assay Introduction Conclusions: The D-100 TM system proved to be a robust and reliable method for HbA 1c

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1 Clin Chem Lab Med 215; aop Stéphane Jaisson*, Nathalie Leroy, Emmanuelle Guillard, Aurore Desmons and Philippe Gillery Analytical performances of the D- TM hemoglobin testing system (Bio-Rad) for assay DOI /cclm Received March 24, 215; accepted April 2, 215 Abstract Background: Glycated hemoglobin ( ) is widely used for the monitoring of glycemic balance in diabetic patients and has also been proposed as a tool for the diagnostic of diabetes mellitus. Accordingly, quantification must be performed using robust, reliable and efficient methods. Here are reported the results of the evaluation of a new high performance liquid chromatography (HPLC) system for quantification, the D- TM system from Bio-Rad Laboratories. Methods: The analytical performances of the method as well as the influence of the most frequent interferences regarding assays (e.g., labile, carbamylated hemoglobin, high HbF) have been tested. Results: Intra- and between-assay CVs were respectively lower than.93% and 1.46% ( results expressed in NGSP units) and lower than 1.67% and 2.27% ( results expressed in IFCC units). The linearity proved to be excellent from 15 mmol/mol (3.5%) to 184 mmol/mol (19.%) (r =.999). The results were well correlated with those obtained by another HPLC method (VARIANT TM II Hemoglobin A1c Program reorder pack NU-Bio-Rad): [VARIANT TM II] = 1.13 [D- TM, mmol/mol]+.637 (r =.993, n = ). The D- TM system provided results consistent with IFCC-assigned external quality control samples and the presence of labile, carbamylated hemoglobin and HbF did not interfere with measurement. *Corresponding author: Dr. Stéphane Jaisson, Laboratory of Paediatric Biology and Research, Department of Biology, University Hospital of Reims, 45 rue Cognacq-Jay, 5192 Reims Cedex, France, Phone: , Fax: , sjaisson@chu-reims.fr Nathalie Leroy, Emmanuelle Guillard, Aurore Desmons and Philippe Gillery: Laboratory of Paediatric Biology and Research, University Hospital of Reims, France Conclusions: The D- TM system proved to be a robust and reliable method for measurement suitable for routine practice in clinical chemistry laboratories. Keywords: diabetes; evaluation; ; HPLC. Introduction, the major form of glycated hemoglobin, is considered the gold standard for the monitoring of glycemic balance in diabetic patients, and is also used in many countries as a diagnostic tool for diabetes mellitus [1, 2]. In addition, values of are correlated with the development of long-term complications in both type 1 and type 2 diabetes [3, 4]. results from the non-enzymatic binding of glucose on N-terminal valine residues of hemoglobin (Hb) β chains. This binding leads to changes in chemicophysical properties of the protein, which have allowed the development of various assays for its quantification. Nowadays, various analytical methods have been developed based on different principles ranging from separative methods [e.g., ion-exchange or boronate high performance liquid chromatography (HPLC) or capillary electrophoresis] to immunological and enzymatic assays. Many efforts have been made by manufacturers to improve the analytical performances of the methods and to guarantee their overall comparability and their traceability to the IFCC reference system [5, 6]. Recently, a new HPLC system intended for quantification has been developed (the D- TM system, Bio-Rad Laboratories) whose analytical performances, throughput and usability are expected to be improved in comparison to previously distributed systems. This β evaluation study aimed at evaluating the analytical performances of this testing device, a special attention being paid to the influence of the most frequent interferences encountered with such HPLC-based methods, and to the usability of the system.

