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

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.

Introduction

Mortality from sickle cell disease (SCD) is high in the first 5 years of life, maximum being the initial 6 months [1]. Screening of newborn children for the presence of sickle hemoglobin (HbS) allows an early clinical intervention in SCD (homozygous sickle cell anemia and double heterozygotes of HbS and beta thalassemia) and reduces the associated morbidity and mortality in young children with this disorder [2]. Therefore, for long the focus of newborn screening (NBS) for hemoglobinopathies has been on SCD. This has resulted in limited experience in screening of other hemoglobin variants that are associated with a high degree of morbidity, especially if co-inherited with HbS [3]. This is more relevant in the Indian context since the incidence of non-sickle hemoglobinopathies that can enhance the morbidity associated with HbS, is high in several communities and geographic areas in this country [4]. Although screening of newborns for sickle cell disease and other hemoglobinopathies is mandatory in many countries [5, 6], very few states in India including those with a high incidence of SCD and other hemoglobinopathies have any statutory requirement for screening of newborns for abnormal Hb. The cost of equipment and reagents, and the associated operational and logistic challenges involved in transporting very small quantities of whole blood samples from newborns residing in remote areas to a central laboratory have made performance of this test difficult on a large scale. The use of dry blood spots (DBS) for transporting newborn samples has resolved the logistic issues to some extent. However, the recommended use of dedicated and especially developed high performance liquid chromatography (HPLC) platforms and software for this purpose that are very expensive has been a deterrent for a wider application of this preventive measure. Therefore, in order to find a cost-effective alternative, we compared the results of screening of DBS from newborns by a dedicated newborn screening HPLC system (Bio Rad Variant NBS) with those obtained by a commonly used and open HPLC system (Bio Rad Variant II). Our data show a 100% correlation between the results obtained by the two approaches. This opens up the possibility of using Variant II HPLC system for screening of hemoglobinopathies in DBS samples from newborns with several associated advantages as discussed below.

Materials and methods

DBS from 7522 newborn children between the ages of 1 day and 4 weeks, referred from hospitals and private clinics in India and abroad from January, 2016 to November 2018, were first examined by Variant NBS system (Bio Rad, Hercules, USA) as per the established protocol. Approximately 20% (1476) of the total samples were received from abroad—all from the Middle East.

DBS was prepared by trained nurses or healthcare workers from heel prick using filter paper cards specially designed for this purpose (supplied by M/s Bio Rad, Hercules, USA) in order to ensure adequate sampling and collection of a fixed amount of blood per spot. The cards, labelled with the identification and demographic information of the babies, were transported to our laboratory at room temperature ensuring that the same reach the laboratory within 24 h of collection. Hemoglobin was eluted from the DBS in deionized water. The hemoglobin solution thus prepared from each newborn was analysed first on the NBS HPLC system.

Eighty six DBS samples that showed the presence of abnormal Hb peaks in the NBS system were subsequently analysed by Variant II system (Bio Rad, Hercules, USA) which is an open HPLC system used by us for routine screening of hemoglobinopathies in anticoagulated blood samples. Twenty of the remaining cases (7436) that yielded normal results in the NBS system were also tested on Variant II system as controls.

All DBS samples were referred to our laboratory officially by the clinician in-charge of the case. Informed parental consent for testing was obtained for each sample prior to its collection.

Results

Hemoglobinopathies detected by Variant NBS system in the 86/7522 (1.14%) DBS samples consisted of HbS (28 cases), HbE (21 cases), HbD (27 cases), HbQ India (9 cases)—all in heterozygous state, and Hb Barts (1 case). Of the samples received from the Middle East (1476/7522) 18 showed hemoglobinopathies (1.22%). Eight hundred fifty six of the newborns were of Indian origin while 620 were non-Indian consisting predominantly of Arabs and very few Pilipino. The incidence of hemoglobinopathies among the newborns from the Middle East was similar (1.22%; 18/1476) to that in the newborns from India (1.12%; 68/6046). The distribution of hemoglobinopathies among the samples received from abroad was, HbS trait = 10, HbE trait = 2 and HbD trait = 6. (Table 1). The incidence of HbS among the two groups was 0.7% (10/1476) and 0.3% (18/6046) respectively.

Table 1.

The incidence and distribution of hemoglobinopathies in different populations examined

Geographic area of origin of newborns	Number of cases	Total number with hemoglobinopathies (% incidence)	Types of hemoglobinopathies (% incidence)
Indian newborns	6046	68 (1.12)	HbS = 18 (0.3); HbE = 19 (0.3); HbD = 21 (0.35); HbQ India = 9 (0.15); Hb Bart’s = 1
Newborns from the Middle East	1476 (Indians = 856; Othersa = 620)	18 (1.22) [Indians 10 (1.17); Others = 8 (1.29)]	HbS = 10 (0.7); HbE = 2; HbD = 6 (0.4)
Grand total	7522	86 (1.14)	HbS = 28 (0.4); HbE = 21 (0.28); HbD = 27 (0.36); HbQ India = 9 (0.12); Hb Bart’s = 1

Please note that the incidences are comparable

^aConsisted predominantly of Arabs, and very few Pilipino

All the 86 samples showing hemoglobinopathies were also examined in parallel by Bio Rad Variant II HPLC system (see examples in Fig. 1a–n). There was a 100% correlation between the results obtained by the two systems in all the 86 cases. Cases with normal results in NBS system showed normal results in Variant II system (Fig. 1a, b). Figure 1c–n show NBS HPLC histogram patterns of HbS, HbE, HbD, Hb Bart’s and HbQ India and their corresponding patterns in Hb Variant II system.

