it would beof utmost importance to assess renal function
to establish optimal dosage. In the elderly the
number of functioning nephrons decreases by
30 – 45%; this is accompanied by the decreaseof glomerular filtration rate. However, creatinine levels change rarely, as the lean body massdecreases. Therefore, net BUN and creatininelevels are not appropriate to estimate renal
function; instead, eGFR calculation is requiredthat incorporates patient’s age.
Low albumin levels
Simultaneously with aging the level of somespecific proteins, particularly that of albumin
decrease (leading to a decrease of total protein levels). This is partly due to impairmentof liver functions and an inappropriate diet. As
albumin is the major carrier protein in blood,
you should not be surprised, if a patient withlow albumin levels presents with low calcium
or hormone levels.
Thyroid function impairment is common
Hypothyreosis is not an inevitable consequenceof ageing; however, it is a quite common phenomenon in aged patients (of note, its signs
and symptoms include weakness, slowness andtiredness that are falsely attributed to old age).
Therefore, it is recommended to screen patients’
TSH levels. Roughly, TSH reference range is comparable to that in younger age. One should remember that medicines used commonly in oldpatients may influence thyroid hormone levels(eg. glucocorticoid hormones suppress TSH,
while lithium inhibits thyroxin secretion).
WHAT SHOULD BE DONE?
Analytes changing with age are challenging as
the doctor should decide whether a laboratory
test result deviates from the ’healthy’ (younger)
reference range due to physiology or due to adisease. Unfortunately, there is no clear-cut answer, just our common advice: one should neverestablish a diagnosis exclusively on laboratory
test results. Instead, laboratory test results canbe used as an aid and prior results along withclinical history should be always considered.
The determination of old age-specific reference
range would be an enormous support for theevaluation. This is, however, not an easy task.
Subjects encounter the doctor (mostly) if they
have complaints; healthy subjects normally
avoid the doctor. The major question with thesubject presenting at the clinic is not that (s)hehas any problem (the answer: yes, (s)he has,otherwise (s)he would not have come); instead:what is the cause of the problem. Indeed, thegoal of laboratory investigation is the exclusion
or reinforcement of a diagnosis in an otherwise
diseased person.
Therefore, it is not a healthy reference rangethat is required for this population; instead, a’non-affected’ reference range that is characteristic for said population (that is representative
for the given subject). (e.g. the ’non-affected’reference range of troponin is different from thehealthy reference range in an old patient withmoderate renal failure.) The routine establishment of such reference ranges even for routine

Page 331

eJIFCC2017Vol28No4pp328-332

Barna Vásárhelyi, Lóránd A. Debreczeni
Letter: Lab test findings in the elderly

laboratory tests, however, is not performed indomestic and foreign laboratories. Furthermore,
the age-adjusted reference range for any analyte is also affected by the analytical environment; therefore, one cannot provide general
exact numbers.
Therefore, it is recommended for physicians
caring for older patients to request as few
laboratory tests as they can. The major risk
with large number of laboratory tests: if morelaboratory tests are performed, the risk of falsepositivity (and associated diagnostic doubts)
is increased. The premise of fewer laboratory
test is that the doctor should be clear with theanticipated information hoped from the test
result, i.e. how the laboratory test result will
improve his/her clinical decision making.
If you still decide to ask a laboratory test, it isrecommended to strictly adhere to standardized sampling conditions.
Unfortunately, the laboratory and age specificreference ranges (that would improve the patient’s assessment) are not commonly available.
The use of individual reference tables available
in literature is also controversial as reference
ranges depend on local laboratory settings including instrument and reagents.

REFERENCES
1. Plebani M. Harmonization of clinical laboratory information – current and future strategies EJIFCC. 2016; 27:
15–22.
2. Myers GL, Miller WG. The International Consortium for
Harmonization of Clinical Laboratory Results (ICHCLR) - apathway for harmonization. EJIFCC. 2016;27:30-36.
3. Ceriotti F Harmonization initiatives in Europe EJIFCC.
2016; 27: 23–29.
4. Armbruster D, Donnelly J Harmonization of clinicallaboratory test results: the role of the IVD industry eJIFCC
2016; 27: 37-47.
5. Lam Q, Ajzner E, Campbell CA, Young A Critical risk result – an update on international initiatives eJIFCC 2016Vol 27 No1 pp 66-76.
6. Tate JR, Koerbin G, Adeli K Opinion paper: deriving
harmonised reference intervals - global activities. EJIFCC.
2016;27:48-65. eCollection.
7. Pulchinelli A, Cury AJ, Gimenes AC: Clinical laboratory findings in the elderly J Bras Patol Med Lab 2012; 48:
169-74.
8. Cavalieri TA, Chopra A, Bryman PN. When outside thenorm is normal: interpreting lab data in the aged. Geriatrics. 1992;47:66-70.
9. Edwards N, Baird C. Interpreting laboratory values inolder adults. Medsurg nursing 2005;14 Harmonization:
220-229.
10. Lauby-Secretan B, Scoccianti C, Loomis D, Grosse Y,
Bianchini F, Straif K; International Agency for Research on
Cancer Handbook Working Group. Body Fatness and Cancer--Viewpoint of the IARC Working Group N Engl J Med.
2016;375:794-8.

