Evaluation of the Menarini Arkray HA 8140 hemoglobin A 1c analyzer

Clinical Chemistry 43:6 968 975 (1997) Endocrinology and Metabolism Evaluation of the Menarini Arkray HA 8140 hemoglobin A 1c analyzer W. Garry John, 1 * Francois Braconnier, 2 Kor Miedema, 3 Carlos Aulesa, 4 and Giampiero Piras 5 We describe a multinational evaluation of the Menarini Arkray HA 8140 hemoglobin (Hb) A 1c analyzer, which utilizes a high degree of automation, including bar code reading, cap piercing, and whole-blood sampling. Within- and between-batch CVs were <2%. Linearity was confirmed throughout the working range of the analyzer. Common Hb variants, including Hb S, Hb C, and Hb F, did not interfere with the Hb A 1c separation, and the potentially interfering labile Schiff base was effectively removed during the chromatographic procedure. The HA 8140 analyzer displayed good correlation to the Bio-Rad Variant analyzer, Tinaquant immunoassay, affinity chromatography, and an optimized in-house HPLC Hb A 1c method. The methods when compared by Altman and Bland plots showed bias (upper, lower 95% confidence limits) of: Variant minus HA 8140 0.99 (0.23, 1.74), Tinaquant minus HA 8140 0.14 ( 0.71, 0.98); affinity minus HA 8140 (after log transformation) 1.13 (0.90, 1.41), and in house HPLC minus HA 8140 (after log transformation) 0.91 (0.82, 1.01). INDEXING TERMS: diabetes mellitus hemoglobin variants HPLC immunoassay. In almost a quarter of a century since the introduction of the first clinically useful method for the estimation of glycohemoglobin (GHb) [1], its measurement has become of central importance in the management of the diabetic 1 Department of Clinical Biochemistry, The Royal London Hospital, Whitechapel, London E1 1BB, UK. 2 Biochemistry Laboratory, Henri Mondor Hospital, 94010 Creteil, France. 3 Central Laboratory, Ziekenhuis De Weezenlande, 8011 JW Zwolle, The Netherlands. 4 Department of Hematology, Hospital de la Valle Hebron, 08035 Barcelona, Spain. 5 Anti Diabetic Centre, S. Michele Hospital, Via Peretti, (09100) Cagliari, Italy. *Author for correspondence. Fax 44 171 377 7777. Received September 20, 1996; revised February 12, 1997; accepted February 25, 1997. patient. 6 The number of requests received by laboratories for its measurement has increased probably to a greater extent than early workers could have estimated. The previously published findings of the Diabetes Control and Complications Trial (DCCT) resulted in highlighting the importance of good glycemic control in delaying the onset of diabetic complications [2], confirming the need for high-quality methods for GHb measurement. Early methods for the measurement of GHb were based on charge separation, either involving minicolumn cation-exchange chromatography [1] or agar gel electrophoresis [3]. Both methods had a number of interferences, most notably the adverse affect of abnormal Hbs. Since these early methods, techniques have been developed that overcome these problems. HPLC techniques have proved to be very successful with ion-exchange separation methods [4]; additionally, methods based on affinity separation [5] and immunoassay [6] have also been described and are widely used in the clinical laboratory. Increasing numbers of requests are being received by laboratories for GHb estimation, while these laboratories are being faced with increased staffing pressures. Therefore there is a need for highly automated GHb analyzers capable of a high sample throughput. The aim of this study was to fully evaluate a new Hb A 1c analyzer, the Menarini Arkray HA 8140, and compare results obtained with those from established methods in clinical use. The HA 8140 analyzer replaces the HA 8121 analyzer; the HA 8121 is also based on ion-exchange separation, but is incapable of accurately measuring Hb A 1c in the presence of abnormal Hbs. The newer HA 8140 also incorporates a bar code reader and cap piercing capability. Materials and Methods hplc Menarini Arkray KDK HA 8140 analyzer. The HA 8140 is a fully automated HPLC analyzer manufactured by Arkray 6 Nonstandard abbreviations: GHb, glycated hemoglobin; DCCT, Diabetes Control and Complications Trial; Hb, hemoglobin; PBS, phosphate-buffered saline; and TPP, tetrapolyphosphate. 968

