CE-HPLC Derived P2 and P3-Peaks in Health and in Hb D-Punjab and HbE States
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CORRESPONDENCE
CE-HPLC Derived P2 and P3-Peaks in Health and in Hb D-Punjab and HbE States Pooja Murgai1,2 • Sanjeev Chhabra2 • Reena Das2
•
Prashant Sharma2
Received: 1 August 2020 / Accepted: 29 September 2020 Ó Indian Society of Hematology and Blood Transfusion 2020
Dear Sir, Diagnostic interpretation of cation-exchange high-performance liquid chromatography (CE-HPLC) focusses on HbA0, HbF and HbA2 along with any variant peaks identified. However, additional chromatographic peaks are also always present, including the P2- and P3-peaks of post-translationally modified HbA0 [1]. The P2-peak represents glycated Hb and an increase helps detect unsuspected diabetes mellitus. It can also corroborate reticulocytosis if reduced. Rare variant hemoglobins like Hb Hope and Hb K-Woolwich also elute in the P2-window (retention time 1.28–1.50 min). The P3% increases on prolonged storage of blood samples and several hemoglobin variants including J-Meerut, Camden, Austin, Fukuyuma and N-Baltimore elute in the P3-window (retention time 1.50–1.90 min) [1–4]. Only limited information is available in literature on the ranges and variations of these two universal peaks in health and in common hemoglobinopathies. In order to characterize these often-ignored Hb subsets, we compared the CE-HPLC findings from 103 healthy adult subjects who had normal CE-HPLC patterns with HPLCs of individuals with the HbD-Punjab variant
Prior presentation This research was presented as a poster in the Indo-US Bilateral Workshop on Thalassemia and Sickle Cell Disease held at PGIMER, Chandigarh on November 5–6, 2016. & Prashant Sharma [email protected] 1
Department of Pathology, Indira Gandhi Medical College, Shimla, Himachal Pradesh, India
2
Department of Hematology, Postgraduate Institute of Medical Education and Research, Level 5, Research Block A, Sector 12, Chandigarh 160012, India
(n = 53) and with HbE (n = 53). All 209 cases were run on the Variant IITM HPLC instrument (Bio-Rad Laboratories, Hercules, USA) using the beta-thal ShortTM programme. D and E variants were verified by automated alkaline pH electrophoresis (Genio-S, Interlab, Rome, Italy) and further corroborated by hemogram findings and the ethnic backgrounds of the patients and family studies to distinguish homozygous from double heterozygous states. We encountered 44 cases of HbD trait, 9 double heterozygous HbD ? b thalassemia, 29 HbE trait, 21 double heterozygous HbE ? b thalassemia and 3 HbE homozygotes. Mean P2% in controls was 3.8 ± 0.7 (range 2.8–7.8) while HbD-Punjab and HbE showed significantly lower P2% of 1.9 ± 1.0 (range 0.0–4.9) and 1.7 ± 1.4 (range 0–4.2), respectively (Table 1, p \ 0.001 by 1-way ANOVA). P2% was inversely proportional to the HbD% and HbE% (Pearson correlation coefficient, r = - 0.854, p \ 0.001). Double heterozygotes for HbD Punjab ? bthalassemia showed especially low P2% (mean 0.3%, range 0.0–1.2) as compared to HbD-traits (mean 2.2%, range 1.3–4.9). P2 was undetectable in all the 3 homozygous HbE cases and significantly lower in dou
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