Journal of Life Sciences 6 (2012) 456-460 Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination Eloísa Urrechaga Laboratory of Clinical Analysis, Hospital Galdakao Usansolo, Galdakao 48960, Vizcaya, Spain Received: July 09, 2011 / Accepted: September 20, 2011 / Published: April 30, 2012. Abstract: Menarini ARKRAY ADAMS ™ A1C HA-8180 is a high pressure liquid chromatography (HPLC) system for the measurement of HbA1C. The analysis time is 48 seconds per sample. The analytical performance was evaluated to verify quality of analysis, according to the criteria established in the recently published documents of consensus on this analyte. Precision and linearity studies were performed according to CLSI’s guidelines. Recovery, the effect of Hemoglobin (Hb) concentration and the presence of coexistent interfering substances were evaluated. The drastic reduction in the time of analysis does not impair the overall quality of the results which were found to be accurate and linear in the clinically significant analytical range and excellent precision (total coefficient of variation bellow 0.6 %). HbA1C measurement is independent of the total concentration of Hb and the presence of Hb chemically modified elutes in the labile A1C fraction. Given the short time of the analysis this is a suitable system for the control of diabetic patients in laboratories with high workflow. Key words: Diabetes, HPLC, HbA1C, HA-8180 analyzer. 1. Introduction Glycohemoglobin (HbA1C) the N-terminal (1-deoxyfructosyl) valine glycation product of hemoglobin (Hb) A0 measurement provides the most important medium- to long-term marker of time-averaged glycemic status. Its relationship to likely clinical outcome in diabetes mellitus has been convincingly demonstrated for both type I and type II subjects in major clinical trials [1, 2]. The recently implemented consensus statement on the worldwide standardization  and the International Federation of Clinical Chemistry (IFCC) reference method for the HbA1C measurement , have contributed substantially to the quality of the HbA1C measurements. An extension of the role of this Hb fraction from monitoring to the screening and diagnosis of diabetes , leads to the possibility of even greater demand for this analysis. Various methods based on its physical, chemical or immunologic characteristics have been used for the measurement of glycohemoglobins . The automation and precision of the high performance liquid chromatography (HPLC) methods has favored their use and the technical improvements has allowed the achievement of shorter analysis times. Along with this, there have come compromises in the chromatographic quality and resolution. Menarini ARKRAY ADAMS ™ A1C HA-8180 (HA-8180) is a fully-automated HPLC device for HbA1C measurement. The most important new feature of this instrument is that the results can be obtained within 48 seconds. We have evaluated the analytical performance of this system to assess Corresponding author: Eloísa Urrechaga, Ph.D., consultant for clinical analysis, research field: analytical hematology. E-mail: [email protected] whether the reduction in the analysis time impairs the quality of the results. Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination 2. Materials and Methods 2.1 Characteristics of the HA-8180 Analyzer The HA-8180 is an automated bench top analyzer [dimensions: 530 (W) × 530 (D) × 530 (H) mm]. The instrument is designed to measure HbA1C (range 9-195 mmol/mol IFCC units; 3%-20% NGSP units), as well as hemoglobins F. Hb variants are not detected. The run time is 48 s per sample that is fast in comparison to the 170 s used by the previous generation (Model HA-8160). The instrument has a capacity of 100 samples per run. Specimens are either primary tubes with cap piercing (patient samples), or tubes for hemolisates (calibrators, controls, patients with small sample volume) placed in specific ARKRAY racks (nine types). There is an option to insert urgent (STAT) samples. The instrument can spin tubes in order to prevent blood sedimentation, automated reagent information codes, self-diagnostic functions, precision controls function and a large colour LCD. In total, 3.4 µL of automatically diluted (standard 1:100; anemic samples 1:50) whole blood is injected. The stainless steel ARKRAY column, maintained at 40 °C in an oven, consists of a prefilter and an analytical column packed with an ion exchange resin (a hydrophilic polymer of methacrylate ester copolymer). Sealing screws are made of PEEK (polyether ether ketone). Elution is achieved in a five-step phosphate buffered gradient with increasing ionic strength. There are three buffers (80A, 80B and 80CV) in aluminium foil packs placed on top of the instrument. Hbfractions are detected with a dual wavelength (420-500 nm) LED-photodiode. At this wavelength, the absorption of oxy- and deoxyhemoglobin is equivalent and thus, ensures a stable signal irrespective of the oxy-/deoxyhemoglobin ratio in the sample. The instrument is calibrated with two calibrators (low and high concentration). Calibration can be performed after power up, but is not required when operation is started from stand by. The reported result is derived from the ratio HbA1C/HbA total, adjusted for 457 calibration and expressed in both IFCC and National Glycohemoglobin Standarization Program (NGSP) units. 