2 2 Jaisson et al.: Evaluation of the D- TM system Materials and methods The analyzer and the reagents used for this evaluation have been provided by Bio-Rad Laboratories (Hercules, CA, USA) and have been used according to manufacturer s instructions. D- TM system The D- TM system consists of a fully-integrated standalone workstation of automated HPLC instrumentation that includes an onboard computer. The software operating the instrumentation is controlled through a graphical user interface via an onboard touchscreen. The principle of the assay is based on the separation of Hb fractions by ion-exchange HPLC. This separation is performed thanks to an easy to install chromatography cartridge with long lifetime (up to 1, tests), preceded by a prefilter. The loading area may receive 1 sample racks, each rack containing 1 positions for whole blood primary capped tubes (5 or 7 ml), with a minimal blood volume of approximately 1 ml (i.e., > 1 cm of blood from the bottom of the tube). For samples with a lower volume, an external dilution may be performed using wash solution (minimal blood volume: 5 μl). Emergency or quality control samples may be loaded via a stat area which is also used for calibrators. The calibration pack allows a one-touch calibration (the reconstitution of calibrators is performed automatically by the analyzer) and a two-point calibration is performed once following the installation of the cartridge. The separation of Hb fractions is performed within 45 s. At each series of assays, the first sample is preceded by flushing and preparation of the system. As a consequence, the first result is obtained after 2 min and 15 s, whereas the following results are obtained each 45 s, allowing a throughput up to 8 samples/h. is eluted just after a peak containing the labile fraction of (LA 1c ) and carbamylated Hb (chb) and before HbA (Figure 1). The result is calculated from the ratio /(all HbA-derived fractions) and are expressed in IFCC units (mmol/mol) and/or in NGSP units (%) after conversion using the IFCC-NGSP master equation [5]. Any Hb fraction that can eluate after HbA peak is excluded. Blood samples Blood samples sent to the laboratory for routine assay have been used for this study. These samples were collected in EDTAcontaining tubes (BD Vacutainer, ref ) and were stored at 4 C before analysis on D- TM system if the assay was not immediately performed. None of these samples were kept after analysis. Quality control (QC) samples (Liquichek Diabetes Controls Trilevel, ref. 171, 172 and 173) and blood samples containing LA 1c, chb, HbF, HbA 2 or Hb variants have been provided by Bio-Rad and prepared as described below. Precision study Intra-assay precision was evaluated by performing 2 assays from the same sample (either frozen ( 8 C) blood samples with different values or quality control samples) within the same series Figure 1: Typical Hb chromatogram obtained using D- TM system.arrows indicate the main hemoglobin fractions, including and HbA in dark characters. Between-assay precision was assessed by measuring in control materials or frozen ( 8 C) blood samples twice a day in duplicate in 2 different series. Linearity study The linearity of the method was evaluated by using the Bio-Rad Lyphocheck Hemoglobin A 1c Linearity Set (ref. 7). This kit was composed of six levels with expected values ranging from 15 to 184 mmol/mol (3.5% 19.%). Each sample has been assayed twice and the regression analysis has been performed using the average value. Correlation study Two thousand samples have been assayed using D- TM system and the HPLC system routinely used in our laboratory (VARIANT TM II, Hemoglobin A1c Program Reorder Pack, NU, Bio-Rad Laboratories), and results were compared using a linear regression analysis and by calculating the ratio of the obtained values. The values were selected to be distributed across the linearity domain (from 19 to 148 mmol/mol, from 3.9% to 15.7%). Samples with

