Samples: Of the 800 subjects tested, 259 (32.4%) were males and 541 (67.6%) were females. The age range of patients was from 5 months to 77 yr with a median age of 25 yr (mean age 24.2 yr). The preponderance of young female patients was due to antenatal patients coming for routine HPLC work-up. Of the total cases, 553 (69.1%) were found to have normal HPLC pattern. Analysis of the retention times and %Hb for HbA0, HbA2, and HbF showed no significant difference with regard to column and/or reagent changes. In this study the influence of haemoglobin stability testing and patient ethnicity were not included.
Retention times and proportions of haemoglobin variants:
The number of observations, %Hb fraction, haemogram findings and the type of Hb variants encountered are shown in the . Separation of cases into transfused and not transfused was not done. Although the sampling was done with due precaution, the HbA and HbF levels shown in the may not be truly representative particularly in the thal major/intermedia group due to frequent transfusion requirement in some of them. As expected, out of 800 samples, β- thalassaemia trait was detected most frequently i.e
., in 145 (18.1%) cases with cut-off value of HbA2
being >3.9 per cent3
and β- thalassaemia major/ β- thalassaemia intermedia was seen in 23 (2.9%) cases. Apart from β-thalassaemia, nine additional variants were encountered; HbS (2.8%), HbE (2.5%) and HbD-Punjab (1.1%) were the most common variants present. Other variants included HbQ-India, Hb-Lepore, δβ-thal/HPFH, HbD-Iran, HbJ-Meerut, Hb-H disease. Possibility of an α-thalassemia trait could be suggested in 11 (1.4 %) cases on the basis of red cell indices and the levels of HbA2
[2.2 (± 0.3)%] which were significantly (P
<0.001) lower than normal population 2.9 ± 0.4. However, results of further molecular testing were not available for a definite conclusion.
Relevant laboratory parameters in relation to normal and abnormal haemoglobin variants
The retention time alone [n = 4 (i.e
., HbS, HbD-Punjab, HbQ-India, HbH)] or in conjunction with %Hb [n = 3 (i.e
., HbJ-Meerut, HbE, δβ-thal trait/HPFH)] or with the peak characteristics [n = 2 (HbD-Iran, Hb-Lepore)] could identify all the 9 haemoglobin variants seen. However, Hb-electrophoresis was resorted to in many doubtful cases to confirm the diagnosis. Family HPLC screening also helped in some difficult cases especially in double heterozygous states. Role of family studies has been emphasized by other studies also4
.Various normal and variant haemoglobins encountered were as follows:
Haemoglobin variants with retention times <1.0 min: One case of HbH disease was detected, which was confirmed by fast-moving band on electrophoresis and a positive HbH inclusion test.
Haemoglobin variants with retention times in the p1 window (0.63–0.85 min): No haemoglobin variants were detected in this window.
Haemoglobin variants with retention times in the F window (0.98-1.20 min)
: At least seven haemoglobin variants (four β- and three α-variants) are expected to elute in this window, all in quantities >10 per cent5
. However, most of our patients with high Hb in the F window were mostly homozygous β-thalassaemia patients or double heterozygous β thalassaemia/haemoglobinopathy patients confirmed by Hb-electrophoresis and family studies. Four cases revealed high HbF levels in the range of 1-10 per cent, two of whom had haemolytic anaemia (Coombs test positive) and the other two were pregnant patients. No variant was found in this window.
Haemoglobin variants with retention times in the p2 window (1.24-1.40 min): HbA1c elutes in the P2 window. When the elution peak was >7 per cent of the total haemoglobin, the patient records were checked for indication of diabetes. We came across two cases of known diabetics with a mean P2 value of 13.2 per cent and retention time of 1.28 min. No haemoglobin variants were detected in this window.
Haemoglobin variants with retention times in the p3 window (1.40-1.90 min)
: A previous study has found nine haemoglobin variants (four α- and five β-variants) with elution peaks in the P3 window6
. We found a single case of possible HbJ-Meerut which was suspected based on its retention time (1.73 min) and Hb-percentage (26.5%).
Haemoglobin variants with retention times in the A0 window (1.90-3.10 min):
Six haemoglobin variants (two α- and four β-variants) have been reported in the A0
. No such variant was found in our study.
Haemoglobin variants with retention times in the A2 window (3.30-3.90 min)
: Four haemoglobin variants had elution peaks in the A2
window- Hb A2
, HbE, Hb-Lepore and HbD Iran. The retention times and %Hb for HbA2
(3.65 min) and HbE (3.73 min) were significantly different (P
<0.001). The retention time for HbD-Iran (3.62 min) appeared to be different from those of HbE (3.73 min) and Hb Lepore (3.5 min). However, statistical analysis could not be done because of less number of cases. In addition, the mean %Hb of HbD-Iran (40.6%) was greater than either of these Hb (HbE- 27.8%, Hb-Lepore- 16.4%). So the values more than 40 per cent in Hb A2
window with a different retention time made us suspect Hb D Iran which was confirmed by starch agarose gel electrophoresis where a band in the SDG region was seen Hb Lepore could be differentiated and identified based on its retention time, %Hb and the characteristic hump on the downward slope of the elution peak6
. δβ-thalassaemia trait presented with a low HbA2
(2.8%) and mildly elevated HbF levels (13.3%).
Haemoglobin variants with retention times in the D window (3.90-4.30 min): Only one haemoglobin variant; HbD-Punjab, with retention times of 4.07 min, was identified in D window. The mean HbA2 values for HbD-Punjab trait (1.7 ± 0.4%) were significantly lower (P<0.001) than the range for HbA2 in the normals.
Haemoglobin variants with retention times in the S window (4.30-4.70 min)
: Only HbS variant, with retention times of 4.41 min, was seen in this window. The mean HbA2
value for HbS trait (3.3 ± 0.3%) was significantly higher (P
<0.001) than the range for HbA2
in the normal samples. A new abnormal haemoglobin Hb D Agri with 2 amino acid substitutions in the same β globin chain [β9 (A6) Ser →Tyr, β121(GH-4) Glu →Gln] has been reported in India which can be mistaken for HbS based on HPLC alone7
. However, a negative sickling and solubility test should raise a suspicion followed by molecular studies for confirmation.
Haemoglobin variants with retention times in the unknown window (4.70–4.90 min): Three cases of HbQ-India were identified with a retention time of 4.7 ± 0.01 min and mean %Hb of 18.5 ± 0.2%.
Haemoglobin variants with retention times in the C window (4.90–5.30 min): No haemoglobin variant was detected in this window.
Effect of iron deficiency anaemia on HbA2 levels: The patients with normal HPLC pattern (total 203 in number where iron studies were available) were divided in two groups depending upon their iron profile. The levels of HbA2 for 186 patients with IDA (2.74 ± 0.34%) was found to be significantly lower (P=0.004) than the 17 patients with normal iron profile (3.03 ± 0.26%). However; there was no significant difference in the levels of HbA2 for 25 β-thalassaemia trait patients, 18 with concomitant IDA (5.49 ± 0.52%) and 7 without IDA (5.09 ± 0.58%).
Effect of megaloblastic anaemia on HbA2 levels: The patients with normal HPLC were divided in two groups depending upon their MCV values (>110fl) and confirmed by further testing. The 19 patients with megaloblastic anaemia showed HbA2 levels (3.32 ± 0.56%) which was significantly higher (P<0.001) than the HbA2 levels (2.89 ± 0.37%) of normal cases with MCV<110fl.