Assessment of an Automatic Liquid Chromatograph for Foetal and Abnormal Haemoglobins

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Abstract
The DIAMAT TM (Bio-Rad) analyser is a microprocessor-operated HPLC system using a silica-based weak cation exchange column with three phosphate buffers of increasing ionic strength as the step gradient mobile phase. A dual wavelength detector measures absorbance at 415 and 690 nm; each sample is completely processed in 8 minutes. The instrument effectively separates and quantifies HbF in a discrete peak. We have verified that the HbF assay is linear up to about 65% values. We calculated within-run imprecision for n = 20 in 3 different haemolysed blood samples; the results are shown below as percent of total haemoglobin: A) Mean = 1.06 SD = 0.048 CV% = 4.5, B) Mean = 1.90 SD = 0.041 CV% = 2.1, C) Mean = 8.93 SD = 0.047 CV% = 0.5. Between-run imprecision for similar samples (n = 18) was: D) Mean = 3.74 SD = 0.20 CV% = 5.5, E) Mean = 9.94 SD = 0.15 CV% = 1.5, Accuracy was assessed in different series by correlating Hb values (y) with those obtained by the alkali denaturation test (x). The regression line equation was y = 1.03 x - 0.33 (r = 0.999, n = 62). The DIAMAT TM instrument also reveals the presence of any HbS and calculates its peak area correctly, as we found by electrophoretic reassay in heterozygous subjects. We also noted the presence of other abnormal haemoglobins characterized by specific retention times.