3 Jaisson et al.: Evaluation of the D- TM system 3 Hb variants or exhibiting chb or LA 1c rates exceeding, respectively, 2.5% and 5%, were excluded from the comparison study. All comparative assays were performed within a 6 h-period, the samples being kept at +4 C until analysis. Accuracy The accuracy of the method has been assessed by analyzing 1 samples deriving from an external quality assurance scheme (Educational Programme 214, European Reference Laboratory for Glycohemoglobin, Winterswijk, The Netherlands). These samples are blood hemolyzates with values assigned using the IFCC reference method in an approved laboratory of the IFCC network. Relative and absolute biases were calculated by comparing the values obtained with D- TM system with the IFCC target values. Interferences Interference of LA 1c was determined by measuring in two blood samples containing increasing proportions of LA 1c. Briefly, a part of each sample has been incubated for 3 h at 35 C in the presence of.5 M glucose in order to generate LA 1c. Samples with intermediate levels of LA 1c were obtained by spiking the initial blood sample with various amounts of the LA 1c -containing fraction. A similar approach has been used for evaluating the interference of chb. Hb carbamylation has been obtained by incubating blood samples for 3 h at 35 C in the presence of 1 mm potassium cyanate. LA 1c and chb rates were estimated using VARIANT TM II system. values of native samples were used as a reference for calculating the relative biases. Interference of fetal hemoglobin (HbF) was evaluated by mixing blood samples with increasing amounts of purified cord blood in order to obtain HbF levels ranging from 5.4% to 2% (HbF rates have been estimated using D- TM system). values obtained in spiked and non-spiked samples were compared. In a qualitative approach, we have analyzed samples containing the most prevalent Hb variants (HbS, HbC, HbD and HbE) and HbA 2, in order to determine the elution profile of these variants and their potential impact in calculation. Results Linearity and precision This method exhibited a good linearity for values ranging from 15 mmol/mol (3.5%) to 184 mmol/mol (19.%). The equation of the linear regression line was (experimental values) =.988 (expected values)+1.155, with a correlation coefficient r equal to.999 (data not shown). Intra-assay coefficients of variation (CVs) were lower than 1.59% for QC samples and 1.67% for patient samples, when calculated from values expressed in IFCC units (mmol/mol). With calculations based on data expressed in NGSP units (%), the intra-assay CVs were respectively lower than.8% and.93% for QC and patient samples (Table 1). Between-assay CVs were lower than 1.86% for QC samples and 2.27% for patient samples when calculated using IFCC units and lower than 1.27% and 1.46%, respectively, using NGSP units. Correlation The comparison of values obtained using D- TM system and VARIANT TM II system showed a good correlation, with the following equation for the linear regression line: y ( VARIANT TM II) = 1.13 ( D- TM )+.637, and a coefficient of correlation r =.993 (Figure 2A). The plot expressing the ratio of values as a function of the best estimated value showed that 98.7% of the ratios were comprised between.9 and 1.1 (i.e., relative biases lower than 1%) and that the average ratio was equal to.97 and did not vary over the range of tested values (Figure 2B). Accuracy The accuracy of the method was assessed by comparing the results obtained using D- TM system in 1 samples deriving from an external quality assurance scheme with IFCC-assigned values (Table 2). The accuracy Table 1: Intra- and between-assay precision of the D- TM system. mmol/mol % Mean (SD) CV, % Mean (SD) CV, % Intra-assay precision (n = 2) Sample (.5) (.4).82 Sample (.7) (.6).93 Sample (.8) (.7).88 Sample (1.1) (.1).91 QC sample (low-level) 31.4 (.5) (.4).8 QC sample (medium-level) 8.4 (.8) (.7).74 QC sample (high-level) (1.1) (.1).69 Between-assay precision (n = 4) Sample (.7) (.7) 1.41 Sample (.7) (.7) 1.8 Sample (1.1) (.1) 1.26 Sample (1.8) (.16) 1.46 QC sample (low-level) 32.3 (.6) (.6) 1.17 QC sample (medium-level) 79.9 (1.3) (.12) 1.27 QC sample (high-level) (1.8) (.16) 1.11 QC, quality control.