Page 332

eJIFCC2017Vol28No4pp328-332

Erratum(1) Pediatric obesity and cardiometabolic disorders: risk factors
and biomarkers
E. Levy, A.K. Saenger, M.W. Steffes, E. Delvin[published in eJIFCC2017Vol28No1pp006-024]

An error occurred in the numbering of the references presented in Table 2 of the review “PediatricObesity and Cardiometabolic Disorders: Risk factors and Biomarkers” Levy E, Saenger AK, Steffes MW,
Delvin E. eJIFCC. 2017 Mar 8;28(1):6-24. eCollection 2017 Mar. PMID: 28439216.
The table should read as follows:
Table 2Cut-off points for defining insulin resistance (IR)Insulin measurement

Population
StudiedAge(years)Gender

HOMA-IR95 percentile

Ref

Immunoassay(Access, Beckman Coulter)

French Canadian

9
13
16

M/F

1.88/2.07
3.28/3.86
3.31/3.10(80)Fluoroimmunoassay(AutoDelfia, Pharmacia)

Brazilian

10-19

M/F

>2.93

(81)

Chemiluminescence
Immunoassay(Immulite, Siemens)

American

11-14

M/F

≥2.7

(82)

Chemiluminescence
Immunoassay(Cobas, Roche Diagnostics)

Spanish

8-18

M/F

≥3.6

(83)

Page 333

eJIFCC2017Vol28No4pp333-333thEditor-in-chief
Gábor L. Kovács
Institute of Laboratory Medicine, Faculty of Medicine, University of Pécs, Hungary

Assistant Editor 				Case Editor			Harjit Pal Bhattoa				Department of Laboratory Medicine		
University of Debrecen, Hungary		Reinhard B. Raggam
Department of Internal Medicine
Division of Angiology, University of Graz, Austria

Editorial Board

Khosrow Adeli, The Hospital for Sick Children, University of Toronto, Canada
Edgard Delvin, CHU Sainte-Justine Research Center, Montréal, Québec, Canada
Nilda E. Fink, Universidad Nacional de La Plata, Argentina
Ronda Greaves, School of Health and Biomedical Sciences, RMIT University, Victoria, Australia
Mike Hallworth, Shrewsbury, United Kingdom
Andrea R. Horvath, Prince of Wales Hospital and School of Medical Sciences, University of New
South Wales, Sydney, Australia
Ellis Jacobs, Abbott, Orlando, FL, USA
Allan S. Jaffe, Mayo Clinic, Rochester, USA
Bruce Jordan, Roche Diagnostics, Rotkreuz, Switzerland
János Kappelmayer, Faculty of Medicine, University of Debrecen, Hungary
Evelyn Koay, National University, Singapore
Tamas Kőszegi, University of Pécs, Hungary
Gary Myers, Joint Committee for Traceability in Laboratory Medicine, USA
Tomris Ozben, Akdeniz University, Antalya, Turkey
Maria D. Pasic, Laboratory Medicine and Pathobiology, University of Toronto, Canada
Maria del C. Pasquel Carrera, College of Chemists, Biochemists and Pharmacists, Pichincha, Ecuador
Oliver Racz, University of Kosice, Slovakia
Rosa Sierra Amor, Laboratorio Laquims, Veracruz, Mexico
Sanja Stankovic, Institute of Medical Biochemistry, Clinical Center of Serbia, Belgrade, Serbia
Danyal Syed, Ryancenter, New York, USA
Grazyna Sypniewska, Collegium Medicum, NC University, Bydgoszcz, Poland
Jillian R. Tate, Queensland Royal Brisbane and Women’s Hospital, Herston, Australia
Peter Vervaart, LabMed Consulting, Australia
Stacy E. Walz, Arkansas State University, USA

				

			

				
		
		

Publisher: IFCC Communications and Publications Division (IFCC-CPD)
Copyright © 2017 IFCC. All rights reserved.
The eJIFCC is a member of the Committee on Publication Ethics (COPE).
The eJIFCC (Journal of the International Federation of Clinical Chemistry)is an electronic journal with frequent updates on its home page. Our
articles, debates, reviews and editorials are addressed to clinicallaboratorians. Besides offering original scientific thought in our featured
columns, we provide pointers to quality resources on the World Wide Web.
Contents may not be reproduced without the prior permissionof the Communications and Publications Division (CPD) of the IFCC.

Produced by:

epub@insoftdigital.com

Published by:www.ifcc.org