Implementing a Reference Measurement System for C-peptide: Successes and Lessons Learned

Transcription

1 Papers in Press. Published June 23, 2017 as doi: /clinchem The latest version is at Clinical Chemistry 63: (2017) Special Report Implementing a Reference Measurement System for C-peptide: Successes and Lessons Learned Randie R. Little, 1* Robert I. Wielgosz, 2 Ralf Josephs, 2 Tomoya Kinumi, 3 Akiko Takatsu, 3 Hongmei Li, 4 Daniel Stein, 5 and Chris Burns 6 BACKGROUND: Assessment of endogenous insulin secretion by measuring C-peptide concentrations is widely accepted. Recent studies have shown that preservation of even small amounts of endogenous C-peptide production in patients with type 1 diabetes reduces risks for diabetic complications. Harmonization of C-peptide results will facilitate comparison of data from different research studies and later among clinical laboratory results at different sites using different assay methods. CONTENT: This review provides an overview of the general process of harmonization and standardization and the challenges encountered with implementing a reference measurement system for C-peptide. SUMMARY: Efforts to harmonize C-peptide results are described, including those by the National Institute of Diabetes and Digestive and Kidney Diseases led C-peptide Standardization Committee in the US, activities in Japan, efforts by the National Institute for Biological Standards and Control in the UK, as well as activities led by the Bureau International des Poids et Mesures and the National Metrology Institute in China. A traceability scheme is proposed along with the next steps for implementation. Suggestions are made for better collaboration to optimize the harmonization process for other measurands American Association for Clinical Chemistry C-peptide is a proinsulin cleavage product released from the pancreas in amounts equimolar to insulin. Although C-peptide is cosecreted with insulin, its half-life is about 10 times longer than that of insulin (1). As a result, measurement of C-peptide can be used as a substitute marker for monitoring endogenous insulin production (2, 3). The Diabetes Control and Complications Trial showed that in subjects with type 1 diabetes, higher C-peptide concentrations were associated with improved HbA 1c, less hypoglycemia, and less retinopathy and nephropathy (4). Stabilization of C-peptide concentrations is therefore being used as a measurable end point for immunomodulatory trials in type 1 diabetes (5, 6). Some studies have also suggested that C-peptide is biologically active and may play a role in preventing and possibly reversing some chronic complications of type 1 diabetes (7 9). It is therefore important that C-peptide measurements be harmonized to a common reference to compare and interpret results across various clinical trials and other research studies. General Process of Harmonization/ Standardization: Components of the Reference Measurement System Harmonization/standardization can be achieved by calibration of all measurement procedures to be traceable to the same reference system. Different calibration hierarchies that can be implemented to achieve measurement traceability are described in the International Organization for Standardization (ISO) :2003 standard (10). In principle, as C-peptide is a well-defined chemical entity, its concentration can be measured in SI units, with a traceability chain linking patient results from routine laboratory procedures, via commutable secondary 1 University of Missouri School of Medicine, Columbia, MO; 2 Bureau International des Poids et Mesures, Sèvres Cedex, France; 3 Bio-Medical Standards Group, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan; 4 National Institute of Metrology, China; 5 Albert Einstein College of Medicine, Yeshiva University, Bronx, NY; 6 National Institute for Biological Standards and Control, South Mimms, UK. * Address correspondence to this author at: Diabetes Diagnostic Laboratory, Rm. M766, Departments of Pathology & Anatomical Sciences and Child Health, University of Missouri School of Medicine, 1 Hospital Dr., Columbia, MO Fax ; LittleR@health.missouri.edu. Received November 17, 2016; accepted April 25, Previously published online at DOI: /clinchem American Association for Clinical Chemistry 7 Nonstandard abbreviations: ISO, International Organization for Standardization; IVD, in vitro diagnostics; JCTLM, Joint Committee for Traceability in Laboratory Medicine; BIPM, Bureau International des Poids et Mesures; IRR, International Reference Reagent; ID, isotope dilution; CRM, certified reference material; LC-MS, liquid chromatography tandem mass spectrometry; isotope-dilution mass spectrometry (ID-MS); NMI, National Metrology Institute; CCQM, Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology; NIM China, National Institute of Metrology in China; NMIJ, National Metrology Institute of Japan; NIBSC, National Institute of Biological Standards and Control. 1 Copyright (C) 2017 by The American Association for Clinical Chemistry

