Measurements of HbA1c by High Performance Liquid Chromatography in D-10 analyzer and Immunological Method by Beckman Coulter AU480 System: A Comparative Study

Background: HbA1c can be measured by different methods. Enzymatic, boronate affinity chromatographic and cation-exchange high performance liquid chromatographic (HPLC) methods are considered as gold standard methods. To make the test cost-effective many laboratories use the immunological method installed in a chemistry analyzer. Objective: To compare the values of HbA1c measured by high performance liquid chromatographic method and immunological method in two laboratory settings. Materials and Methods: This observational and comparative study was conducted in Bangladesh Institute of Health Sciences (BIHS) and Enam Medical College, Savar, Dhaka. HbA1c levels measured by HPLC in BIHS were compared with that measured by immunological method in Enam Medical College, Savar, Dhaka. p values <0.05 were considered significant. Results: Statistically no difference was observed in mean HbA1c% between two methods in <7.0% HbA1c group and 7.1-10.0% HbA1c group. Significant statistical difference was found in mean HbA1c >10.0% group, but it was within acceptable limit. Conclusion: HbA1c can be measured by immunological method installed in an automated chemistry analyzer to make the test costeffective. But HPLC method is preferable.

Diabetes mellitus is a syndrome of impaired carbohydrate, fat and protein metabolism caused by either lack of insulin secretion or decreased sensitivity of the tissues to insulin. 1 It is characterized by hyperglycemia resulting from the body's inability to utilize glucose for energy. In type 1 diabetes, the pancreas cannot produce insulin and glucose in the blood cannot get entry inside the cell to be metabolized to provide energy. In type 2 diabetes, either the pancreas cannot produce enough insulin or the tissues of the body are insensitive to insulin. The complications of diabetes, involving the eyes, kidneys, nerves and blood vessels of heart, brain, extremities are common to both types of diabetes. 2 Currently more than 5% of the world populations are affected by diabetes and it is getting more and more epidemic. 3 only by the measurement of HbA 1c every two to three months. [4][5][6] HbA 1c can also be translated to average blood glucose (eAG) to get the approximate plasma glucose level. 7 HbA 1c c a n be measured by immunological, enzymatic, boronate affinity chromatographic and cation-exchange high performance liquid chromatographic (HPLC) methods. 8 It can also be measured by electrophoresis. Enzymatic and boronate affinity chromatographic and HPLC methods are considered as gold standard methods to measure HbA 1c . But it is not possible to install these methods in all laboratory settings, especially in the developing countries like ours. Measurement of HbA 1c by electrophoresis is also cumbersome and costly. For HbA 1c measurement, many laboratories use the immunological method installed in a chemistry analyzer to make the test cost-effective. We designed this study to compare the values of HbA 1c measured by HPLC method and immunological method in two laboratory settings.

Materials and Methods
This observational and comparative study was done in the department of Clinical Biochemistry, Bangladesh Institute of Health Sciences (BIHS), Dhaka and the department of Biochemistry, Enam Medical College, Savar, Dhaka during the period of July 2011 to December 2011. Sixty specimens for measurement of HbA 1c levels by HPLC method in Bangladesh Institute of Health Sciences were included for this study. Twenty specimens had HbA 1c level <7.0%, 20 had 7.1 to 10% and 20 were of >10%. These specimens were also measured for HbA 1c levels in Enam Medical College Hospital laboratory by immunoassay method. Results by these two methods were compared by appropriate statistical analyses. 3 We used the Bio-Rad D-10 TM Hemoglobin A 1c program which is intended for the percent determination of HbA 1c in human whole blood. The D-10 Hemoglobin A 1c P r ogram is based on chromatographic separation of HbA 1c on a cation exchange cartridge. Separation is optimized to minimize interferences from hemoglobin variants, labile A 1c and carbamylated hemoglobin. The D-10 TM Hemoglobin A 1c program also offers automatic sampling from a primary whole blood tube, followed by sample dilution and an analysis time of three minutes per sample.