Clinical Chemistry 43, No. 6, 1997 969 KDK (formerly Kyoto Daiichi, Kagaku) Kyoto, Japan; the instrument is distributed by Menarini Diagnostics (Florence, Italy) and their subsidiary companies. The HA 8140 is at present unique among HPLC analyzers in that all operations are fully automated. The automatic feed to the analyzer holds up to a maximum of 100 samples at any one time. Bar coded samples are placed in racks that will hold up to 10 samples; additionally, the analyzer may be continuously fed with samples. After reading the bar code, the analyzer samples directly from the primary sample tube; if this tube incorporates a rubber stopper, the instrument will pierce the cap, thereby eliminating the need to remove it. The HA 8140 samples 3 L of whole blood from the bottom of the sample tube; the blood is automatically hemolyzed with a tetrapolyphosphate (TPP) buffer at ph 6.0, and the hemolyzed sample is held in a thermal-jacketed loop for 2 min at 48 C. Incubation in TPP has been shown to effectively remove the labile Schiff base [7]. After this time the loop is switched and the hemolysate injected onto a methacrylic acid and methacrylate copolymer column (Micronex A 1c -HSII column unit; Sekisui Chemical Co., Tokyo, Japan) thermostatically controlled at 40 C. Separation is achieved with discrete addition of three phosphate buffers (ph 4.8) containing 6% inorganic phosphate; these are supplied by the manufacturer. Buffer is added in the order A, C, B; then A again. This will regenerate the column, switching is under computer control, and results are available in a further 2 min. A true analytical throughput of 15 samples per hour is achieved. Results are presented as %Hb A 1 and %Hb A 1c ; these are calculated from the peak areas of the different Hb fractions as a percentage of the total Hb. Extrapolation of the peaks to baseline is under the control of the analyzer s computer; the calculation of the percent Hb A 1c may include a minor peak present as a shoulder eluting before the main Hb A 1c fraction. This shoulder increases/decreases with the Hb A 1c and can be seen with several HPLC methods that have a longer elution time. If a small or a capillary sample is received, the HA 8140 analyzer will automatically switch to analyzing hemolysates that are manually diluted 1:150 in the buffer provided. This is achieved by leaving the first four positions in the rack empty; the analyzer will automatically treat the sample in position five as a hemolysate. The analyzer is not calibrated; results are based on the natural color of the Hb in the fractions measured at a wavelength of 415 nm (blanking wavelength 500 nm) as they elute from the column. The analyzer does have the capability of automatically correcting results after standardization with a reference material when this becomes available. A typical chromatogram is shown in Fig. 1. Variant Hb A 1c analyzer. After initial pretreatment, samples were loaded onto the Variant automated Hb A 1c analyzer (manufactured by Toya-Soda, Tokyo, Japan), which is distributed by Bio-Rad Labs., Hemel Hempstead, UK. All Fig. 1. Separation obtained with normal adult Hb (Hb A). The Hb A 1c peak may, as in this case, include a shoulder. reagents used were supplied by Bio-Rad, and analyses performed according to the manufacturer s instructions. In-house cation-exchange chromatography. Samples were prepared by adding 45 L of whole blood to 2.5 ml of hemolyzing solution (acid potassium phthalate 25 mmol/l; KCN 8 mmol/l; Triton X-100 10 ml/l, ph 5.5), mixed vigorously, sonicated (2 min), and then incubated for 45 min at 25 C to eliminate the labile fraction. Chromatography was performed with a HPLC cationexchange system with a PolyCat A (diameter 4 50 mm) column. A two-buffer gradient was used for separation. The starting buffer consisted of 0.04 mol/l Bis Tris, 0.004 mol/l KCN, and 5 ml/l Triton X-100 adjusted to ph 6.65; the second buffer was the same except for addition of 0.2 mol/l NaCl. This was a modification of a method previously described [8, 9]. The precision (CV) of this method was 2.1% for Hb A 1c of 10.8%, and the range of results found in a nondiabetic population was 3.7 4.4% Hb A 1c. immunoturbidimetry All reagents used in this latex particle immunoassay were supplied by Boehringer Mannheim (Lewes, UK) and performed according to the manufacturer s instructions. The method is calibrated with calibrators whose values have been assigned by HPLC; the method was performed on an Hitachi (Tokyo, Japan) 717 analyzer, which gave an imprecision of 4.6% at 9.8% Hb A 1c.

