Role of HPLC in the clinical laboratory
HPLC is a form of column chromatography, used to separate, identify, and quantify each component in a mixture. HPLC is a valuable tool with clinical applications in analytical chemistry and pharmaceutical industry. In clinical laboratories, this technology is widely used in HbA1c measurement and detection of hemoglobinopathies. HbA1c represents the fraction of hemoglobin bound to glucose, and presence of a coincidental hemoglobinopathy can affect A1c measurement in various ways, and this depends on the method used for detection. Modern automated devices used to quantify HbA1c are able to separate and quantitate the different hemoglobin fractions. Therefore, the evaluation of both hemoglobinopathy mode and HbA1c mode of the analyzers is of clinical relevance. The Bio-Rad Variant II Turbo, installed across SRL reference labs, is a fully automated ion-exchange HPLC system for mid- to high-volume laboratories, which provides a comprehensive solution for HbA1c testing; it delivers quality results in 97 seconds. There is no known interference in the presence of Hemoglobin S, C, D, or E traits and HbF concentrations up to 25 percent. This method is NGSP-certified and traceable to diabetes control and complications trial. It provides rapid separation without compromising the level of resolution of the HbA1c peak from potential interfering compounds, i.e., labile HbA1c and carbamylated HbA1c.
HPLC has also proved to be an accurate and simple technique for quantitation of HbA2, HbF, and other hemoglobin subtypes. The Bio-Rad Varant II Beta thalassemia short program is being used at SRL Laboratories for the presumptive identification of many abnormal hemoglobin variants. This method is capable of separating more than 45 commonly encountered hemoglobin variants within 6.5 min, based on the characteristic retention time. The simplicity of the sample preparation, superior resolution of the method, and accurate quantitation of hemoglobin concentration, combined with complete automation, makes this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory. Both percentages of HbA2 and HBF are calibrated for the highest levels of precision and accuracy. Each chromatogram shows peaks of Hb A0, A2, and HbF along with C window, D window and S window. Other relevant tests like family studies and molecular testing are required in a few cases.
Nowadays, HPLC is a very valuable tool in the clinical laboratory. In the future, more and more HPLC methods will replace old photometric assays and also immunoassays, since these methods fulfill the claims in clinical chemistry – high precision, accuracy, sensitivity, and specificity.