2 Special Report reference materials (in serum) and reference measurement procedures to a primary reference material (pure substance) value assigned for the mass fraction of C-peptide in the material. This traceability would allow clinical results to be comparable across measurement systems, location, and time and is essential for patient care, research translation, and disease prevention and control. Establishing traceability for calibrator and control materials for in vitro diagnostic (IVD) devices became mandatory in Europe with the implementation of the European Union Directive on IVD devices. Although this is a European Union Directive, it had a worldwide effect on clinical laboratory measurements because these devices have a worldwide market. The ISO standard 17511:2003 provides the framework for calibration traceability in laboratory medicine (10). There are also several reviews of the traceability process and examples of traceability chains for specific measurands (11 14). The Joint Committee for Traceability in Laboratory Medicine (JCTLM) is an international committee created in 2002 by the Bureau International des Poids et Mesures (BIPM), IFCC, and International Laboratory Accreditation Cooperation (15). The JCTLM has established a database containing lists of higher-order reference materials, higher-order reference measurement procedures, and accredited reference laboratory services that have been reviewed and found to meet the requirements of the harmonized standards that support the IVD Directive (16). The production and value assignment of reference materials, the development of reference measurement procedures, and the provision of reference measurement services may, and often does, occur in different laboratories. However, it is essential that these organizations work together so that each component of the traceability chain can be considered and traceability can be established throughout the reference system. Efforts to Harmonize C-peptide Results ACTIVITIES SPONSORED BY THE NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES In 2002, the National Institute of Diabetes and Digestive and Kidney Diseases in the US organized a C-peptide standardization committee and funded an international comparison study of C-peptide assays. The Diabetes Diagnostic Laboratory at the University of Missouri coordinated this effort. The goal of the initial studies was to assess the degree of equivalence of C-peptide results and to determine whether C-peptide results could be harmonized. The majority of C-peptide assays have been calibrated for many years with the pure WHO International Reference Reagent (IRR) for C-peptide (IRR 84/510), yet these assays produced results that differed among measurement procedures (17). Additional off-line calibration with WHO IRR 84/510 did not successfully improve equivalence of results. However, matrix-appropriate patient samples could successfully be used to calibrate assays and greatly reduce variability of results among methods (18). Samples for these studies were obtained with Institutional Review Board approval from the University of Missouri Health Sciences. An isotope dilution (ID)-LC-MS measurement procedure using a purified 13 C, 15 N-labeled reference standard had been described and was used to assign reference values to serum calibrators (19, 20). However, the pure reference standard was not developed into a certified reference material (CRM). Fig. 1 shows the between-lab/ between-method variability before and after calibration with serum-based secondary reference material value-assigned by the ID-LC-MS reference measurement procedure. In 2008, the C-peptide committee initiated yearly meetings during the annual meeting of the AACC with manufacturers of C-peptide assays. In 2010, the group discussed the need to develop a suitable primary reference standard, and all agreed that this was an important step. However, no standard became available over the next 5 years. The C-peptide committee agreed that it was important to move forward with harmonization, and the manufacturers agreed that the candidate reference measurement procedure should be internationally recognized by being listed in the JCTLM database. Following this recommendation, the ID-LC-MS candidate reference measurement procedure was established at a second site. A comparison study confirmed the equivalence of results from the 2 sites (20), and the reference measurement procedure was then listed in the JCTLM database in 2014, having been reviewed by the independent experts and found to be compliant with ISO 15193:2009 requirements (21). By July 2014, the C-peptide committee had shown that variability among assay methods could be reduced by using serum-based secondary reference materials with values assigned by the JCTLM-listed ID-LC-MS reference measurement procedure. Manufacturers were asked to begin the recalibration process and adjust their calibration to the matrix-appropriate secondary reference materials that had values traceable to the pure labeled C-peptide standard used to calibrate the ID-LC-MS reference measurement procedure. Because C-peptide is considered an exempt assay by the US Food and Drug Administration, manufacturers are not required to resubmit the entire assay with a new calibration in the US. Therefore, the time frame for Food and Drug Administration approval of a recalibration is relatively short. In 2015, it became clear that other efforts (discussed in more detail below) to standardize C-peptide measurements were ongoing, in addition to the National Institute of Diabetes and Digestive and Kidney Diseases C-peptide Standardization Committee s activities, and this would raise further issues to be solved before a global harmonization scheme could be implemented. 2 Clinical Chemistry 63:9 (2017)