970 John et al.: Evaluation of Menarini Arkray Hb A 1c analyzer affinity chromatography In addition to measuring Hb A 1c, we measured total GHb by affinity chromatography (Pierce and Warriner, Chester, UK). This method has been previously described [10], and a precision of 1.5% at a concentration of 18.7% GHb was reported. variant hb identification The different variant Hbs were identified either by isoelectric focusing on thin polyacrylamide gel (ph 6 9) as previously described [11], or by HPLC with cation-exchange chromatography, and structurally studied by specific methods of protein biochemistry. samples Samples were collected into bottles containing EDTA; additionally, samples were collected into lithium heparin and fluoride oxalate to investigate the effect of different anticoagulants. Samples were collected by venesection from healthy volunteers or from patients having blood samples collected for medical reasons; permission was sought before venesection and samples were analyzed blind in accordance with local ethical recommendations. effect of the labile schiff base Two EDTA whole-blood samples with %Hb A 1c values within the nondiabetic and diabetic ranges were centrifuged, and the plasmas removed and set aside. The red cells from both samples were divided into three aliquots and suspended in phosphate-buffered saline (PBS), ph 7.0, and in PBS containing 20 mmol/l glucose and 50 mmol/l glucose. The suspensions were incubated at 37 C for 4 h. After this, the red cells were again separated by centrifugation and resuspended in their own plasma; Hb A 1c analysis was performed in duplicate on these samples within 15 min of resuspension. effect of hemoglobin f Ten samples were analyzed for %Hb A 1c. Two 500- L aliquots of these samples were taken; one aliquot was supplemented with 100 L of cord blood, the other with 150 L of cord blood. These samples were again analyzed for %Hb A 1c. Results imprecision Within-batch. Samples collected from two diabetic patients and a nondiabetic subject were analyzed 20 times within a single analytical batch. The results obtained are shown in Table 1. Between-batch. The above three samples were analyzed 20 times over 2 weeks; samples were stored at 4 C during this period. Only one batch of mobile phase was used during this study, but the analyzer was switched off/on, and purged between each analysis. Additionally, control samples were analyzed twice daily during this study. The results obtained are shown in Table 1. linearity Linearity was investigated with a sample with a low Hb A 1c (2.4%) and one with a high Hb A 1c (14.2%). Hemolysates (1:150) were made of the samples in hemolyzing buffer. These two hemolysates were then mixed with each other in different proportions to produce a range of Hb A 1c results; expected results for the dilutions were calculated from the results obtained on the high and low samples. The regression between the measured %Hb A 1c (y) and the calculated value (x) was y 1.03x 0.12; r 1.0. method comparison Samples collected from nondiabetic and diabetic subjects and analyzed for Hb A 1c on the HA 8140 were additionally analyzed for Hb A 1c with the Bio-Rad Variant HPLC analyzer, a published in-house HPLC method, and a latex particle immunoassay, and total GHb was measured by affinity chromatography. The regressions obtained are shown in Fig. 2. The methods were also compared by using Altman and Bland plots; these produced mean bias (lower, upper 95% confidence limits) of: Bio-Rad Variant minus HA 8140 0.99 (0.23, 1.74), Tinaquant minus HA 8140 0.14 ( 0.71, 0.98). Altman and Bland comparison of the HA 8140 and the in-house method and affinity chromatography were performed after log transformation of the data. This was done because the bias was not constant (Fig. 2b and 2d); the bias increased with increas- Table 1. Imprecision for Hb A 1c on the HA 8140 analyzer with blood collected from diabetic and nondiabetic subjects and with commercial quality-control (QC) material. Diabetic Nondiabetic QC QC Within-batch imprecision Mean, %Hb A 1c 10.83 4.51 10.77 4.49 SD, %Hb A 1c 0.21 0.05 0.29 0.06 CV, % 1.7 1.1 2.68 1.24 Between-batch imprecision Mean, %Hb A 1c 10.58 4.39 9.47 4.30 SD, %Hb A 1c 0.20 0.09 0.12 0.05 CV, % 1.9 2.0 1.23 1.12 Samples from diabetics and nondiabetics were analyzed 20 times. QC samples were analyzed either 30 (within-batch) or 76 (between-batch) times.