2.2 Samples and Controls Blood specimens were obtained from patients whose diabetic control was being assessed routinely and were used in this study according to the hospital’s ethics guidelines. Whole blood samples were obtained in the course of routine analysis and collected in EDTA anti coagulant tubes (VacutainerTM Becton-Dickinson, Rutherford, NJ, USA). Quality-control materials used throughout the evaluations were provided by Menarini Diagnostics (Glyco Hb Control, Menarini Diagnostics, Firenze, Italy). 2.3 Protocol of Evaluation The analyzer was calibrated at the beginning of the evaluation, according to manufacture’s instructions in NGSP units (%). Precision and linearity studies were performed. Sample carryover, precision and linearity studies, the effect of Hemoglobin (Hb) concentration and the presence of coexistent interfering substances were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and the manufacture’s recommendations. Carryover between blood samples with high and low HbA1C concentrations was checked using the method of Broughton et al. , processing samples of low (4.6 %) and high (12.8 %) concentration in triplicate and sequentially, examining the first and third results of each tripletfor systematic deviation. Within run precision (intra assay) was studied at two concentrations, using pooled blood from non diabetic and poorly controlled diabetic adults; these samples were assayed 10 times. Mean value and coefficient of variation (CV) of the measurements were calculated. Total precision was investigated using CLSI EP-5 458 Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination A2 protocol  with control materials (Glyco Hb Control, Menarini Diagnostics, Firenze, Italy). With this protocol, on 20 working days, a low and a high samples are assayed in duplicate twice a day in an analytical run, with at least 10 samples. EP-5 defines four parameters for the precision, all listed in Table 1. Linearity was estimated in accordance with protocol CLSI EP6 . Linear regression analysis was applied to compare expected and observed analytical values using results from proportional mixtures of high (12.5 % ) and low (5.0 %) HbA1C samples. The manufacturer’s claim is that the optimized total chromatographic area should be between 1,000 and 4,000 units. We therefore investigated the effect of varying hemoglobin concentration on chromatographic quantification. One sample was centrifuged for 10 minutes at 300 rpm to separate blood cells from plasma. Blood cells and plasma were mixed at the ratios 9:1, 8:2 … 1:9; the set of samples obtained had a broad hematocrit range. The labile fraction is separated as a distinct peak on the column. The following steps were taken to confirm the effective separation of the labile Schiff base peak. Glucose incubation: pooled blood with a HbA1C concentration 5.0% was poured into six tubes; a glucose solution was added to these test samples at increasing ratios to achieve final glucose concentrations up to 55.5 mmol/L. These samples were incubated at 37º C for two hours and then analyzed. Carbamylated Hb elutes form column in the same peak along with the labile Schiff base fraction. The following steps were taken to confirm the effective separation of this interfering chemically modified. Sodium Cyanide incubation: pooled blood with a HbA1C concentration 5.0% was poured into six tubes; a Sodium Cyanide solution was added to these test samples at increasing ratios up to 10 mmol/L Sodium Cyanide concentrations). The samples were incubated for 2 h at 37 ºC to achieve in vitro carbamylation of the hemoglobin before analysis and then analyzed. The effect of HbF concentration was investigated adding blood cord to samples before the analysis. Comparison of HA-8180 against our current routine method (Menarini/ARKRAY ADAMS A1c HA-8160, Menarini Diagnostics, Firenze, Italy) was carried out applying Passing-Bablok Linear Regression  and Bland-Altman Analysis , using samples from 170 diabetic and nondiabetic subjects being routinely monitored (range 4.7 % - 14.3 % HbA1C). 3. Results Precision within Laboratory study is summarized in the Table 1; the results of the different analytical characteristics are summarized in the Table 2. 