Glycated Hemoglobin Evaluation by Means of Ion Exchange Chromatography and Immunoassay in Normal and Hemoglobinopathy Patients

Aims and Objectives: High performance liquid chromatography which is basically an Ion Exchange Chromatography, is the most popular as well as expensive method for glycated hemoglobin estimation but biased results have been observed in hemoglobinopathies. Sometimes results cannot be reported also. In such situation the laboratory adopted immunoassay method and successfully reported the results. Therefore, a comparative analysis of glycated hemoglobin results of normal and abnormal hemoglobin were essential as high performance liquid chromatography is the most sought after method.

Study Design: Number of patients with normal adult hemoglobin compared were 120. Patients with Hb-AE thalassemia were 21. Patients with β-thalassemia trait were 36. Hb-E-β thalassemia patients were 6, β-thalassemia homozygous 10 and HbSS patients tested were 4. The glycated hemoglobin values obtained were from euglycemic to severe diabetic range. The instruments used for measurement are D10 BIORAD and Cobas Integra 400plus.

Data Analysis: Deviations calculated from results of same patient obtained from two system. Regression coefficients of each group were calculated. Trend analysis was done and graphically presented.

Results and Discussion: Regression analysis of normal, HbAE&β-variant patients show coefficients are 0.988,0.976 &0.961 respectively proving excellent comparability of two methods. But trend analysis of deviations show right shift of 85%HbAE& 86.1%β-variants .Similar bias was observed in external quality assessment results also. The immunoassay results were comparable with boronate affinity chromatography and enzymatic estimation method. The deviation in HPLC observed only when in external assessment sample a window was seen. Therefore, observations of the trend of patients and EQAS sample indicates low bias of HbA1c estimation by HPLC method in D10.In severe forms of Hemoglobinopathy i.e. HbSS, Hb-E-β and β-thalassemia homozygous 75%,67% & 60% results could not be obtained from D10. The acceptability of immunoassay results checked by comparing glucose fasting and estimated average glucose calculation values.

Conclusion: Immunoassay, the second popular method is reliable, less expensive method and results remain unaffected in hemoglobinopathies.