Chromatography Techniques

Biography

Biography: Luljeta Hetemi

Abstract

Introduction: The purpose of this study was to: follow-up patients with diabetes  type ll  through testing of: glycated hemoglobin (HbA1c) derivatives, but the importance of the HPLC method during HbA1c testing in: diagnosis, monitoring, follow-up, access  to diabet type ll  therapy also obtained results from: diabetes ,pre  diabetes, patient insulin insertion, monotherapy, but also findings of abnormal HgB forms in diabetic patients, such as hemoglobinopathies, and during the research we found that the diagnostic orientation through a HbA1c  test with HPLC  is in some pathologies, which was both preventive and diagnostic in other pathologies.
The research lasted over 1 year at the Olive medical & laboratory diagnostic center.

Method:HPLC(high performance  liquid chromatography) G8 Tosoh,an exclusive distributor :

Keis group pharmaceutical Kosovo.For  the first  time in our  country we performed HbA1c  with HPLC.
The G8  Analyzer is the next generation of Tosoh’s leading HPLC testing systems for fast and accurate HbA1c results. The analyzer dilutes the whole blood specimen with Hemolysis & Wash Solution and then injects a small volume of the treated specimen onto the TSKgel Glyco HSi Variant Column. Separation is achieved by utilizing differences in ionic interactions between the cation exchange group on the column resin surface and the hemoglobin components in a step gradient elution. The hemoglobin fractions (designated as A1a, A1b, F, LA1c , SA1c, A0, and H-V0, H-V1, H-V2) are subsequently removed from the column material by performing a step-wise elution using Elution Buffers HSi Variant 1, 2, and 3 that have specific salt and pH concentrations.
The separated hemoglobin components pass through the LED photometer flow cell where changes in absorbance are measured at 415nm. The G8 software integrates and reduces the raw data, and then calculates the relative percentages of each hemoglobin fraction. The print-out consists of the numerical results and the chromatogram. This represents the changes in absorbance versus retention time for each peak fraction. An analysis requires only 1.6 minutes.The study was conducted on 150 patients, of which 80% were  diabet type ll, age 40-65 years, with HbA1c ranging from 6.9-11%, HbA from 6-8, HbF of over 1 % -8% (in 48% of cases was HbF positive), the control group were non-diabetic, suspect, dyslipidemia, patients with no signs of type II diabetes, the reason for testing being because they had high insulin resistance, or moderate , some of these patients were  with polycystic ovary syndrome. In addition to the absolute accuracy of the HPLC method in testing HbA1c, in this study we found HbF positive of patients with type II diabet, which is leading us to anemia, and the cause of this anemia is thought to be impairment of renal function, such as diabetic nephropathy - complication of type II diabet, but a still unclear mechanism between abnormal HbF congenital and type II diabet  will to remain uninvolved as we were not able to test all the patients for Hb electrophoresis.