Lorraine Phelan

Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of α thalassaemia

Aims: To assess the accuracy and precision of measuring haemoglobin A2 by high performance liquid chromatography (HPLC) in the presence and absence of sickle cell trait, with or without α thalassaemia trait. Methods: The haemoglobin A2 percentage and the haemoglobin A2 plus S percentages were determined by HPLC on 82 normal controls and 78 patients with sickle cell trait, respectively; the α thalassaemia status of each patient was determined by polymerase chain reaction. Red cell indices and haemoglobin A2 and S percentages were compared in patients with two, three, or four α genes. Results: Of the 78 patients with sickle cell trait, 17 were heterozygous for α+ thalassaemia (−α3.7/αα) and 13 were homozygous (−α3.7/−α3.7). Microcolumn chromatography showed that the haemoglobin A2 percentage was slightly, but significantly, higher than normal in sickle cell trait. HPLC determinations of haemoglobin A2 percentage in patients with sickle cell trait are precise but inaccurate, the percentage being appreciably overestimated. The measured haemoglobin A2 percentage is stable for one week, but inaccuracy increases by two weeks in most samples. Despite this inaccuracy, there are significant differences in the HPLC “haemoglobin A2 percentage” between groups of individuals with two, three, and four α genes. Conclusions: Haemoglobin A2 determinations by HPLC are precise but inaccurate. Nevertheless, there are significant differences in the haemoglobin A2 percentage in subjects with two, three, and four α genes. Although there is some overlap between groups, this can be useful, together with the red cell indices, in predicting the likelihood of coexisting α thalassaemia.

Increased haemoglobin A2 percentage in HIV infection: disease or treatment?

An elevated haemoglobin A2 percentage has been reported in HIV-infected patients, possibly attributable to therapy. In cross-sectional and cohort studies we have established that A2 is often elevated in untreated patients; a further rise during treatment is attributable specifically to zidovudine. The haemoglobin A2 may be high enough to lead to a misdiagnosis of beta thalassemia trait if there is a lack of awareness of this unexpected effect of HIV infection and its treatment.

Reassessment of a simple chemical method using DCIP for screening for haemoglobin E

Background: Haemoglobin E is a variant haemoglobin that can lead to considerable morbidity in compound heterozygous states with β thalassaemia. Therefore, its detection is important because it permits antenatal counselling. The parts of the world where haemoglobin E is prevalent are resource poor and detection can therefore be problematical. A simple visual test using 2,6-dichlorophenolindophenol (DCIP) has been developed in Thailand, but its use has not become widespread. This test has now become available in kit form. Aims/Methods: To evaluate the new DCIP test kit for the detection of haemoglobin E. Results: Seventeen of 18 samples from individuals with haemoglobin E gave positive results and one gave an equivocal result. False positive or equivocal results were seen in three of 21 individuals with other disorders of globin chain synthesis but were not seen in normal subjects. Conclusions: This study evaluated the sensitivity, specificity, and reproducibility of the kit and confirmed the usefulness of the DCIP test as a screening test for haemoglobin E. In countries with limited health resources, its use would reduce the number of samples requiring referral to a central laboratory for definitive tests.

Hb NIIGATA (β1(NA1)Val®Leu) IN A ROMANIAN INDIVIDUAL RESULTING FROM ANOTHER NUCLEOTIDE SUBSTITUTION THAN THAT FOUND IN THE JAPANESE

We present a new case of Hb Niigata that we named Hb Niigata(C), observed in a woman from Romania, with a mutation different from that described in Japanese (GTG→CTG instead of GTG→TTG). This single nucleotide substitution replaces the valine residue for leucine at codon 1 and causes retention of the N-terminal methionine leading to an elongated β chain. This mutation was without any hematological consequences.

Diabetes mellitus diagnosed following request for haemoglobin electrophoresis

A general practitioner requested a full blood count and haemoglobin electrophoresis on a 38-year-old Somalian woman with � known anaemia� . The haemoglobin concentration was normal, although there was borderline microcytosis. High performance liquid chromatography (HPLC) was performed as per standard protocol in our laboratory. The trace (lower image) showed the presence of haemoglobins A, A2 and F quantified at 80%, 2AE4% and 2AE4% respectively. In addition, there was a prominent peak of glycosylated haemoglobin (arrowhead, compared with a normal trace, upper image), quantified at 10AE9%. The patient’s general practitioner was contacted. The patient was not known to be diabetic but she did have a history of dizziness, weight loss, thirst and urinary frequency. Appropriate samples were subsequently obtained and these showed a fasting blood glucose of 16AE9 mmol ⁄ l and HbA1c of 11AE4%, confirming the diagnosis of diabetes mellitus. This case illustrates that using HPLC rather than haemoglobin electrophoresis as the primary technique for investigating suspected haemoglobinopathies may lead to the diagnosis of unsuspected diabetes mellitus. Haemoglobinopathy laboratories should therefore draw the attention of clinical staff to an increase of glycosylated haemoglobin in any patient who is not known to be diabetic.

Haemoglobin A2′ and its significance in β thalassaemia diagnosis

We have studied, haemoglobin A2′ (A2 prime), a δ chain variant haemoglobin occurring in a small percentage of individuals of African ancestry. In heterozygotes, the percentage of haemoglobin A2′ was found to be slightly lower then the percentage of haemoglobin A2, suggesting that the variant δ chain is synthesized at a reduced rate. When quantifying haemoglobin A2 for the diagnosis of β thalassaemia heterozygosity, it is essential to add together the A2 and A2′ to give ‘total haemoglobin A2’. However, it not necessary to use a different reference range for total haemoglobin A2 in A2′ heterozygotes. When using microcolumn chromatography, A2′ was found to be measured with A2. On the high‐performance liquid chromatography instrument studied, haemoglobin A2′ fell in the haemoglobin S window but its retention time differed from that of haemoglobin S.

The frequency of detection of unexpected diabetes mellitus during haemoglobinopathy investigations.

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.

Inaccuracy of high-performance liquid chromatography estimation of haemoglobin F in the presence of increased haemoglobin A1c

Increasingly high‐performance liquid chromatography is being used for identification and quantification of normal and variant haemoglobins. In many laboratories, the Beta Thal Short programme of the Bio‐Rad Variant II instrument is used for this purpose. We noted that a factitious elevation of haemoglobin F was sometimes observed in diabetic patients and therefore carried out a systematic study of this phenomenon. We found discrepant results in 41% of samples from diabetic patients but in no normal volunteers. This factitious elevation could be predicted from a retention time for haemoglobin F of more than 1.15 min, the normal retention time being 1.08–1.15 min. Haemoglobinopathy laboratories need to be alert to the possibility of this erroneous result.