Hemoglobin, globin and heme assessment
Assessment of the hemoglobin is an important assay to define RBC characteristics. Altering the hemoglobin switch from adult hemoglobin to fetal hemoglobin (HbF) has been used to improve RBC physiology in hemoglobinopathies. Similarly, compounds have been developed that will alter hemoglobin such as Voxelotor.
The figure shows hemoglobin assessment of a sickle cell patient on hydroxyurea, a HbF stimulator before (left) and after (right) treatment with voxelotor. All hemoglobin species are altered by voxelotor.
The HPLC assessment of hemoglobin and globin in the RBC lab is superior as shown by a recent analysis of a hemoglobin variant. Hemoglobin La Desirade (HbLD) is a hemoglobin variant characterized by amino acid Alanine (Ala) replacing Valine (Val) at position 129 in the beta globin chain. In both standard Electrophoresis (CE) and HPLC, HbLD runs like normal HbA. This poses a diagnostic problem. When combined with HbS, patients with HbLD/S could be misdiagnosed as sickle cell trait (HbA/S). Sickle cell trait cells will sickle under low oxygen and sickling is also reported for HbLD/S. The rate of sickling may differ as the levels of HbS in these HbLD/S heterozygotes is significantly increased as compared to sickle cell trait. This suggests that HbLD/S poses a higher risk for sickling which may become apparent at high altitude (lower PO2).
In contrast to published data on HPLC and clinical lab standard CE analysis, the RBC lab HPLC hemoglobin and globin assessment allows separation of HbLD from HbA, which is further confirmed by the detection of an altered beta globin species. Only 10 μl of fresh or frozen blood is needed for these assays.
HPLC Hb separation profile of blood from a HbSS patient (red), HbA normal (black) and HbLD (green).
HPLC globin separation profile of blood from a HbSS patient (red), HbA normal (black) and HbLD (green).
Heme assessment
Heme is a highly hydrophobic compound. When released from hemoglobin into plasma it will partition in several pools. The RBC lab provides assessment of “bound” and “unbound” heme in biological samples. Combined with assessment of haptoglobin and hemopexin, it provides a detailed picture of this compound which play a potential role in oxidative damage and inflammatory processes.