3 Reference Measurement System for C-peptide Special Report Fig. 1. Between-lab/between-method variability before (A) and after (B) calibration with serum-based secondary reference material with assigned values. Each symbol represents a different assay method. The solid line represents the reference measurement procedure results. ACTIVITIES OF THE BIPM AND NATIONAL METROLOGY INSTITUTE OF CHINA National Metrology Institutes (NMIs) provide CRMs, develop reference measurement procedures, and provide reference measurement services for many areas of chemical and biological measurement, including laboratory medicine. To demonstrate the accuracy and equivalence of the measurement services and values assigned to reference materials, the NMIs participate in regular interlaboratory comparisons, which are named key comparisons Clinical Chemistry 63:9 (2017) 3

4 Special Report coordinated through the BIPM s Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM), and have the code CCQM-KXX, where the XX is a number identifying a particular comparison, with the results publicly available in the BIPM key comparison database ( In 2014, the BIPM, together with the National Metrology Institute of China (NIM China), organized CCQM- K115 to compare NMI capabilities to measure the mass fraction of C-peptide in a pure C-peptide material. C-peptide was chosen as the material for the comparison for several reasons: It was a model system for mass fraction assignments of straight chain peptides with molar mass of 5000 g/mol; CRMs for C-peptide and reference measurement procedures for C-peptide measurement were already being provided by at least 1 NMI [National Metrology Institute of Japan (NMIJ)] and listed in the JCTLM database; and the material would be available in sufficiently large quantities for it to be valueassigned by independent methods by the BIPM and NMIs, notably by mass balance, amino acid analysis corrected for peptide impurities, and quantitative NMR corrected for peptide impurity content. The CCQM-K115 material was purchased from a peptide material manufacturer and not purified further by the BIPM, as the material was being used to demonstrate measurement capability equivalence among NMIs, rather than being prepared as a high purity calibration material; this enabled the BIPM to distribute 25 mg of material to each participant in the comparison. The homogeneity and stability of the material were studied at the BIPM, following ISO Guide 34 guidelines (22), and were found to be suitable for a CCQM comparison. C-peptide as a solid material is a salt, and so in addition to the C-peptide component, it is expected to contain counter ions, peptide impurities, and water. Ten NMIs participated in the key comparison. The reference value for the comparison was decided during the April 2016 meeting of the CCQM Protein Analysis Working Group, and was based on mass balance values of all impurities and their measurement uncertainties, allowing a key comparison reference value of mg/g to be assigned with an expanded uncertainty of 6.2 mg/g (k 2). The BIPM identified and quantified 70 peptide impurities in the material using high-resolution mass spectrometry. Amino acid analysis methods performed by the NMIs were in general found to agree with the reference value within their stated uncertainties, provided peptide impurities had been appropriately accounted for in the amino acid analysis, with expanded uncertainties of approximately 50 mg/g (k 2) for amino acid methods reported on this particular material (23). Beginning in 2017, NIM China will provide this material for use as a CRM. ACTIVITIES OF THE JAPANESE COMMITTEE FOR CLINICAL LABORATORY STANDARDS AND THE NMIJ An extensive standardization project for clinical chemistry in Japan was initiated by the Japanese Committee for Clinical Laboratory Standards and the NMIJ with the support of the New Energy and Industrial Technology Development Organization in The aim of this project was to establish traceability chains to higherorder reference materials in the field of clinical chemistry in Japan. This large-scale project involved major Japanese IVD manufacturers, and it provided the opportunity for a comparison study of results from different manufacturers assays. More than 20 measurands were included in the project, with C-peptide as 1 of the analytes of interest, with 7 manufacturers participating in the harmonization/standardization study. Although the pure C-peptide solution (prepared from WHO IRR 84/510 or a commercial reagent) had failed to harmonize kit results, the use of serum calibrators, prepared by spiking pure C-peptide material into C-peptide depleted serum, successfully reduced between-method variability. Calibrators prepared from patient serum were also shown to reduce variability to the same extent (24). The project s end point was a demonstration of harmonization. The project also succeeded in making Japanese manufacturers aware of the importance of higher-order reference materials for standardization. The NMIJ initiated an effort to develop CRMs for proteins and peptides at the same time as the national project. To establish a CRM of C-peptide having