4. Discussion HbA1C is a high volume request test in the Clinical Laboratory and therefore efficiency is required. Fully automation, high turn around time for results, robustness of the instrument and low costs are prerequisites. Efforts of manufactures contributed to Table 1 Precision study. Total precision was investigated using CLSI EP-5 guideline with commercial controls HbA1C 6.0% and 11.5%. HbA1C 6.0% Within run Between run Between day Total CV % 0.52 0 0.18 0.57 HbA1C 11.5% Within run Between run Between day Total CV % 0.24 0.14 0.1 0.29 Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination 459 Table 2 Summary of the results. The procedures are described in Materials and Methods section; fresh whole blood samples from non diabetic and poorly controlled diabetic adults were employed. Carry over (HbA1C 12.4% and 4.9%) Precision within run Linearity 0% Mean 4.2% CV 0.4%; Mean 10.5% CV 0% y = 1.0 x - 0.1, r = 0.999; analytical range 5.0% - 12.5% Total Hb concentration range 14 - 6 mmol/L NO effect HbA1C 5.4% Labile A1C fraction 4.5% NO effect HbA1C 5.4% Carbamylated Hb 5.3% NO effect HbA1C 5.4% HbF 6% NO effect HbA1C 5.4% Correlation with ADAMS HA-8160 results y = 1.07 x - 0.11 (95 % CI intercept -0.2- 0.01; 95% CI slope 1.05-1.09; P < 0.01); r = 0.997; mean difference -0.27 improvements of the analyzers creating faster systems. On the other hand, therapeutic strategies rely on reproducible and unbiased methods. These analytical and clinical requirements become even more important now, when HbA1C is on the threshold of becoming applicable for screening and diagnosis of diabetes . A high level of reproducibility in glycohemoglobin measurement is a fundamentally important requirement in the provision of laboratory support for the process of diabetes monitoring. Changes in results obtained between patient visits to the physician must reflect the pathology of the disease and its response to treatment rather than analytical uncertainty. In this respect, biological variation generally has dictated the desirable targets for analytical performance of laboratory methods . However, it has been shown that the situation in persons with diabetes is more complex, being affected by both clinical control and sampling time interval, and a practical working CV for analytical reproducibility in long-term monitoring of 2.1% has been proposed . The analyzer achieved this stringent target in our evaluations; between-run CVs of 0.2%-0.4%, within-run CVs of 0.2%, and total CVs of 0.4%-0.7% the evaluation revealed excellent reproducibility, far below the most stringent requirements of 2% . The results were reliable within a wide range of total Hb values; this feature shows the robustness of the system, so the patient could be controlled with confidence in different clinical situations. Labile A1C, the concentration of which varies with blood glucose concentrations at the time of blood collection, is an intermediate in the production of the corresponding stable moiety. The isoelectric point of the labile fraction closely approximates that of its stable counterpart, which leads to little or no separation of the two, giving falsely increased results in many methods that rely on charge separation unless sample pretreatment steps are taken to remove the labile fraction [15, 16]. Technological advances have enabled modern analyzers with high resolution capabilities to handle these potentially interfering components, thus obviating the need for pre- or on-column treatment to destroy this fraction before analysis. The presence of uremia in patients whose long-term glycemic status is being assessed is not an uncommon finding in those whose renal function has deteriorated; it can provide yet another potential interference with some, although not all, glycohemoglobin methods, because in most of the commercially available systems carbamylated hemoglobin elutes in the HbA1C labile fraction [17-19]. We have confirmed that the labile fraction is separated on the Menarini 8180 system, irrespective of its concentration and molecule Schiff base or carbamylated hemoglobin so the HbA1C remains reliable. Nevertheless the presence of Hb variants or δβ thalassemia (with HbF > 6 %), can affect the results. 5. Conclusion The drastic reduction in the time of analysis does not 460 Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination impair the overall quality of the results. The results were accurate and linear in the clinically significant analytical range. The values were independent of the total concentration of Hb and the presence of Hb chemically modified Hb eluting in the labile fraction. Given the short time of the analysis this is a suitable system for the control of diabetic patients in laboratories with high workflow, mainly in areas with low incidence of hemoglobinopathies. 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