Clinical Chemistry 43, No. 6, 1997 971 Fig. 2. Regression of Hb A 1c results obtained with the HA 8140 analyzer and (a) the Bio-Rad Variant analyzer; (b) an optimized in-house HPLC method; (c) latex particle immunoassay; and (d) affinity chromatography. ing results: in-house HPLC minus HA 8140 0.91 (0.82, 1.01), affinity chromatography minus HA 8140 1.13 (0.90, 1.41). anticoagulants Ten subjects, 6 diabetics and 4 nondiabetics, had samples collected into different anticoagulants, and each sample was analyzed for Hb A 1c. The mean Hb A 1c result obtained with EDTA was 6.88%, whereas the mean result (mean difference from EDTA) for lithium heparin was 6.94% (0.06%) and for fluoride oxalate was 6.97% (0.08%). The results obtained on the different anticoagulants did not differ significantly (P 0.05) from each other. reference range Blood was collected from 399 subjects into an EDTA tube and a fluoride oxalate tube. Hb A 1c was measured on the EDTA tube. Subjects all denied being diabetic, and random blood glucose measured on the fluoride oxalate sample was 6.5 mmol/l in all cases. Results displayed a normal distribution, which produced a nondiabetic reference range of 3.7 5.1% Hb A 1c. abnormal hbs Hb F. The Hb A 1c results before and after addition of cord blood to 10 samples containing normal adult Hb are given in Table 2. Addition of the Hb F did not affect the

972 John et al.: Evaluation of Menarini Arkray Hb A 1c analyzer Table 2. Hb A 1c results obtained with the HA 8140 analyzer before and after addition of Hb F. Hb A 1c,% Fetal blood Original result 100 L 150 L 4.5 4.4 4.6 7.1 6.1 6.4 8.3 8.4 8.3 6.5 6.2 6.2 11.6 10.8 11.1 4.9 4.8 4.8 7.2 6.7 6.4 9.0 8.8 8.3 5.9 6.0 6.0 8.9 8.2 7.4 Mean Hb F 0.4 10.0 17.1 Hb A 1c result obtained, the increased Hb F peak being seen well in advance of the Hb A 1c peak (Fig. 3a). Variant Hbs. The HA 8140 system incorporates separation conditions that will detect many Hb variants; a number of the variants were studied in this evaluation. With the common Hb variants such as Hb S, Hb C, and Hb D Punjab, the abnormal Hb fraction eluted as a single fraction after the Hb A 0 peak; the variant was identified by the analyzer by producing a variant Hb message, and the abnormal fraction indicated by a hatched peak (Fig. 3b and 3c). Analysis of samples that did not contain Hb A (e.g., Hb SS and Hb SC) has shown that the glycated variant Hb peak is well separated from the Hb A 1c peak, and will not be included in the calculated %Hb A 1c. The %Hb A 1c is automatically calculated as a ratio of the Hb A 1c peak compared with total Hb minus the variant Hb fraction. When no Hb A is present in the sample, as for example in the variant Hb SC, an abnormal separation message is printed and no result displayed (Fig. 3e). One common Hb variant (Hb E) did not always separate. In this case a shoulder that appears behind the Hb A 0 peak can be easily identified (Fig. 3d); no error message is printed. The major peak includes Hb A 0 and Hb E 0, and the glycated Hb E fraction elutes separately from the Hb A 1c fraction, thereby producing an artificially low %Hb A 1c result. The presence of the Hb E variant is easily recognized from the chromatogram produced by the analyzer; the chromatographs should always be examined to eliminate a wrong result being generated. Several uncommon Hb variants were also investigated; Table 3 shows whether the HA 8140 detects the abnormal Hb. Separation of the major fractions of Hb A and variant Hb will allow the correct calculation of %Hb A 1c ;asinall cases the glycated variant fraction elutes separately from the Hb A 1c peak. If no Hb A is present in the sample, an abnormal separation message is printed and no result displayed. effect of the labile schiff base The results obtained by incubating in 20 mmol/l and 50 mmol/l glucose are shown in Table 4. There was a trend to increased results in the glucose-incubated samples, but the largest