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Prevention of Excess Glycosylation of Haemoglobin in Filter Paper Blood Spots Taken for Glycosylated Haemoglobin Measurement

Glycosylated haemoglobin (HbAl) assay is now widely used as a measure of diabetic control, but problems with sample collection and storage limit the overall usefulness of the measurement. These factors are of importance in screening programmes, and for patient care in remote areas. In hospital practice, analysis of samples is not usually possible during an out-patient clinic and prior home-blood sampling would enable a recent result to be available for the patient's visit. Sending blood samples is inconvenient, and filter paper" spots would be preferable. However, further glycosylation of haemoglobin during the storage and transport of dried blood must be prevented. We describe here a simple and cheap method for impregnation of filter paper which inhibits further glycosylation of haemoglobin, enabling clinically suitable HbAl determination up to 4 weeks after sampling. Blood spots were taken from 16 diabetic and four non-diabetic subjects onto Blood Collection Cards (Sigma Chemical Co., St Louis, MI, USA), which were previously impregnated with citrate buffer (0,5 molar, pH 4,5) containing semicarbazide (60 mmol/L) and aniline (25 mmol/L) to trap glucose present in the sample. Filter paper blood spots were left to air dry and stored in plastic bags at room temperature until assayed. Parallel blood samples were taken into standard EDTA tubes for reference measurement. Red blood cell haemolysates (1:20 dilution in distilled water) were prepared from the reference samples and stored at 4°C for 18 h to remove the unstable aldimine fraction. HbAl was measured using the Glyco-Gel Test KitTM (Pierce Chemical Co., Rockford, IL, USA) affinity chromatography method (betweenbatch CV 3,5%). For dried blood analysis 9 mm