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. 2021 Jul;37(3):430-435.
doi: 10.1007/s12288-020-01352-2. Epub 2021 Feb 3.

Screening of Dry Blood Spots from Newborns by Two High Performance Liquid Chromatography (HPLC) Systems: A Comparison of Their Ability to Diagnose Both Sickle and Non-sickle Hemoglobinopathies

Manisha Ramani Daruwalla  1 , Amar Das Gupta  1 , Ravikiran Pawar  1
Affiliations

Screening of Dry Blood Spots from Newborns by Two High Performance Liquid Chromatography (HPLC) Systems: A Comparison of Their Ability to Diagnose Both Sickle and Non-sickle Hemoglobinopathies

Manisha Ramani Daruwalla et al. Indian J Hematol Blood Transfus. 2021 Jul.

Abstract

Screening of newborns for the presence of sickle hemoglobin (HbS) is aimed at reducing the morbidity and mortality associated with sickle cell disease in early childhood. The high cost and limited availability of dedicated high performance liquid chromatography (HPLC) systems specially designed for screening of dry blood spots (DBS), however, restrict a wider application of this preventive approach. Therefore, we examined the ability of a commonly used HPLC system for detection of hemoglobinopathies in DBS samples in order to find an alternative for the dedicated newborn screening (NBS) HPLC system. DBS samples from 7522 newborns were first examined by Variant NBS HPLC system (Bio Rad, USA) for the presence of hemoglobinopathies. Positive samples were then analysed by Variant II system (Bio Rad, USA), another platform commonly used for hemoglobinopathy screening of anticoagulated blood samples. Eighty six newborns (1.1%) showed the presence of hemoglobinopathies (HbS 28, HbE 21, HbD 27, HbQ India 9 and Hb Barts 1) by Variant NBS system-all in heterozygous state. There was 100% correlation between the two sets of results obtained by the two HPLC systems. Newborns with HbQ India showed an additional Hb peak in HPLC resulting from combination of the abnormal alpha globin chain of HbQ India with the normal gamma chain of HbF-'HbF Q India'. Variant II HPLC system, used for routine hemoglobinopathy screening in anticoagulated blood, can also be used for screening DBS samples. This obviates the need for a dedicated NBS system for hemoglobinopathy screening in newborns. We also demonstrated that both the systems are equally competent in detecting non-sickle Hb variants in DBS samples.

Keywords: HPLC for diagnosis of hemoglobinopathies; Hemoglobinopathies in newborns; NBS HPLC system; Newborn screening for hemoglobinopathies.

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Figures

Fig. 1
Fig. 1
HPLC chromatographs of DBS samples from normal newborns (a, b) and from those with hemoglobinopathies (c–n) on Variant NBS and Variant II systems. c, d HbS trait; e, f Hb E trait; g, h HbD trait; i, j Hb Bart’s; k, l: HbQ India in the newborn showing an additional hybrid Hb peak at 3.84 min, and m, n HbQ India in the mother of one of the newborns. The abnormal Hb peaks have been marked by arrows. The corresponding retention times are mentioned in the chromatographs
Fig. 1
Fig. 1
HPLC chromatographs of DBS samples from normal newborns (a, b) and from those with hemoglobinopathies (c–n) on Variant NBS and Variant II systems. c, d HbS trait; e, f Hb E trait; g, h HbD trait; i, j Hb Bart’s; k, l: HbQ India in the newborn showing an additional hybrid Hb peak at 3.84 min, and m, n HbQ India in the mother of one of the newborns. The abnormal Hb peaks have been marked by arrows. The corresponding retention times are mentioned in the chromatographs
Fig. 1
Fig. 1
HPLC chromatographs of DBS samples from normal newborns (a, b) and from those with hemoglobinopathies (c–n) on Variant NBS and Variant II systems. c, d HbS trait; e, f Hb E trait; g, h HbD trait; i, j Hb Bart’s; k, l: HbQ India in the newborn showing an additional hybrid Hb peak at 3.84 min, and m, n HbQ India in the mother of one of the newborns. The abnormal Hb peaks have been marked by arrows. The corresponding retention times are mentioned in the chromatographs
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