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

Nine newborn children showed the presence of HbQ India trait (Hb peak retention time of ~ 1.4 min) in Variant NBS system (Fig. 1k, l). The presence of HbQ India in 5 of these 9 newborns was also verified by the presence of HbQ India peak in one of the two parents in Variant II system. Blood samples were not available for testing in the parents of the remaining cases. Furthermore, DBS prepared from the parents with HbQ India trait were also reverse-tested by the NBS system and in all of them the abnormal Hb, i.e. HbQ India eluted at or near 1.4 min as in the cases of their children (Fig. 1m, n). Interestingly, an additional abnormal Hb peak was observed in HbD window (retention time 1.01–1.07 min) in NBS system in all newborns with HbQ India trait. In the Variant II system this peak eluted in the retention time range of 3.8 min (in 5 cases) to 3.88 min (in the remaining 4 cases) (Fig. 1l). HbQ India eluted at ~ 4.7 min in all cases in Variant II as expected. The abnormal peak at retention time of 3.8 min in the newborns possibly represents a hybrid Hb (‘HbF Q India’) resulting from the combination of the abnormal alpha globin chain of HbQ India with the normal gamma chain of HbF that is available in abundance in neonatal red cells. Only the parents showed an additional small post-HbQ peak at RT of 4.82–4.84 min, known to be observed in adults with alpha chain variants such as HbQ India. The absence of this peak in the newborns could be due to the small quantities of HbQ India.

Discussion

The low incidence of hemoglobinopathies (~ 1%) in a cross sectional population of resident and non-resident newborns of Indian origin observed by us is similar to that reported in other screening programmes [5, 6]. In a newborn screening programme in the United Arab Emirates (UAE) Al Hosani et al. found the incidence of HbS disease to be 0.04% (0.07% for UAE citizens and 0.02% for non-UAE citizens). The incidence of sickle cell trait was 1.1% overall (1.5% for UAE citizens and 0.8% for non-UAE citizens) [5]. All our cases of HbS were traits and we found an incidence of 0.37% (0.3% in native Indian newborns and 0.6% among foreign Indians) for this hemoglobinopathy which is lower than that reported from the UAE (1.1%). In a study conducted in the north eastern state of Tripura in India, however, Upadhyaye et al. reported a very high incidence of HbE in heretrozygous (9.3%) and homozygous states (3.3%) in cord blood samples [6] reflecting the high incidence of HbE in the population in north east India. Regional biases in the incidence and distribution of hemoglobinpathies will obviously be reflected in the spectrum of hemoglobinopathies observed in the newborn in such screening programmes.

That all newborns with hemoglobinopathies in this study were heterozygotes is interesting. The reason for the same, however, is not clear. An active prenatal diagnostic programme for hemoglobinopathies at some of the hospitals and maternity centres from where these samples were received could have contributed to this trend.

The small abnormal Hb peak seen at RT of 1.04–1.07 min (in HbD region) in the Variant NBS system and at 3.80–3.88 min in Variant II systems in the newborn children with HbQ India (Fig. 1l) possibly represents a hybrid Hb that results from combination of the abnormal alpha chain of HbQ India with the normal gamma chain of HbF (HbF Q India). It would be interesting to look for this peak in adults with compound heterozygotes for HbQ India and other hemoglobinopathies associated with elevated HbF, e.g. homozygous beta thalassemia, delta-beta thalassemia, hereditary persistence of fetal Hb, and HbS disease etc. A detailed search of the published literature on this subject failed to throw up any such example. We have observed the presence of similar hybrid Hb peaks in HPLC in double heterozygotes of HbQ India (an alpha chain variant) with HbD Punjab (a beta chain variant) (unpublished), possibly composed of the variant alpha chain and the abnormal beta chain.

The hump in the descending limb of HbA2 and the post-HbQ India small peak in Variant II HPLC system in the parents of newborns with HbQ India (Fig. 1m, n) are known associations with this hemoglobinopathy as explained in earlier publications [7, 8]. These findings provide a strong clue for the diagnosis HbQ India from HPLC chromatogram itself.

Our data show that it is possible to use the more commonly used Variant II HPLC system for screening of newborn children for the presence of inherited hemoglobinopathies using either anticoagulated blood samples or DBS instead of the Variant NBS system that is designed for DBS only. Therefore, laboratories having Variant II HPLC system can also use the instrument for newborn screening for hemoglobinopathies. This approach also provides a cost effective alternative to and a backup method for NBS screening of hemoglobinopathies by HPLC through better utilization of the equipment.