α‐Haemoglobin pool measurement: a useful biomarker for evaluation of β‐thalassaemia intermedia? – response to Huang and Li (2017)

We thank Drs Huang and Li for their interesting comments pertaining to our recently published article (Vasseur et al, 2017). Their first concern is about the exact genotypes of the 50 healthy volunteers initially recruited and the values of their α-Hb pool measurements. Of the 50 healthy volunteers recruited, 1 had every clinical, enzymatic and haematological data missing, and 11 had one of the tested most common α or β abnormal genotypes. The 38 healthy volunteers without detectable abnormalities of α- and β-globin genes were then used as controls. Table 1 summarises the β and α globin genotypes of all healthy volunteers. All healthy volunteers were asymptomatic and no origin criteria were used. Individual origins were not documented but, as a group, the controls well reflected patient ethnic diversity. So, it is not surprising that amongst 50 healthy volunteers re-examined, 11 had a common α or β abnormal genotype (22%) which was an exclusion criterion. Furthermore, this observation is in agreement with previous studies in normal individuals, which reported that the −α3·7 deletion is observed in almost every studied ethnic group with a frequency up to 19% (Nava et al, 2006) and the carrier frequency of ααα (anti 3·7) triplication is present in 0·05–5%, depending on the studied population and ethnic group (Goossens et al, 1980; Nava et al, 2006; Giordano et al, 2009). The second concern is the question why is there still an α-Hb pool in Hb H disease patients with only one α-globin gene active? As we have noted in our article, the term ‘α-Hb pool’ corresponds to the soluble α-Hb that is not bound to β-chain in red blood cells (RBCs) but could be linked to α-haemoglobin stabilizing protein (AHSP). We previously conducted a competitive in vitro study by adding AHSP/α-Hb complex to the glutathione S-transferase (GST)-AHSP column; 20% of the α-Hb within the AHSP/α-Hb complex were indeed transferred to the AHSP bound to the column. These results show that the AHSP bound to the column can trap both free α-Hb and a large fraction of α-Hb within the AHSP/α-Hb complex in the haemolysate (Vasseur et al, 2011). We therefore believe that the α-Hb pool we measured in Hb H could correspond to α-Hb linked to AHSP in the haemolysate. Previously published work has been shown that the α-Hb complexed with AHSP could exist as obligatory intermediates during Hb A synthesis (Weiss & dos Santos, 2009). So it is not surprising that, even in genetic conditions with a very low alpha/non-alpha ratio a small amount of alpha pool could be detected (Weiss & dos Santos, 2009). It would indeed be interesting to determine the α-Hb pool in subjects with six active α-globin genes, but we have not encountered such cases. Their third point states that ‘more β-thal intermedia cases needed for study to enhance the classification system of the severity of this disorder by using the free α–Hb pool in the scoring system’. We agree with this comment. The discussed work was designed as a preliminary proof of concept. Finally, we are in full agreement with Drs Huang and Li regarding the development of an economic and accurate automated test for the α-Hb measurement in the future. However, the validation and acceptance of such a test in the clinic will require an effective laboratory-industry partnership. This work was supported by the Institut National de la Santé et de la Recherche Médicale (Inserm), France, by the Assistance Publique-Hôpitaux de Paris and by the University of Paris Est Créteil (UPEC), France. This study is supported by PRCT 2012-2016 (program of ‘Recherche Clinique Translationnelle’), funded by Inserm and by French Ministry of Health (DGOS). V.B-C, F.G and C.V conceived and designed the study, and wrote the paper.