traceability to SI, the NMIJ developed isotope-dilution mass spectrometry (ID-MS) (25, 26) methods for amino acid analysis and amino acid CRMs (27). The certification process of C-peptide candidate CRM consisted of impurity quantification, and homogeneity and stability testing. The concentration of C-peptide was determined by 2 independent amino acid analyses using liquid phase and gas phase hydrolyses. These analyses used ID-MS with 13 C- and 15 N-labeled amino acids as internal standards and NMIJ CRM amino acids to enable SItraceable measurements. This material is a high purity lyophilized synthetic peptide produced in compliance with ISO Guide 34 (28). The provision of the first lot was started in 2011, and the second lot, NMIJ CRM 6901-b, is now available. The need for serum calibrators having traceability to higher-order reference material was widely recognized after the national project. The reference measurement procedure for serum C-peptide measurement using both ID-MS and the pure C-peptide CRM made it possible to achieve traceable measurements for the value assignment of the serum calibrator. However, to cover the reference interval of serum C-peptide, particularly in the low concentration range, it was necessary to improve the sensitivity of LC-MS measurements. The combination of im- 4 Clinical Chemistry 63:9 (2017)

5 Reference Measurement System for C-peptide Special Report munoaffinity purification and precolumn derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was shown to result in a 20-fold improvement in sensitivity. The ID-MS measurement was performed using C-peptide CRM 6901-b as the calibrant and 2 H 8 - Val 2 C-peptide as the internal standard by the double isotope dilution method. The method validation studies showed a linear response in the to 2.9-ng interval on the column that covered the reference interval. The relative SDs within and between days were both 4.0%, and the results of spike and recovery experiments showed mean recoveries between 99% and 108%. The comparison study of serum C-peptide measurements between the ID-MS and a chemiluminescence enzyme immunoassay by Lumipulse (Fujirebio) showed high correlation across the interval examined [( ) nmol/l, r ] (29). Both the C-peptide CRM- and ID-MSbased reference measurement procedure enabled traceable measurements of serum C-peptide, and both the material and the measurement procedure have been listed in the JCTLM database. The NMIJ also participated in the CCQM-K115 key comparison. The value submitted by NMIJ for the comparison, 808 mg/g (k 2) and an expanded uncertainty of 48 mg/g, agreed with the reference value (23). This provided confidence in the NMIJ value-assignment procedures, and value and measurement uncertainty on the much purer C-peptide material that NMIJ had issued as CRM 6901-b and is listed in the JCTLM database. ACTIVITIES OF THE NATIONAL INSTITUTE OF BIOLOGICAL STANDARDS AND CONTROL AND THE WHO It is now well accepted that calibrators prepared in a sample matrix and value-assigned using a reference measurement procedure will be important in improving the between-method agreement of C-peptide measurements in patient samples. However, it was also recognized by the UK and the WHO in 2010, that having made a reference material available to manufacturers for nearly 30 years, and with no certainty that certified matrixbased reference materials for C-peptide in serum would be available in the near future, there was an ongoing responsibility to this community to maintain the availability of such a reference material for the continuing development, calibration, and monitoring of C-peptide immunoassays. Because stocks of the existing WHO reference reagent (84/510) were exhausted, a timely replacement was therefore required and an international project was initiated. It should be added that it was not the intention of this project to prepare a material to be used as a primary standard to calibrate the ID-LC-MS reference measurement procedure. Indeed, any study to prepare a WHO reference material intended to be used to calibrate immunoassays is usually conducted differently than one designed to produce a primary standard to calibrate a reference measurement procedure. For example, the reference material is typically lyophilized in glass ampoules in low amounts, and it is not designed to be weighed out of the ampoule. In addition, WHO International Standards are prepared in large batches and are thoroughly evaluated for their long-term stability and fitness for purpose in immunoassay systems. However, it was recognized by the National Institute of Biological Standards and Control (NIBSC) and WHO that the anticipated uses of this material may change (e.g. from a primary calibrator for immunoassays to a stable external reference to monitor within-assay performance) once the complete reference measurement system (consisting of a primary reference material/calibrators, reference measurement procedures, and secondary matrix-based reference materials) had been successfully implemented. With this in mind, an international collaborative study was initiated in 2013 and was conducted in 3 phases. Phase I involved the assignment of a value in SI units to a primary calibrator, containing a larger amount of C-peptide, using amino acid analyses. Phase II applied this value to the calibration