ABSTRACT Aims To establish the frequency of detection of previously undiagnosed diabetes mellitus as a result of detection of an increased glycated fraction of haemoglobin during high performance liquid chromatography (HPLC) for haemoglobinopathy diagnosis. Methods A prospective study was carried out over a 3-month period. During that period a total of 2094 patient samples were received for haemoglobinopathy investigation and were included in the study. Results Fifty samples were found to have an apparent increase in the glycated haemoglobin fraction and of these 38 were found to be from patients with known diabetes. Previously undiagnosed diabetes was discovered in 11 patients and it is likely that the twelfth patient also had diabetes. Conclusions The detection of evidence of undiagnosed diabetes during HPLC haemoglobinopathy investigations is not rare, there being four cases per month in this study. This incidental observation should be reported to clinical staff. A proportion of haemoglobin A undergoes posttranslational modification including glycosylation (non-enzymatic addition of glucose to the aminoterminal valine of the b chain) or glycation (less specifically, addition of carbohydrate to a protein). 1 Glycosylation occurs throughout the life-span of the red cell, at a rate determined by the ambient glucose concentration. Glycated haemoglobin is known as haemoglobin A 1 , of which 60e80% is glycosylated haemoglobin, haemoglobin A 1c . 1 In patients with diabetes mellitus, the proportion of haemoglobin that is haemoglobin A 1c has been found to be useful as an indication of the degree of hyperglycaemia during the preceding 3 months. In addition, since there are few other causes of an elevated haemoglobin A 1c , the finding of an elevated proportion can be useful in diagnosis. Glycated haemoglobin includes a labile fraction, which responds rapidly and transiently to raised blood glucose levels, and a stable fraction, to which the labile fraction is converted. 1 It is the stable fraction that is useful in judging long-term control of diabetes. Haemoglobin A 1c can be quantified by a variety of methods, of which high-performance liquid chromatography (HPLC) is increasingly used. When suspected haemoglobinopathies are investigated by cellulose acetate electrophoresis, the glycated fraction is not resolved and no specific abnormality is apparent in diabetic patients. However, when the technique used for such investigations is HPLC, the presence of a glycated fraction may be apparent. Our laboratory has previously drawn attention to the possibility that this may lead to the diagnosis of previously unsuspected diabetes mellitus. 2 Early diagnosis and good control of diabetes mellitus is important in reducing the end-organ damage that is characteristic of this disease, and it is therefore important for haematologists to alert clinical staff to the probability of this diagnosis when an increased proportion of glycated haemoglobin is observed. We therefore carried out a study to determine the frequency with which this is observed, and as a result of this study we developed a policy for notification of an increased glycated fraction to clinical staff. MATERIALS AND METHODS All tests were performed on peripheral blood samples anticoagulated with EDTA. HPLC for establishing relevant reference ranges and for haemoglobinopathy investigations was performed on a Bio-Rad Variant II instrument (Bio-Rad Laboratories, Hemel Hempstead, UK) using the b-Thalassaemia Short Program. Haemoglobin A 1c was quantified by HPLC using a Tosoh A1c 2.2 instrument. One hundred and two samples were obtained from fully informed young healthy volunteers of north European origin, in order to establish a reference range for haemoglobin A 2 . Data from the same samples were used to establish a reference range for peak 2 (P2), this being the peak with a retention time slightly longer than that of haemoglobin F, which we had previously noted to be increased in patients with an increased proportion of haemoglobin A 1c . The instruction manual of the Variant II states 'Diabetic specimens typically exhibit an elevated P2 peak'. In order to investigate the relationship between P2 on the Bio-Rad Variant II and haemoglobin A 1c on the Tosoh A1c 2.2, 93 samples from either healthy volunteers or patients with an elevated glycated fraction were studied in parallel on the two instruments. Results of all patients investigated by our haemoglobinopathy laboratory were surveyed over a 3-month period and when a sample was found to have a P2 fraction of 6% or greater, haemoglobin A 1c was measured and we investigated whether or not a diagnosis of diabetes mellitus had previously been established. If a review of request forms and laboratory records did not disclose this diagnosis, we contacted relevant clinical staff and requested review of clinical notes. If the patient had not been identified previously as suffering from diabetes mellitus, further tests were advised in order to confirm the diagnosis. Subsequently we followed-up the results of these confirmatory tests. Tests done were those usually carried out by the relevant clinical staff in order to confirm a diagnosis of diabetes