increase was only 0.3% Hb A 1c in the nondiabetic sample; this increase was well within the precision of the method. Discussion The introduction of GHb measurements has revolutionized the clinical management of diabetic patients and provided objective guidelines for diabetes research. Additionally, the publication of the DCCT [2] has highlighted the need for high-quality, reliable GHb methods; the increasing number of requests and the requirement for improved turnaround time for results has led to a need for methods that are capable of a high specimen throughput and that require limited manual intervention. The Menarini Arkray KDK HA 8140 analyzer is unique among HPLC analyzers in that all aspects of analysis have been automated: bar code reading, primary sample tube and cap piercing, automatic hemolyzing, and analysis. Described here is a multinational evaluation of this HA 8140 Hb A 1c analyzer; investigators in each country using their own analyzer contributed a specific aspect to the study. The Hb A 1c analyzer gave good within- and betweenbatch precision. Both comply with the within- and between-batch imprecision of 5% suggested by the National Diabetes Data Group [12], and a maximum CV of 5% with an optimum CV of 2% published by Larsen et al. [13] as desirable for the assay. The analyzer was found to produce good linearity through the operating range, and when compared with several GHb methods widely used in clinical laboratories showed a high degree of correlation. Results obtained on the HA 8140 analyzer were found to be negatively biased when compared with the Bio-Rad Variant analyzer, the Variant analyzer displaying results 1.0% Hb A 1c higher than the HA 8140, this bias being constant throughout the range as shown by a slope of 0.95. The constant bias found suggests that this difference may be due to the calibration factor used by the Variant analyzer; the HA 8140 analyzer does not use standardization (although it has the ability to be calibrated). Good agreement (r 0.99) was also found with an in-house HPLC method, the conditions of which had been optimized for Hb A 1c separation. In this case the HA 8140 results were higher than those obtained with the optimized HPLC, the mean bias (after log transformation) being 0.91; this may reflect a cleaner separation obtained with a slower optimized system. The best regression was found between the HA 8140 and the latex particle immunoassay method (y 0.99x 0.06), even though these two methods involve different technologies. This good agreement is not totally surprising, as the immunoassay method is calibrated by using HPLC. When Hb A 1c results obtained with the HA 8140 are compared

Clinical Chemistry 43, No. 6, 1997 973 Fig. 3. Hb separation obtained with the HA 8140 in patients with (a) increased Hb F; (b) sickle cell trait; (c) Hb A/Hb C; (d) Hb A/Hb E (shoulder arrowed); (e) Hb S/Hb C. The glycated fraction of the variant Hb elutes separately from the Hb A 1c peak. with total GHb results measured by affinity chromatography, a difference in the results obtained is apparent; this reflects the fact that different glycated fractions are being measured. The difference in the results obtained with the two methods increases as the measured result increases. This relation has previously been identified [10], and may reflect increased glycation at sites on the Hb molecule other than the N-terminal valine; this occurs at high GHb concentrations. The differences in the slopes and intercepts of the comparison methods highlight the differences in calibration or in the type of fraction measured (i.e., total GHb or Hb A 1c ), supporting the need for standardization of methods. In the US, there has been a national attempt to overcome these result differences; this has been achieved by adopting a standardization protocol written by the AACC Subcommittee on Glycohemoglobin. The problem of result variability is currently a topic under investigation by the IFCC working party on GHb stan-