by HPLC of a candidate standard formulated at lower C-peptide content and lyophilized in 2700 glass ampoules. In phase III, the candidate standard was compared with the first IRR (84/510) by current immunoassays to assess its suitability to serve as an international standard. In this phase, participants were also requested to determine the C-peptide concentration of 16 serum and 16 urine samples to assess the impact of the introduction of the candidate preparation on the routine measurement of C-peptide in human samples and to also address commutability considerations. Laboratories were in good agreement in phases I and II, and calibration of the candidate standard by HPLC gave a final estimate of content of 8.64 g per ampoule. The candidate standard also appeared to be sufficiently stable to serve as an international standard based on the analysis of accelerated thermal degradation samples. The candidate standard was also shown to have appropriate immunological activity in the immunoassay systems included in the study. C-peptide is measured clinically in serum/plasma and urine, and so, using a difference in bias approach (27) in 10 of these immunoassay systems to assess the commutability of the candidate standard with serum and urine samples, it was also shown that, except for 2 of these assays, the candidate standard demonstrated commutability with respect to the serum and urine samples included in the study (30). The first international standard for C-peptide is now available from NIBSC. CHALLENGES IN IMPLEMENTING THE STANDARDIZATION PROCESS FOR C-PEPTIDE In 2015 it was clear that 3 different pure materials were being evaluated for C-peptide. Clinical Chemistry 63:9 (2017) 5

6 Special Report The NMIJ has a high purity material currently available as a CRM and listed in the JCTLM database. The material is lyophilized and can be reconstituted to produce a 100-mg/L solution with an expanded uncertainty of 5 mg/l (k 2). In addition, the NMIJ proposed another ID-MS method as a potential higher-order reference measurement procedure for standardization of serum C-peptide; this method is listed in the JCTLM database (29). The BIPM and NIM China initiated an international comparison in 2014, circulating a C-peptide material to participating NMIs, designed to assess the measurement capabilities of participating NMIs, to measure the mass fraction of C-peptide in C-peptide material expressed in milligrams per gram. The NIBSC had prepared the first WHO International Standard for Human C-peptide NIBSC code, 13/ 146, and this is now available, replacing the IRR 84/510, stocks of which are exhausted. Each ampoule of the material contains 8.64 g of C-peptide with an expanded uncertainty of 0.43 g. Because the material is solely intended to be used as a reference material for immunoassays, it was sent to manufacturers of C-peptide immunoassay methods and shown to be commutable with serum samples for 8 of 10 commercially available immunoassays (30). The fact that there were several independently developed reference materials for C-peptide in circulation in 2015, with different intended uses, led to confusion at the C-peptide Manufacturer Meeting organized by the National Institute of Diabetes and Digestive and Kidney Diseases C-Peptide Standardization Committee and held at the July 2015 AACC annual meeting in Atlanta, GA. The intended uses of the different C-peptide reference materials and comparison studies were later clarified; however, the coexistence of several different calibration materials, as well as 2 different calibration hierarchies for C-peptide, within which pure C-peptide was used for direct calibration of IVD kits or only as a calibrator for the reference ID-MS measurement procedures, was unsettling to IVD manufacturers at a time when they were initiating recalibration of their assays to the National Institute of Diabetes and Digestive and Kidney Diseases C-Peptide Standardization Committee s reference measurement procedure. This led to the issue being discussed at the JCTLM members and stakeholders meeting held at the BIPM in December 2015, which led all parties to clarify the status of their activities and work together to find recommendations for going forward. Current Status of the Reference Measurement System for C-peptide and Recommendations for Facilitating Implementation Fig. 2 illustrates the calibration hierarchy (traceability chain) that the work of Little et al. (18) has showed would be necessary to ensure equivalence of C-peptide kit results from different IVD manufacturers. A fully operating reference system for an analyte is expected to have primary reference material, primary calibrators, reference procedures, commutable matrix reference materials, and laboratories operating reference measurement procedures and providing these as measurement services. To ensure the quality and accuracy of the measurement values, regular participation in interlaboratory comparisons is recommended for those laboratories providing reference values, as well as maintenance of appropriate quality systems. The conformity of these systems with written standards can be independently verified by third-party peer review or accreditation. The reference measurement system for C-peptide as depicted in Fig. 2 is in development, and as such, the current status of and the proposed next steps for the various components of the system are outlined below. 