4 4 Jaisson et al.: Evaluation of the D- TM system A B 1.2 VARIANT TM II y=1.13x r=.993 Ratio D- TM /VARIANT TM II D- TM Best estimated mean Figure 2: Comparison of values obtained with D- TM and VARIANT TM II systems (n = ). (A) Correlation plot with the corresponding linear regression line. (B) Plot showing the ratio of D- TM to VARIANT TM II values as a function of the best estimated value (average of the values obtained with both systems). proved to be excellent since the absolute biases did not exceed 1 mmol/mol and the relative biases were comprised between 2.7% and +2.4% (the average relative bias being equal to +.17%). These results fit with the NGSP certification criteria where 92.5% of the results must have biases comprised within ±6% in comparison with the target value. Analytical interferences The chromatogram shown in Figure 1 indicates that LA 1c and chb are coeluted just before the peak, suggesting a potential interference of these two Hb fractions on quantification. However, the analysis of samples presenting elevated rates of these fractions demonstrated that LA 1c and chb did not interfere with measurement by D- TM system (Tables 3 and Table 4). Indeed, the quantification of was not influenced by the presence of LA 1c until 5.2% of this fraction, with relative biases varying randomly between 4.9% and +7.4%. Similar results were obtained with chb levels (until 3.3% of chb), the corresponding relative biases being comprised between 5.% and %. HbF, HbA 2 and Hb variants The interference of HbF on quantification was evaluated by assaying samples spiked with increasing quantities of HbF (until 2% HbF). results were not modified by the presence of HbF given that relative biases were lower than 4.8% (Table 5). The influence of HbA 2 and of the most prevalent Hb variants has also been evaluated in a qualitative approach. The aim of this approach was to determine where these Table 2: Comparison of results obtained using D- TM system with IFCC-assigned values for 1 external assurance quality samples. Control sample Absolute bias, IFCC target Measured mmol/mol values values Relative bias, % Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Table 3: Influence of the labile fraction of HbA 1C (LA 1C ) on measurement by D- TM system. LA 1c, % a b Relative bias, % Sample Sample a LA 1c rates have been estimated using VARIANT TM II system; b Average value (n = 2).

5 Jaisson et al.: Evaluation of the D- TM system 5 Table 4: Influence of carbamylated hemoglobin (chb) on measurement by D- TM system. chb, % a b Relative bias, % Sample Sample a chb rates have been estimated using VARIANT TM II system; b Average value (n = 2). variants were eluted in regard to and HbA peaks and if they could alter the fitting of these peaks and the calculation of. As shown on the chromatograms, all Hb variants as well as HbA 2 were eluted after HbA peak (Figure 3). As any peak located after HbA is not integrated in the calculation of, the variants should not interfere with quantification. Moreover, for some peaks eluted close to HbA peak, the software applies a specific fitting of HbA peak taking into account the presence of the variant. Besides, no additional peak which could correspond to the glycated forms of Hb variants has been observed in these chromatograms. Usability and ergonomics The D- TM system proved to be a very easy-to-use analyzer with a simple installation and replacement of buffers (even during a run) and column/prefilter. This system is Table 5: Influence of fetal hemoglobin (HbF) on measurement by D- TM system. HbF, % a b Relative bias, % Sample Sample a HbF rates have been estimated using VARIANT TM II system; b Average value (n = 4). able to perform assays at a high throughput (45 s per sample). The maintenance of the system is limited and the lifetime of the analytical column is very long (1, injections, and injections for the prefilter), with no intermediate calibration (the system is calibrated only once after the installation of the column). The performances of the column after 1, injections have been evaluated by analyzing 7 samples using this column and a new one (data not shown). The comparison of values showed a good correlation which confirm that the column keeps its performances during the course of its lifetime. Indeed, the ratios of values (values obtained using the new column vs. those obtained using the old column) were comprised between.98 and 1.3 with an average ratio of 1.1. The traceability of reagents is ensured by a Radio Frequency Identification (RFID) system. The system is controlled by an intuitive software composed of a friendly interface for QC tracking and result review, and a large storage capacity. An on-board advisor, which is fully configurable by the user, allows a quick validation of results by flagging results that need further review or by performing automatic resampling. Discussion is now used for the monitoring of glycemic balance of diabetic patients as well as a diagnostic tool for diabetes diagnosis in many countries [2]. Thus, quantification must be performed using robust, accurate but also convenient methods in clinical chemistry laboratories. Many systems have been developed for assay based on