974 John et al.: Evaluation of Menarini Arkray Hb A 1c analyzer Table 3. Separation characteristics of the common Hb variants studied. Variant Error message Identifiable Correct result Common variants Hb A/S Variant Hb Yes Yes Hb A/E None Yes No Hb A/C Variant Hb Yes Yes Hb S/C Abnormal separation Yes No result Hb D Punjab Variant Hb Yes Yes Less common variants Hope Abnormal separation Yes No result Lepor None No No G Norfolk Abnormal separation Yes No result Korle Bu Variant Hb Yes Yes Hasharon Abnormal separation Yes No result dardization; this group is attempting to produce a primary calibration material and a reference method for the measurement of Hb A 1c. The reference range for results in nondiabetics in this study was 3.7 5.1% Hb A 1c, lower than that quoted for the Bio-Rad Diamat analyzer (4.3 6.1% Hb A 1c ) used in the DCCT [2]. This difference is confirmed by the 1.0% bias found when the HA 8140 was compared with the Bio-Rad Variant analyzer. Additionally, the National Glycohemoglobin Standardization Program has recently certified one of the HA 8140 analyzers used in this study, and the conversion factor of y DCCT 0.96x HA 8140 1.30 given is similar to that found in this study for the regression between the HA 8140 and the Bio-Rad Variant. The effectiveness of the HA 8140 analyzer s ability to remove the labile fraction by incubating the sample in TPP [7] was investigated by incubating red cells in PBS containing 20 mmol/l and 50 mmol/l glucose. Previous investigations of incubation conditions by one of the investigators (W.G.J., unpublished data) and others [14] have confirmed that red cells suspended for this time in these glucose concentrations reproducibly produce an increase in the labile fraction by 2% and 8% measured GHb, respectively. The samples incubated in glucose in this study showed only a small increase (0.3%) in measured Hb A 1c ; this increase may actually be due to the formation of the stable ketoamine. This increase is within the precision of the method, and in clinical terms is negligible considering the expected increase in labile Table 4. Hb A 1c results obtained with the HA 8140 analyzer measured in samples incubated in PBS and in various glucose concentrations. Nondiabetic sample Diabetic sample Mean Hb A 1c, % Difference Mean Hb A 1c, % Difference PBS 4.2 9.6 20 mmol/l glucose 4.3 0.1 9.7 0.1 50 mmol/l glucose 4.5 0.3 9.8 0.2 fraction that will have occurred after the glucose incubation. The predecessor to the HA 8140 analyzer (the HA 8121) was adversely affected by abnormal Hbs; it was therefore important to identify if, as claimed, the HA 8140 has overcome this problem. In this study a number of samples containing common and uncommon Hb variants were analyzed for Hb A 1c. The results obtained indicate that with the most commonly encountered abnormal Hbs, i.e., Hb S and Hb C, the HA 8140 can be confidently used to measure Hb A 1c. Another commonly encountered Hb variant, Hb E, is not always separated completely from Hb A 0. When separation is not achieved, this variant can easily be recognized by inspecting the chromatogram produced. Hb F, a variant that commonly interferes with GHb measurement, did not interfere with this analyzer, which can be used to measure Hb A 1c even in the presence of very high concentrations of Hb F. Of course, the presence of Hb variants may cause a decreased red cell survival, thereby increasing the Hb turnover, which leads to a decreased exposure time of this protein to glucose, resulting in a decreased percentage being glycated [15]. We thank Menarini Diagnostics, Florence, Italy, for their support during this study, and for their subsidiaries in France, Spain, The Netherlands, and the UK (Biomen Ltd.) for use of the HA 8140 analyzers. References 1. Abraham EC, Huff TA, Cope ND, Wilson JB, Bramsome ED Jr, Huisman THJ. 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