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Routine Serum Creatinine Measurements: How Well Do We Perform? Liesbeth Hoste1†, Kathleen Deiteren2†, Hans Pottel1, Nico Callewaert2 and Frank Martens2*

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Routine Serum Creatinine Measurements: How Well Do We Perform? Liesbeth Hoste1†, Kathleen Deiteren2†, Hans Pottel1, Nico Callewaert2 and Frank Martens2* Hoste et al. BMC Nephrology (2015) 16:21 DOI 10.1186/s12882-015-0012-x RESEARCH ARTICLE Open Access Routine serum creatinine measurements: how well do we perform? Liesbeth Hoste1†, Kathleen Deiteren2†, Hans Pottel1, Nico Callewaert2 and Frank Martens2* Abstract Background: The first aim of the study was to investigate the accuracy and intra-laboratory variation of serum creatinine measurements in clinical laboratories in Flanders. The second purpose was to check the effect of this variation in serum creatinine concentration results on the calculated estimated glomerular filtration rate (eGFR) and the impact on classification of patients into a chronic kidney disease (CKD) stage. Methods: 26 routine instruments were included, representing 13 different types of analyzers from 6 manufacturers and covering all current methodologies (Jaffe, compensated Jaffe, enzymatic liquid and dry chemistry methods). Target values of five serum pools (creatinine concentrations ranging from 35 to 934 μmol/L) were assigned by the gold standard method (ID-GC/MS). Results: Intra-run CV (%) (n = 5) and bias (%) from the target values were higher for low creatinine concentrations. Especially Jaffe and enzymatic dry chemistry methods showed a higher error. The calculated eGFR values corresponding with the reported creatinine concentration ranges resulted in a different CKD classification in 47% of cases. Conclusions: Although most creatinine assays claim to be traceable to the gold standard (ID-GC/MS), large inter-assay differences still exist. The inaccuracy in the lower concentration range is of particular concern and may lead to clinical misinterpretation when the creatinine-based eGFR of the patient is used for CKD staging. Further research to improve harmonization between methods is required. Keywords: Creatinine, External quality assessment, Glomerular filtration rate Background recommendations, manufactures already made efforts The calculation of the estimated glomerular filtration to standardize their Scr measurements to have calibra- rate (eGFR) using mathematical formulas has been tion traceable to the isotope dilution gas chromatog- encouraged as a simple, rapid and reliable way of asses- raphy/mass spectrometry (ID-GC/MS) gold standard sing kidney function. A problem related to the use of method. However calibration traceability does not ad- creatinine-based eGFR formulas is the existing diversity dress non-specificity which remains of concern. of methods to determine serum creatinine (Scr). Small In the Jaffe reaction creatinine forms a coloured prod- analytic changes in Scr can create major shifts in the uct after addition of alkaline picrate. However, proteins, distributions of eGFR, which then cause large differences glucose and substances with a ketone group are known in the eGFR-based chronic kidney disease (CKD) classifi- to interfere [2]. Many manufacturers tried to improve cation of patients [1]. So it is clear that control of la- the performance characteristics of the Jaffe reaction by boratory analysis of Scr and worldwide standardized compensating for these interferences (eg. rate blanking Scr measurements are necessary. According to the and subtraction of a fixed factor to compensate for non- National Kidney Disease Education Program (NKDEP) specific reactions). Since 1970 enzymatic assays were developed to improve creatinine specificity. These en- * Correspondence: [email protected] zymatic methods have generally fewer interferences than †Equal contributors 2Department of Clinical Chemistry and Toxicology, AZ Groeninge Hospital, Reepkaai 4, B8500 Kortrijk, Belgium Full list of author information is available at the end of the article © 2015 Hoste et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hoste et al. BMC Nephrology (2015) 16:21 Page 2 of 9 the Jaffe methods and therefore result in more accurate Methods staging of CKD [3]. The higher cost of the enzymatic Scr Preparation of serum pools assay is the main reason why the use of Jaffe or compen- The creatinine samples of different concentrations were sated Jaffe assays is still in practice [4]. Recently Piéroni prepared in AZ Groeninge Hospital Kortrijk, Belgium by et al. demonstrated substantial improvement in the cali- pooling fresh leftovers of patient serum samples. The bration, traceability and precision of the enzymatic samples of coagulated blood were centrifuged (10 min at methods, reaching the NKDEP recommendations [5]. 