To the Editor,
Substitution of glutamic acid by lysine at codon 26 in the beta globin gene results in hemoglobin (Hb) E . In Hb E trait the percentage of Hb E is around 30%. In homozygous Hb E this percentage is around 90 or more whereas in Hb E/β thalassemia it is about 40%–60% . This is the commonest Hb variant in Southeast Asia and in India it is common in the north-eastern regions . In cation exchange high performance liquid chromatography, Hb E elutes along with Hb A2 . In homozygous Hb E, no Hb A will be seen, whereas a small amount of Hb F (fetal Hb) may be present. Usually the amount of Hb F is <5% but may go up to 15% .
In the present study we evaluated chromatograms of 70 patients with homozygous Hb E disorder. Only patients with no history of blood transfusion in the previous 3 months were included in the study. EDTA anticoagulated blood was loaded on to the D-10 analyzer (Bio-Rad) where the Hbs are separated based on their ionic interactions with the cartridge and detected on the basis of their absorbance at 415 nm. The chromatograms were analyzed for the presence of Hbs E, A and F. The results are summarized in Table 1. Our patients varied in age from 4 to 66 years, of whom there were 47 males and 23 females. Their Hb levels varied from 40 to 134 g/L. Hb A was seen in all cases and varied from 2.9% to 16.2%. Hemoglobin F was seen in all cases and varied from 1.2% to 15.6%. Hemoglobin E+ A2 varied from 86.6% to 110%. Regression analysis of Hb A with other variables (total Hb level, Hb F, E+A2) was done using Vassarstats (online resource). Significant p-value was taken to be <0.05. On regression analysis a negative correlation between Hb A and total Hb level (p-value of 0.02) as well as Hb F (p-value 0.003) was seen. There seems to be no relationship with amount of Hb E (p-value 0.39).
Means of various Hbs in homozygous Hb E disorder (Hb E elutes with A2 and hence it is depicted as E+A2).
|Hemoglobins||Mean (standard deviation)|
|Hb E+A2%||99.36 (4.99)|
|Hb F%||4.02 (2.70)|
|Hb A%||7.57 (2.54)|
|Hb g/L||97.9 (1.95)|
It is interesting to note the presence of Hb A in all 70 cases whereas; we do not expect Hb A in untransfused homozygous Hb E disorders. In our patients it varied from an insignificant 2.9 to as much as 16.2%. We presume that this Hb A may have resulted from adducts of Hb E falling in the Hb A peak. Adducts of sickle Hb causing raised Hb A2 has been reported in literature . Such adducts causing falsely raised Hb A in homozygous Hb E disorders have not been reported previously in literature. In homozygous Hb E disorders it may raise a suspicion of previous transfusion whereas in Hb E/Beta thalassemia it may result in one thinking of Hb E/Beta+ thalassemia mutation rather than a Hb E/Beta0 thalassemia mutation. It might go undiagnosed in Hb E trait as Hb A is expected to be present. However, this could be an instrument related issue as there are no reports of this occurrence in other analyzers [6, 7]. The second problem associated with homozygous Hb E disorder is the amount of Hb E present. In 32 of our cases the percentage reported exceeded 100% with the maximum being 110%. This creates a peculiar situation where the total Hb along with Hb F and Hb A and minor Hbs exceeds 100%. There are two ways of computing peak percentages. One is the standardless method (peak are of Hb/total area×100) which assumes the total Hb to be 100% and the other is by use of standard calibrators. Two levels of calibrators are used for Hb A1C, Hb F and A2 and the result is integrated in to the value got above using the regression equation [area%=intercept+slope(peak area/total area×100)]. As Hb E falls in A2 peak, the results reported by this method are higher than the standardless method. Hence homozygous Hb E disorders may have Hb E of more than 100% and Hb E/Beta thalassemia may be diagnosed with Hb E above 80% if the clinical and blood picture matches with the findings.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
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