HPLC method
The samples are automatically diluted on the D-10 and injected into the analytical cartridge. The D-10 delivers a programmed buffer gradient of increasing ionic strength to the cartridge where the hemoglobin is separated based on their ionic interactions with the cartridge material. The separated hemoglobin then passes through the flow cell of the filter photometer where changes in the absorbance are measured at 415 nanometer wavelength. The D-10 software performs reduction of raw data collected from each analysis. Two-level calibration is used for quantification of the HbA 1c values. A sample report and a chromatogram are generated for each sample. The A 1c area is calculated using an exponentially modified Gaussian (EMG) algorithm that excludes the labile HbA 1c and carbamylated peak area from the A 1c peak area. Immunoassay method 9 We used the Beckman Coulter AU480 analyzer. The absolute HbA 1c and total hemoglobin (THb) values generated as part of the HbA 1c assay are intended for the quantitative determination of hemoglobin A 1c concentration as a percentage of total hemoglobin in human whole blood.
The assay for percent HbA 1c involves the use of four reagents: Total hemoglobin R1, HbA 1c R1 antibody reagent, HbA 1c R 2 a g glutinator and hemoglobin denaturant. In the pre-treatment step the whole blood is mixed with hemoglobin denaturant in a 1:41 dilution and incubated for a minimum of five minutes at room temperature. The red blood cells are lysed and the hemoglobin chain is hydrolyzed by the protease present in the reagent. Total hemoglobin is measured via the conversion of all hemoglobin derivatives into alkaline hematin in the alkaline solution of a non-ionic detergent. Addition of the pre-treated blood sample to the total hemoglobin reagent results in a green solution which is measured at 600 nm wavelength. HbA 1c is measured in a latex agglutination inhibition assay. An agglutinator, consisting of a synthetic polymer containing multiple copies of the immunoreactive portion of HbA 1c , causes agglutination of latex coated with HbA 1c s p ecific mouse monoclonal antibodies. In the absence of HbA 1c in the sample, the antibody-coated microparticles in the HbA 1c R1 and the agglutinator in the HbA 1c R 2 w i ll agglutinate. Agglutination leads to an increase in the absorbance of the suspension. The presence of HbA 1c in the sample results in a decrease in the rate of agglutination of the HbA 1c R 1 a n d the agglutinator in the HbA 1c reagent R2. The increase in absorbance is therefore inversely proportional to the concentration of HbA 1c in the sample. The increase in absorbance due to agglutination is measured at 700 nm wavelength.

Statistical analysis
Statistical analyses were performed by Paired t test, Bland-Altman plot and Pearson's test using MedCalc ® version 11.4 for Windows. p values <0.05 were considered significant. Table I shows the mean HbA 1c % and comparison in different HbA 1c levels by two methods. In < 7.0% HbA 1c group it was 6.27 ± 0.48% by HPLC method in D-10 analyzer and 6.11 ± 0.62% by immunological method in Beckman Coulter AU480 system. In 7.1-10% HbA 1c group it was 8.24 ± 0.83% by HPLC method in D-10 analyzer and 8.16 ± 1.07% by immunological method in Beckman Coulter AU480 system. In >10.0% HbA 1c g r oup it was 12.66 ± 2.06% by HPLC method in D-10 analyzer and 11.98 ± 2.29% by immunological method in Beckman Coulter AU480 system. 64 The bias of HbA 1c measured by immunological method against the HbA 1c m e asured by HPLC method was 2.6% for HbA 1c <7.0%, 0.7% for HbA 1c range of 7.1 -10% and 5.4% for HbA 1c >10% (Fig 2). The correlation coefficients of HbA 1c measured by HPLC and immunoassay were 0.671 (p=0.0017) for HbA 1c <7.0%, 0.588 (p= 0.0050) for HbA 1c range of 7.1 -10.0% and 0.870 (p< 0.0001) for HbA 1c >10.0%. Casewise performance of the two methods in different HbA 1c ranges is presented in Fig 3.

Discussion
In this study we found no significant difference in HbA 1c values between the two methods (HPLC in D-10 analyzer and immunological method in Beckman Coulter AU480 system) in < 7.0% and 7.1-10% ranges of HbA 1c levels. But there was significant difference in HbA 1c values between the two methods (HPLC in D-10 analyzer and immunological method in Beckman Coulter AU480 system) in >10.0% range. But in these cases the percentage of difference against HPLC method was 5.4% which was very close to acceptable limit of 5%. Moreover the correlation coefficients between these two methods were strong in different HbA 1c ranges. It was also evident from the casewise performance of the two methods that there was no systemic underestimation or overestimation, values of HbA 1c w e re randomly scattered around the HbA 1c values measured by HPLC methods. So, it can be concluded that by having proper and regular calibration and by undertaking strict quality control measures in accordance with good laboratory practices HbA 1c c a n also be measured by immunological method installed in a chemistry analyzer to make the test cost-effective. However, more studies in different laboratory settings are recommended.