3000 rpm) immediately after arrival in the laboratory. The importance of standardized creatinine measure- The creatinine concentration was determined enzymati- ments to calculate eGFR in a correct way has also led to cally on a Cobas 6000 (Roche) and the samples were the development of improved creatinine-based eGFR stored at 4°C (max 3 days). According to the creatinine equations. Two of the most popular eGFR formulas, the concentration the serum was added to a specific frozen original pediatric Schwartz equation [6] and the original pool (n = 5) and allowed to freeze at −20°C on top of the MDRD (Modification of Diet in Renal Disease) [7] equa- already frozen material. All pools were prepared within tion, were derived using Scr levels measured by the one week. Once the needed volume was reached, the kinetic Jaffe method. This method is known to overesti- pools were thawed at room temperature and homoge- mate ID-GC/MS-traceable Scr up to 20%. After the nized (30 min, roller mixer). Afterwards the thawed widespread standardization of creatinine measurement pools were centrifuged (10 min, 3000 rpm). No pellet methods both equations were re-expressed for ID-GC/ was visible. MS standardized (compensated Jaffe or enzymatic) Scr The combined serum samples were tested for viral ser- [8-10]. Also the popular CKD-EPI (Chronic Kidney Dis- ology and found negative for hepatitis B surface antigen, ease Epidemiology Collaboration) equation [11] was de- hepatitis C virus, human immunodeficiency virus and veloped to be used only with ID-GC/MS standardized syphilis. In all pools bilirubin was <53 μmol/L, triglycer- Scr. Unfortunately, the restricted use of these formulas ides <1.92 mmol/L and no hemolysis was observed [12]. for specific creatinine methods has not been an overrid- One mL aliquots of the homogenized pools were frozen ing concern in some studies. at −80°C until shipping on dry ice to the reference la- Our study was performed after the widespread boratory (ID-GC/MS) and to the 22 participating clinical standardization of creatinine measurement methods laboratories. and reports on the status of standardization in clinical The study was approved by the local Ethical Commit- laboratories in Belgium – Flanders. The first aim of the tee of the AZ Groeninge Hospital Kortrijk, Belgium (ref- present study was to investigate the intra-run variation erence number B39620140695). Since the pools were and accuracy (bias) of commonly used Scr assays in prepared using remains of patient serum samples that Flanders. It was our goal to include all current method- were anonymized, it was not necessary to obtain written ologies including Jaffe, compensated Jaffe and enzym- informed consent. atic assays (dry or liquid chemistry). Our interest was to study how results can vary if patients are followed in different laboratories that work with various assays or Target assignment of serum pools instruments eg. in the lab of the general practitioner or Because the ID-GC/MS method is believed to be with- in the lab of the specialized doctor in the hospital. The out interference of other substances it is considered second purpose was to investigate the effect of the the best method for determining creatinine concentra- variation in Scr determination on eGFR values and on tions. The target values were assigned by an ID-GC/MS the CKD classification for some specific patient cases. reference method approved by the international Joint Five fresh frozen serum pools were prepared in a large Committee on Traceability in Laboratory Medicine concentration range (35 to 934 μmol/L) by minimal (JCTLM) (Department of Analytical Chemistry of Prof. processing. Therefore the properties of the serum pools Dr. Thienpont at the University of Ghent, Belgium) are comparable with authentic clinical samples (com- [13,14]. Since it is a highly laborious and costly method, mutable). This is a major advantage over the non- only a few highly specialized laboratories worldwide are commutable control materials (eg. lyophilized) typically offering this method. The measurement protocol consisted used in (inter)national proficiency schemes. In contrast of analysis of each sample in triplicate on 3 independent to the study of Piéroni et al. [5], we specified that the occasions (separate sampling, sample preparation, inde- routine settings were used instead of performing cali- pendent calibration and mass-spectrometry measure- bration just before the run of the study samples. The ment). Calibration mixtures were always prepared from participants were also asked to report the results like three independently prepared working solutions. The they usually do. This approach
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