Measurement of glycated haemoglobin in whole blood by a novel fluorescence quenching assay.
We describe a method for the specific measurement of glycated Hb (GHb) by fluorescence quenching. Whole blood is added to lysing solution, then the lysate is mixed with eosin-boronic acid solution and reacted for at least 5 min at room temperature. The quenching of the fluorescence of the eosin-boronic acid solution is proportional to the concentration of GHb present. Total Hb concentration was measured by absorbance and the GHb expressed as a percentage of the total Hb. Comparison with a commercial high-performance liquid chromatography (HPLC) system for HbA1c showed: %GHb=1.30 (SD 0.04) %HbA1c + 1.36 (SD 0.30), S(y/x) 0.803, n=95, r=0.965 (SD=standard deviation). Intra-assay coefficients of variation were <2.5% (for GHb concentrations in the range 6-20%) and inter-assay coefficients of variation were <4.1% (10 assays on six samples with GHb concentrations in the range 6-20%). Linearity of response was demonstrated by dilution. The effect of adding exogenous glucose, bilirubin and triglycerides was tested on samples with low, medium and high GHb concentrations. No significant interference was found. Variation of haematocrit over the range 0.4-0.6 also had no significant effect on percentage GHb. Preliminary results with samples containing variant Hb (HbAS and HbAC) indicated good agreement with HPLC for these samples also.
Blincko
S
S
Netria, St Bartholomew's Hospital, London, UK. R.Edwards@mds.qmw.ac.uk
Anzetse
J
J
Edwards
R
R
D001897:Boronic Acids / Q000378:metabolism; D002624:Chemistry, Clinical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D005470:Fluorometry; D006400:Hematocrit; D006444:Hemoglobin C / Q000032:analysis; D006451:Hemoglobin, Sickle / Q000032:analysis; D006454:Hemoglobins / Q000032:analysis*; D006801:Humans; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013053:Spectrophotometry
D001897:Boronic Acids; D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D006444:Hemoglobin C; D004801:Eosine Yellowish-(YS)