1. TRACEABILITY CHAIN ISO outlines various calibration hierarchies (traceability chains) that can be used to obtain comparable results that agree within their measurement uncertainties. To ensure that values are assigned accurately to IVD kits, it is vital that the reference materials used for this calibration step are commutable. Little et al. (17, 18) have demonstrated that matrix-based reference materials are necessary within the calibration hierarchy as proposed in Fig. 2. Historically, and in the absence of reference measurement procedures for assigning values of C-peptide concentrations in serum-based reference materials, a calibration hierarchy based on pure standards of C-peptide has been used, as depicted in Fig. 3. In this scenario, the primary reference material is to be used directly by manufacturers for assay calibration. The historical use of the WHO IRR for C-peptide (84/510) for direct calibration of IVD kits has meant that for many years a different calibration hierarchy has been in operation. However, for some measurands such as C-peptide, this process has been clearly shown to be ineffective (17). It is now clear that the existence of 2 different calibration hierarchies will not improve the equivalence among C-peptide results from different IVD manufacturers. As a result, upon the establishment of a complete and sustainable reference measurement system for C-peptide to enable manufacturers to calibrate their kits using commutable matrixbased reference materials, it can be recommended that the practice of establishing the traceability of C-peptide results to the WHO International Standard should be discontinued. The status of the international standard might then change to act as a stable external reference to monitor within-assay performance. 6 Clinical Chemistry 63:9 (2017)

7 Reference Measurement System for C-peptide Special Report Fig. 2. Traceability scheme for C-peptide based on a calibration hierarchy that achieves SI traceability and requires both pure primary reference material and primary calibrator, as well as a commutable (matrix-based) secondary calibrator. 2. PRIMARY REFERENCE MATERIAL A primary reference material (pure C-peptide), available from the NMIJ, is listed in the JCTLM database and was produced in compliance with ISO (also ISO Guides 34 and 35). NMIJ demonstrated its capabilities to value assign a C-peptide pure material in the international comparison coordinated by the BIPM and NIM (CCQM-K115), and, as a result, the material is suitable as a primary reference material/calibrator for the ID-MS reference measurement procedure as shown in Fig. 2. The NMIJ material has not been developed or evaluated for its suitability for direct calibration of immunoassays. The WHO International Standard from NIBSC is not suitable for use as a primary reference material for the ID-MS reference measurement procedure. This material was produced as a reference material for the calibration of immunoassays, following WHO guidelines, and the calibration hierarchy model depicted in Fig. 3. The material was characterized through an international collaborative study, coordinated by NIBSC, which included the immunoassay manufacturers of C-peptide kits. The results of this study indicated that the standard is commutable for several, but not all, immunoassay methods. 3. REFERENCE MEASUREMENT PROCEDURES Two ID-MS reference measurement procedures are published in the JCTLM database for the measurement of C-peptide in serum, 1 from NMIJ and the other from the Diabetes Diagnostic Laboratory. Both procedures cover similar ranges and report similar achievable relative expanded uncertainties within the reference range for C-peptide of about 10%. Method validation work at the Diabetes Diagnostic Laboratory demonstrated that their reference measurement procedure operated in 2 different laboratories could lead to agreement within 5% of reported values, within the measurement uncertainty of the procedures (20 ). However, the NMIJ and Diabetes Diagnostic Laboratory results, although well correlated (r ), currently exhibit a bias of approximately 20%, which is outside the combined measurement uncertainty of both methods. The 2 ID-MS measurement procedures differ in their source of primary calibrator, Clinical Chemistry 63:9 (2017) 7

8 Special Report Fig. 3. Historical (and current) traceability scheme for C-peptide, to WHO pure C-peptide material. extraction procedure, labeled internal standard, and mass spectrometric analysis (with or without a derivatization step). The possible causes of bias are currently under investigation by both laboratories. When the sources of bias between the 2 ID-MS methods are identified and corrective action is initiated, the information listed in the JCTLM database will be updated accordingly. In the meantime, a note will be added advising users of the discrepancy between these 2 methods. In addition, the JCTLM procedures for the review of new reference measurement procedures will be reviewed, as it pertains to the situation in which reference measurement service data from the IFCC External Quality assessment scheme are not available to demonstrate method result equivalence. 