different technologies, the most common ones being HPLC and immunological assays. Following the development of successive HPLC systems devoted to quantification over the last decades, Bio-Rad Laboratories have recently implemented a new HPLC system in clinical chemistry laboratories, the D- TM system, which benefited from different improvements in terms of performance, usability and throughput. Herein, we have evaluated the performances of this new testing system. Precision studies showed good performances since intra- and between-assay CVs were lower than 1.46% in NGSP units, and 2.27% in IFCC units. These CVs are consistent with the current recommendations for such systems [2, 7], being higher when obtained using IFCC values [8]. The linearity proved to be excellent over the clinical range of values and the evaluation of accuracy based on the use of control samples deriving from an external quality assurance scheme demonstrated the

6 6 Jaisson et al.: Evaluation of the D- TM system HbF HbS HbC HbA 2 HbD HbE Figure 3: Chromatograms obtained with samples exhibiting abnormal percentages of fetal hemoglobin, elevated HbA 2 or the most prevalent hemoglobin variants (HbS, D, C and E). The grey peak corresponds to peak whereas peaks corresponding HbF, HbA 2 or Hb variants are indicated by arrows. traceability of the method to the IFCC reference measurement procedure. Moreover, the results obtained with D- TM system were well correlated with results obtained using the VARIANT TM II system (r =.994), with a low proportion (1.3%) of outliers which corresponded to relative bias above than 1%. None of the most frequent analytical interferences encountered using assays (i.e., LA 1c and chb) had a significant impact on values. In the same way, elevated percentages of HbF (until 2%) did not interfere with determination using this system. For evaluating the potential influence of Hb variants or elevated percentages of HbA 2, we used a qualitative approach to determine where these Hb fractions were eluted. All the tested Hb variants (i.e., Hb S, C, D and E) as well as HbA 2 were eluted after HbA. Taking into account that all the fractions eluted after HbA are not included in the calculation formula and that, for some of them which are closely eluted with HbA (e.g., HbA 2 or HbE), a specific fitting of HbA peak is performed for taking into account the shoulder, it could be expected that these Hb variants do not interfere with quantification. However, additional experiments are needed to confirm the ability of the system to provide reliable results in the presence of Hb variants, especially by performing a large scale and quantitative study based on the comparison with affinity chromatography-based methods [9]. In conclusion, the D- TM system exhibited good analytical performances for such a assay, with improvements in terms of usability and ergonomics, making this system suitable for a routine practice clinical chemistry laboratories performing large assay series.

7 Jaisson et al.: Evaluation of the D- TM system 7 Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Financial support: This study was supported by Bio-Rad Laboratories which provided the D- TM system as well as the corresponding reagents, quality control samples, and blood samples containing LA 1c, chb, HbF, HbA 2 and Hb variants, in the frame of a contract established with the University Hospital Center of Reims and the University of Reims Champagne-Ardenne. Employment or leadership: This beta site evaluation was conducted under a contract established between the University Hospital Center of Reims and Bio-Rad Laboratories (Hercules, CA, USA). Bio-Rad Laboratories has played no role neither in the interpretation of data, nor in the writing of this publication. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. References 1. The International Expert Committe. International Expert Committee report on the role of the A 1c assay in the diagnosis of diabetes. Diabetes Care 9;32: Sacks DB, Arnold M, Bakris GL, Bruns DE, Horvath AR, Kirkman MS, et al. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem 211;57:e The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329: UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998;352: Hoelzel W, Weykamp C, Jeppsson JO, Miedema K, Barr JR, Goodall I, et al. IFCC reference system for measurement of hemoglobin A 1c in human blood and the national standardization schemes in the United States, Japan, and Sweden: a method-comparison study. Clin Chem 4;: Jeppsson JO, Kobold U, Barr J, Finke A, Hoelzel W, Hoshino T, et al. Approved IFCC reference method for the measurement of HbA1c in human blood. Clin Chem Lab Med 2;4: Little RR, Rohlfing CL, Sacks DB. Status of hemoglobin A1c measurement and goals for improvement: from chaos to order for improving diabetes care. Clin Chem 211;57: Weykamp CW, Mosca A, Gillery P, Panteghini M. The analytical goals for hemoglobin A 1c measurement in IFCC units and National Glycohemoglobin Standardization Program Units are different. Clin Chem 211;57: Lin CN, Emery TJ, Little RR, Hanson SE, Rohlfing CL, Jaisson S, et al. Effects of hemoglobin C, D, E, and S traits on measurements of by six methods. Clin Chim Acta 212;413: