Clinical Chemistry 43:4 644 – 648 (1997) Automation and Analytical Techniques Capillary electrophoresis system for hemoglobin A1c determinations evaluated Cees J.A. Doelman,1* Carla W.M. Siebelder,1 Wim A. Nijhof,1 Cas W. Weykamp,1 Jacques Janssens,2 and Theo J. Penders1 chromatography) for measuring glycohemoglobin, or immunological techniques [3–5]. All methods have their own advantages and well-known limitations [3–5], e.g., interferences from fetal hemoglobin (Hb F), carbamylated and acetylated hemoglobins, labile Hb A1c fractions, or hemoglobin variants. Capillary electrophoresis (CE) is a modern analytical technique that separates molecules on the basis of their charge and their hydrodynamic volume . The CE method described here for separating hemoglobin derivatives and hemoglobin variants makes use of a dynamic coating technique that allows rapid separation (#4 min) of hemoglobin variants and derivatives at pH 4.5. This proprietary coating principle was developed by Analis (Namur, Belgium) . Here we describe our evaluation of this new method, especially as developed for Hb A1c measurement. We examined some potential interferents and compared the results with those by cation-exchange HPLC. Hb A1c is the analyte of choice for monitoring metabolic control in patients with diabetes mellitus. Here we present a new analytical technique for measuring Hb A1c, capillary electrophoresis. The Hb A1c determination is not influenced by the labile Hb A1c fraction or by carbamylated or acetylated hemoglobin derivatives. Also, hemoglobin variants (Hb F, Hb S, and Hb C) do not interfere. This new application of capillary electrophoresis seems to be a valuable analytical tool for measuring Hb A1c in the clinical laboratory. diabetes mellitus hemoglobin variants INDEXING TERMS: • glycohemoglobin • Diabetes mellitus is one of the most prevalent chronic diseases of humans. Not only are affected patients predisposed to late complications (e.g., retinopathy, neuropathy, and nephropathy), but also they are at notable risk for cardiovascular disease. All of these complications are hypothesized to be the effect of chronic glycosylation of proteins and cellular structures . Measurement of Hb A1c (the reaction product of glucose and the N-terminal valine of the b-chain of hemoglobin) has been used to monitor the metabolic control of patients with diabetes mellitus. The amount of Hb A1c present is related to the risk of long-term diabetic complications, as clearly shown in the Diabetes Control and Complications Trial (DCCT) . Accordingly, Hb A1c has become a generally accepted marker for follow-up of diabetic therapy. Several analytical methods currently available measure either Hb A1c or glycohemoglobins (;60% of which is Hb A1c). These methods are based on either differences in electrical charge (HPLC, electrophoresis) for measuring Hb A1c, specific binding (affinity Materials and Methods specimens Blood samples (anticoagulated with EDTA) were obtained from diabetic and nondiabetic patients who entered our outpatient clinic. All procedures involving patients were in accordance with the Helsinki Declaration of 1975, as revised in 1983. Blood samples with a high Hb F content were derived from cord blood. Carbamylated and acetylated hemoglobin were synthesized in vitro as described by Weykamp et al. . Blood with a high percentage of labile Hb A1c fraction (Schiff base) was made by incubating washed erythrocytes in a 100 mmol/L glucose solution for 24 h. To prepare the samples for assay, we used 20 mL of EDTA-anticoagulated blood mixed with 100 mL of saponin-containing “hemolyzer” reagent (provided by Analis). 1 Department of Clinical Chemistry and Hematology, Queen Beatrix Hospital, Beatrixpark 1, 7101 BN Winterswijk, The Netherlands. 2 Analis S.A., 14 Rue Dewez, B 5000 Namur, Belgium. *Author for correspondence. Fax 131543524265. Received May 29, 1996; revised October 29, 1996; accepted November 12, 1996. electrophoresis Capillary zone electrophoresis was performed on a Beckman P/ACE System 5000 (Beckman, Brea, CA) with a 25 644 645 Clinical Chemistry 43, No. 4, 1997 mm (i.d.) 3 24 cm fused-silica capillary at 25 °C. Proprietary patented reagents (malic acid buffers, pH 4.5)  were obtained from Analis. Before sample injection, the capillary was first rinsed with initiator solution (containing a polycation, albumin, pH 4.5) for 0.3 min under 13.8 kPa (20 psi) pressure, followed by 1.00 min of prerinsing with buffer solution containing a polyanion (chondroitin sulfate, pH 4.5), at the same pressure. Sample was injected for 8 s at 5 kV; this was followed by a 10-s injection with buffer solution at 3.5 kPa (0.5 psi) to rinse the outside of the capillary. The capillary was then transferred to another vial containing buffer solution, in which the electrophoresis was performed. Negatively charged molecules (chondroitin sulfate, pH 4.5) in the buffer solution bind to hemoglobin. Electrophoresis was performed with a constant current of 52 mA for 4 min with the negative electrode at the detector site. Detection was executed with a UV/VIS absorbance detector at 415 nm. Peak integration for peak area measurement was performed by a Beckman System Gold chromatography data system (vers. 8.10); peak area percentages corrected for velocity were used. After electrophoresis, the capillary was rinsed with 1 mol/L NaOH for 2 min at 13.8 kPa. The relative apparent mobility of each peak was calculated according to the method described by Harris . Because of a lower refractive index of the hemolysate sample, the “hemolyzer” peak was used as an internal standard. Within-run variability was determined by analyzing 20 times three different patients’ samples (containing low, medium, and high concentrations of Hb A1c) in one run. Between-run variability was determined by analyzing the same three samples once a day on 20 working days (stored samples). The comparison method, cation-exchange HPLC with a Bio-Rex 70 column (Bio-Rad, Veenendaal, The Netherlands), has been described elsewhere . In brief, 4 mL of hemolysate (packed cells diluted 1:3 in distilled water) was injected onto the Bio-Rex 70 HPLC column, which was operated at 25 °C with a flow rate of 1.5 mL/min. We eluted the sample for 5 min with buffer A (8.0 mmol/L potassium cyanide dissolved in a 113 mmol/L sodium phosphate buffer, pH 6.77) and then for 15 min with buffer B (564 mmol/L sodium phosphate buffer, pH 6.42). Detection was performed by measuring absorbance at 410 nm. Between each run the column was equilibrated for 15 min with buffer A. Linear regression analysis of the CE and HPLC results was followed by an outlier detection procedure described previously . Results The electrophoresis pattern of hemoglobin from a nondiabetic healthy volunteer is shown in Fig. 1. The hemolyzer, Hb A1c, and Hb A0 peaks are eluted at 2.63, 3.01, and 3.29 min, respectively. Apparent mobilities (relative Fig. 1. Hemoglobin CE pattern from a nondiabetic healthy volunteer showing complete separation between Hb A1c and Hb A0. The hemolyzer peak is eluted at 2.63 min, the Hb A1c peak at 3.01 min, and the Hb A0 peak at 3.29 min. to the internal standard, i.e., the hemolyzing reagent) are shown in Table 1. Reproducibility. Within-run variabilities were determined by assaying three different blood samples. Low (4.3%), medium (7.0%), and high (10.5%) Hb A1c samples gave within-assay CVs of 1.7%, 2.9%, and 1.4%, respectively. The between-run variabilities for these samples were 3.7%, 3.3%, and 1.9%, respectively. However, the aging of the blood samples over the 20 working days (1 month, total) of the between-assay reproducibility study resulted in an extra peak (relative apparent mobility 1.18) between the peaks for Hb A1c and Hb A0. Interferences. The results for in vivo carbamylated hemoglobin are shown in Fig. 2. No interferences were observed from carbamylated or acetylated hemoglobin or from the labile Hb A1c fraction, whose relative apparent mobilities are shown in Table 1. Investigation of hemoglobin variants S (Fig. 3, top) and F and C (Fig. 3, bottom) also showed no apparent interference. Hb S and Hb C peaks corresponding to Hb S1c and Hb C1c were visible at 3.07 min and between 3.29 and 3.74 min, respectively. Table 1. Relative apparent mobilities of hemoglobin variants and derivatives. Relative mobilitya Hb F Labile Hb A1c HbCarb Hb A1c HbAcet Aging peak Hb A0 Hb S Hb C a Mean SE 1.05 1.09 1.08 1.13 1.16 1.18 1.24 1.33 1.46 0.013 0.001 0.001 0.011 0.007 0.015 Relative to the mobility of the hemolyzer peak. No. of samples 6 8 28 40 1 26 40 1 1 646 Doelman et al.: Capillary electrophoresis determination of Hb A1c Fig. 2. Hemoglobin CE pattern of blood containing in vivo carbamylated hemoglobin; the carbamylated hemoglobin (eluted at 2.88 min) is well separated from Hb A1c (eluted at 3.01 min). Blood was obtained from an insulin-dependent diabetic patient having a serum urea concentration of 54 mmol/L. None of these potentially interfering substances, including the sample-aging peak mentioned above, influenced the CE assay of Hb A1c. Results of the comparison between CE and Bio-Rex 70 HPLC gave a good linear correlation (Fig. 4): CE Hb A1c 5 Fig. 4. Correlation between the Bio-Rex 70 HPLC results and those by CE (n 5 100). The two samples that have a much larger deviation from the regression line than the others both contained carbamylated hemoglobins, which seem to interfere in the Bio-Rex 70 method. 21.41 1 1.02 HPLC Hb A1c (r 5 0.98). No outliers were detected. Discussion Fig. 3. Electrophoresis patterns of heterozygous Hb S (upper panel) and Hb C and Hb F (lower panel). (Top) Hb S is eluted at 3.47 min, Hb A1c at 2.99 min in this run. The peak at 3.07 min might be Hb S1c. (Bottom) Hb F elutes at 2.74 min and Hb C at 3.74 min. The Hb A0 peak is shown at 3.29 min. The DCCT clearly showed that the risk of late complications of diabetes mellitus is related to the percentage of Hb A1c in a patient’s blood. The Bio-Rex 70 HPLC method, described by Goldstein et al.  and having been shown to be very accurate, precise, and useful for calibrating different methods , was used to calibrate all of the Hb A1c methods used in that study. The therapy goals of diabetic therapy (i.e., Hb A1c values as low as reasonably achievable) defined in the DCCT study are therefore related to the Bio-Rex 70 HPLC results . The AACC subcommittee on standardization of Hb A1c analyses has chosen to use this method as an anchor and recommends that routine assays be calibrated in terms of the Bio-Rex 70 HPLC method . The CE separation technique is rapid, uses low amounts of reagents, and is easily automated. Although separation of hemoglobin variants and derivatives in uncoated fused-silica capillaries is limited by adsorbance of proteins to the capillary wall and by variable rates of electroosmotic flow, some methods for separating hemoglobin variants this way have been described [13, 14]. Those investigators tried to overcome the problems by performing electrophoresis at a relatively high pH, which induces a strong and constant electroosmotic flow; this reduced the resolution, however, so long capillaries had to be used, which increased the analysis time [13, 14]. Others have shown that capillary isoelectric focusing with coated capillaries could also be used to separate hemoglobin variants [15–18], but only two of these publications demonstrated the possibility of Hb A1c determination Clinical Chemistry 43, No. 4, 1997 (they did not report complete separation of Hb A1c from Hb A0) [17, 18]. The CE method used here is performed at an acidic pH (4.5) and is based on an ion-pairing effect between hemoglobin and a negatively charged molecule in the running buffer solution. The equilibrium of this ion pairing depends on the charges carried by the hemoglobin molecule and on the accessibility of these charges. At acidic pH, the amino group of hemoglobin is more positively charged and more accessible than is the amino group of glycohemoglobin. Thus, the glycohemoglobin is eluted first because it is less strongly attached to the negatively charged molecule. At the working pH of 4.5, the electroosmotic flow is low, unstable, and highly variable from capillary to capillary. We overcame this difficulty by applying a dynamic coating to the capillary. This coating is made in two steps. The capillary is rinsed with buffer containing a polycation, which binds to the negatively charged silica surface of the capillary; this approach can diminish or even reverse the electroosmotic flow, depending on the nature of the polycation and its concentration. In the second step, the capillary is rinsed with buffer containing a polyanion, which adds a layer of negative charges over the polycation layer. As a result, the internal surface of the capillary will present a controlled and reproducible high number of charges from the polyanion, thereby controlling the electroosmotic flow. The hemoglobin complexed with the negatively charged molecule is then electrophoresed over the coated capillary. The double-layer coating is removed after each run by a simple rinse with NaOH  The principle of coating the capillary makes possible rapid separation of these hemoglobin variants (within a few minutes)—which is the new development in this assay. It is generally accepted that to be useful an assay for Hb A1c should not be influenced by Hb F or by carbamylated or acetylated hemoglobin derivatives; hemoglobin variants should be detected but should not interfere. Analysts should also be aware that hemoglobinopathies may reduce erythrocyte life span, which will result in artifactually low Hb A1c values both in nondiabetic and diabetic subjects . Knowledge of the presence of hemoglobin variants in a patient’s sample or of other factors that reduce erythrocyte life span is therefore necessary. An Hb A1c method that identifies samples containing hemoglobin variants would be useful. In this study we evaluated these potentially interfering substances. The fact that Hb F and carbamylated and acetylated hemoglobins do not interfere in Hb A1c measurement (Fig. 2) seems to be the main advantage of the CE assay. The most common hemoglobinopathies Hb S and Hb C also don’t appear to interfere. Probably, the N-terminal glycated products of Hb S and Hb C are also separated from Hb A0 and Hb A1c. The question, of course, is which percentage—that of Hb A1c vs Hb A0, or of Hb S1c/Hb C1c vs Hb S0/Hb C0— corresponds best with 647 the metabolic control of the patient. This should be investigated in a diabetic population with heterozygous hemoglobinopathies. The CE results correlated very well with those by the Bio-Rex 70 HPLC method (Fig. 4). Two samples visually out of line but not statistical outliers contained carbamylated hemoglobins; we concluded, therefore, that Hb A1c measurement in the Bio-Rex 70 HPLC system is influenced by carbamylated hemoglobin. Also, the negative intercept at 1.4% in the correlation plot indicates that other hemoglobin derivatives (e.g., carbamylated or acetylated forms) are coeluting with Hb A1c in the cation-exchange HPLC system. A comparable difference was observed by Turpeinen et al. , comparing the Diamat method (cation-exchange chromatography) and HPLC with PolyCAT A; they found that the PolyCAT A values were 2–3% lower than the Diamat values. (The Diamat assay is easily calibrated to the Goldstein et al. Bio-Rex 70 HPLC method.) From these results, one might conclude that the CE method described here is also somewhat biased (by ;1%) in comparison with the PolyCAT A HPLC. We speculate that the integration method of the chromatograms (valley-to-valley for PolyCAT A) and electrophoresis patterns (forward horizontal for CE) might account for this 1% difference. For lack of a more satisfactory explanation, perhaps a study should be performed on comparability of the integration techniques in the various Hb A1c assays. In any event, this situation underlines the lack of a “golden” reference assay and the need for Hb A1c standardization. Somewhat disappointing was the high interassay variability. The sample aging produces a peak between Hb A1c and Hb A0, which during 1 month progressively influences the outcome of the Hb A1c measurement (data not shown). Therefore, the interassay variability was increased, especially at low Hb A1c values. This should not present problems in routine analyses, however, because the procedure calls for Hb A1c to be measured within 1 week after blood collection. The question arises as to whether this CE assay will be used in a routine setting for Hb A1c measurement in clinical laboratories. Until now, the test has been used as a reference test for other routinely used Hb A1c assays. By the time multichannel CE systems are available and the throughput of samples is substantially increased, the assay might be more suited for routine use. However, CE equipment is rather expensive, whereas other Hb A1c assays, e.g., immunoassays, can be run on routine clinical chemistry analyzers. Nonetheless, because of its fast and complete separation of Hb A1c from Hb A0 and from hemoglobin derivatives and variants, the CE method might be useful for clinical laboratories. The cost effectiveness of CE vs the Bio-Rex 70 method is presented in Table 2. The main disadvantage of the Bio-Rex 70 HPLC method is the low throughput of samples (only 5 per day vs 40 per day by CE). This makes running the CE method, 648 Doelman et al.: Capillary electrophoresis determination of Hb A1c Table 2. Cost-effectiveness of CE and Bio-Rex 70 HPLC assays of Hb A1c. Bio-Rex 70 HPLC Equipment, $ per year Maintenance, $ per year Sample throughput Tests per day Tests per year Technician, $ per year Reagents, estimated, $ per year Costs per sample, $ 3000 1200 5 1000 48 000 600 52.80 CE 6000 6000 40 8000 48 000 23 500 10.45 The HPLC method is not suitable for routine use because of its low throughput (5 samples a day). 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