4. COMMUTABLE MATRIX CRMs Implementation of this part of the traceability chain is currently foreseen through value assignment of serum samples with the reference measurement procedure(s), as currently no CRMs for C-peptide in serum are available or listed in the JCTLM database. The serum samples need to be sufficiently homogeneous and stable to meet their intended use and for them to be considered reference materials. The provision to industry of reference measurements on patient samples provides an alternative acceptable mechanism to a commutable CRM. 5. REFERENCE MEASUREMENT SERVICES At least 3 laboratories have operated reference measurement procedures for C-peptide and could provide these as reference measurement services to the IVD industry. JCTLM listing of a reference measurement service provider requires the listed laboratory to use a JCTLM-listed reference measurement procedure, participate regularly in interlaboratory comparisons for reference laboratories (e.g., IFCC quality assessment scheme), and be accredited to ISO and for the service. Currently no laboratories are listed in the JCTLM database as providing C-peptide reference measurement services. As the reference system for C-peptide is implemented, it is expected that to continue to demonstrate the accuracy of the reference measurement system, reference laboratories will participate in interlaboratory comparisons. 6. EXTERNAL QUALITY ASSESSMENT FOR CLINICAL LABORATORIES Accuracy-based external quality assessment schemes for C-peptide will enable the equivalence of results on patient samples to be monitored and the success of the 8 Clinical Chemistry 63:9 (2017)

9 Reference Measurement System for C-peptide Special Report harmonization process to be demonstrated. Assessment scheme providers should be encouraged to adopt accuracybased systems as has been done for HbA 1c (31). A pilot serum survey ( wild card challenge ) recently took place in the College of American Pathologists 2016 survey (2016-B ING) for C-peptide. Conclusions References From the above description, it is clear that different organizations, laboratories, and individuals involved in harmonization efforts are often separated by distance and funding mechanisms. This can cause lack of coordination among the parties involved that can hinder the process of achieving global harmonization. Clearly, effective communication and coordination of activities are important for progression through the harmonization process. The recently established International Consortium for Harmonization of Clinical Laboratory Results provides a forum for communication to support planning and coordination of harmonization activities among different stakeholders at an early stage (13). The current C-peptide situation is a good example where better communication would have led to cooperation and earlier implementation of a reference system for harmonization of medical laboratory measurement procedures. The JCTLM, in addition to overseeing a listing of reference materials, measurement procedures, and laboratory services, can provide a forum for interaction among all organizations and laboratories involved with harmonization activities. The most recent JCTLM meeting held in Sèvres, France, from November 30 to December 1, 2015 enabled discussion of C-peptide harmonization activities with better communication among all interested parties regardless of their countries of origin and funding streams. Unfortunately, the time frame for successful implementation of harmonization of clinically important analytes is long. Development of reference materials and measurement procedures is difficult, time-consuming, and needs to be sufficiently funded if it is to be successful. It is important that the goals for a given analyte be decided at the beginning of a harmonization activity, including the model for the calibration hierarchy to be applied, as well as interaction between all bodies that will eventually contribute to a sustainable reference measurement system for an analyte at an early stage in the development of that system. Better communication will certainly help achieve this coordination. The JCTLM stakeholders meeting was useful in bringing various organizations together once a problem had been identified. The International Consortium for Harmonization of Clinical Laboratory Results coordinating activities for early collaboration is also a practical way to avoid such problems arising in the future. A new reference measurement system for C-peptide measurements is proposed, and we have identified some next steps required to successfully implement and sustain that system. In addition, C-peptide harmonization history provides a good case study of the challenges for the harmonization process, and the collaborations and interactions required to make it a success. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: R.R. Little, NIH/NIDDK (Grant Number 1UC4DK ); Funding support to NIM China from the International Science & Technology Cooperation Program of China (No. 2014DFA31810) for the K115/P55.2 comparison. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. 