HPLC Applications

HPLC is considered the “Gold Standard” technology in the follow-up of the plasma glucose concentration of diabetic patients over time, via the measurement of HbA_1c (= glycated hemoglobin fraction).

This technique was used in the ground breaking DCCT (Diabetes Control and Complications Trial) study. To follow-up the plasma glucose concentrations over time the “HbA_1c analysis mode” should be used. In this mode predetermined windows are set in the software to detect the presence of Hemoglobins A_1a, A_1b, F, l-A_1c, s-A_1c and A₀.

Calibration against DCCT (NGSP) or IFCC allows the quantitation of the stabile A_1c fraction, with other windows allowing presumptive identification of various variant hemoglobins.

In March 2009 the WHO convened the present consultation (including experts in diabetology, biochemistry, immunology, genetics, epidemiology and public health) in order to update the 1999 and 2006 reports “Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia” with the place of HbA_1c in diagnosing diabetes, based on the now available evidence. 

The official recommendation states:

HbA_1c can be used as a diagnostic test for diabetes providing that stringent quality assurance tests are in place and assays are standardized to criteria aligned to the international reference values, and there are no conditions present which preclude its accurate measurement.

An HbA_1c of 6,5% is recommended as the cut point for diagnosing diabetes. A value of less than 6,5% does not exclude diabetes diagnosed using glucose tests.

The use of HbA_1c can avoid the problem of day-to-day variability of glucose values, and importantly it avoids the need for the person to fast and to have preceding dietary preparations. A further major factor concerns costs and availability of HbA_1c assays in many countries.

In view of the above and the advances in technology over recent years, the WHO consultation members agreed that HbA_1c may be used to diagnose diabetes providing that appropriate conditions apply, i.e. standardized assay, low coefficient of variation and calibration against IFCC standards. Furthermore, each country should decide whether it is appropriate for its own circumstances. The choice of diagnostic method will depend on local considerations such as cost, availability of equipment, population characteristics, presence of national quality assurance system etc.

In the aid of diagnosing β-thalassemia and hemoglobinopathies the “β-thalassemia analysis mode” should be used. In this mode predetermined windows are set in the software to detect the presence of Hemoglobins F, A₀, A₂, D+, S+ and C+. Calibration allows the quantitation of HbF and HbA₂, with all other windows allowing the presumptive identification of various variant hemoglobins.