1. Bonser AM, Garcia-Webb P. C-peptide measurement and its clinical usefulness: a review. Ann Clin Biochem 1981;18: Palmer JP, Fleming GA, Greenbaum CJ, Herold KC, Jansa LD, Kolb H, et al. C-peptide is the appropriate outcome measure for type 1 diabetes clinical trials to preserve -cell function: report of an ADA workshop, October Diabetes 2004;53: Greenbaum CJ, Mandrup-poulsen T, McGee PF, Battelino T, Haastert B, Ludvigsson J, et al. Mixed-meal tolerance test versus glucagon stimulation test for the assessmentof -cellfunctionintherapeutictrialsintype1 diabetes. Diab Care 2008;31: Lachin JM, McGee P, Palmer JP for the DCCT/EDIC Research Group. Impact of C-peptide preservation on metabolicandclinicaloutcomesinthediabetescontroland Complications Trial. Diabetes 2014;63: Hagopian W, Ferry RJ, Sherry N, Carlin D, Bonvini E, Johnson S, et al. Teplizumab preserves C-peptide in recent-onset type 1 diabetes: 2-year results from the randomized, placebo-controlled Protégé trial. Diabetes 2013;62: Moran A, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, et al. Interleukin-1 antagonism in type 1 diabetes of recent onset: two multicenter, randomized double-masked, placebo-controlled trials. Lancet 2013; 381: Luppi P, Kallas A, Wahren J. Can C-peptide mediated anti-inflammatory effects retard the development of microvascular complications of type 1 diabetes? Diabetes Metab Res Rev 2013;29: Marx N, Silbernagel G, Brandenburg V, Burgmaier M, Kleber ME, Grammer TB, et al. C-peptide levels are associated with mortality and cardiovascular mortality in patients undergoing angiography: the LURIC study. Diab Care 2013;36: Clinical Chemistry 63:9 (2017) 9

10 Special Report 9. Ekberg K, Brismar T, Johansson BL, Lindström P, Juntti- Berggren L, Norrby A, et al. C-peptide replacement therapy and sensory nerve function in type 1 diabetic neuropathy. Diabetes Care 2007;30: In vitro diagnostic medical devices measurement of quantities in biological samples metrological traceability of values assigned to calibrators and control materials. ISO 17511: Geneva: International Organization for Standardization; Vesper HW, Thienpont LM. Traceability in laboratory medicine. Clin Chem 2009;55: Braga F, Panteghini M. Verification of in vitro medical diagnostics (ICD) metrological traceability: responsibilities and strategies. Clin Chim Acta 2014;432: Miller WG, Tate JR, Barth JH, Jones GRD. Harmonization: the sample, the measurement, and the report. Ann Lab Med 2014;34: Greenberg N. Update on current concepts and meanings in laboratory medicine standardization, traceability and harmonization. Clin Chim Acta 2014;432: Jones GRD, Jackson C. The Joint Committee for Traceability in Laboratory Medicine its history and operation. Clin Chim Acta 2016;453: Bureau International des Poids et Mesures, Joint Committee for Traceability in Laboratory Medicine (JCTLM). (Accessed November 2016). 17. Wiedmeyer HM, Polonsky KS, Myers GL, Little RR, Greenbaum CJ, Goldstein DE, Palmer JP. International comparison of C-peptide measurements. Clin Chem 2007;53: Little RR, Rohlfing CL, Tennill AL, Madsen RW, Polonsky KS, Myers GL, et al. Standardization of C-peptide measurements. Clin Chem 2008;54: Rogatsky E, Balent B, Goswami G, Tomuta V, Jayatillake H, Cruikshank G, et al. Sensitive quantitative analysis of C-peptide in human plasma by 2-dimensional liquid chromatography mass spectrometry isotope-dilution assay. Clin Chem 2006;52: Stoyanov AV, Connolly S, Rohlfing CL, Rogatsky E, Stein D, Little RR. Human C-peptide quantitation by LC-MS isotope-dilution assay in serum or urine samples. J Chromat Separation Techniq 2013;4: In vitro diagnostic medical devices measurement of quantities in samples of biological origin requirements for content and presentation of reference measurement procedures. ISO 15193: 2009 (E). Geneva: International Organization for Standardization; Reference material producers ISO guide 34 standard application document. Australia: National Association of Testing Authorities; Josephs RD, Li M, Song D, Westwood S, Stoppacher N, Daireaux A, et al. Metrologia 2017;54(Technical Suppl): Research report, Program for development of measurement standard and intellectual infrastructure, New Energy and Industrial Technology Development Organization (NEDO), in Japanese. 25. Kato M, Kato H, Eyama S, Takatsu A. Application of amino acid analysis using hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for peptide and protein quantification, J Chromatogr B Analyt Technol Biomed Life Sci 2009;877: Kinumi T, Ichikawa R, Hirokazu A, Takatsu A. Traceable amino acid analyses of proteins and peptides by isotope-dilution mass spectrometry using pre-column derivatization reagent. Anal Sci 2010;26: Kato M, Yamazaki T, Kato H, Eyama S, Goto M, Yoshioka M, Takatsu A. Development of high-purity certified reference materials for 17 proteinogenic amino acids by traceable titration methods. Anal Sci 2015;31: Kinumi T, Goto M, Eyama S, Kato M, Kasamam T, Takatsu A. Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses. Anal Bioanal Chem 2012;404: Kinumi T, Mizuno R, Takatsu A. Quantification of serum C-peptide by isotope-dilution liquid chromatographytandem mass spectrometry: enhanced detection using chemical modification and immunoaffinity purification. J Chromatogr B Analyt Technol Biomed Life Sci 2014; : Moore M, Ferguson J, Dougall T, Rigsby P, Burns C. WHO International Collaborative Study of the Proposed 1st International Standard for human C-peptide. WHO/ BS/ /197773/1/WHO_BS_ _eng.pdf (Accessed November 2016). 31. CAP Hemoglobin A1c (5 challenge) GH5-A 2016 Participant Summary, Clinical Chemistry 63:9 (2017)