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Objective: The capillary zone electrophoresis (CZE) and highperformance liquid chromatography (HPLC) methods were compared in terms of HbA2 measurement for the assessment of hemoglobinopathies. Materials and Methods: CZE was compared with HPLC for the evaluation of patients without hemoglobinopathy (n=321), with β-thalassemia trait (n=113), and with common (HbD-Punjab, E, C, S/A, and S/S) and rare (HbS/D, O-Arap, Lepore, G-Coushata, Setif, Hamadan, Q-Iran, and H) variants (n=21). The reference range for HbA2 was determined by CZE. Results: Among patients without hemoglobinopathy, the median (2.5th-97.5th percentiles) values were 97.4% (97.0-98.0%) and 97.5% (96.6-98.4%) for HbA (p=0.060) and 2.4% (1.6-3.0%) and 2.5% (1.6-3.1%) for HbA2 (p<0.001) by HPLC and CZE, respectively. The reference range for HbA2 was 1.6-3.1% by CZE. In the comparison of methods for HbA2, there was a constant error of 0.255 (confidence interval: 0.062-0.448) and bias of 0.10% (limit of agreement: 0.33- 0.53), and higher values were obtained with CZE. A strong correlation was observed between the methods (r=0.782). Interrater agreement was almost perfect for clinical diagnosis (=0.911). The two methods detected and identified the common variants similarly. All rare variants, except HbH by HPLC and HbS/D by CZE, were detected as separate peaks by both methods. Conclusion: The two methods were in agreement regarding the preliminary identification of β-thalassemia patients. Different Hb variants were detected by both methods but with possible methodological interference for HbA2 measurements. CZE is a reliable and simple alternative for the evaluation of hemoglobinopathies. The standardization of HbA2 measurements should be prioritized as more techniques become available in routine laboratory practice [ABSTRACT FROM AUTHOR]

Objective: To report the diagnostic challenges of newborn screening for abnormal haemoglobins. Setting: Cord blood samples from 13 hospitals in southwest Jamaica taken in 2008–2019. Methods: Blood spots, collected from the umbilical cord, were analysed by high pressure liquid chromatography (HPLC) to reveal phenotypes for HbSS and HbCC, but genotype confirmation may require parental studies or gene sequencing. Such cases that were successfully traced were analysed in this follow-up study. Results: HPLC screening of 121,306 samples detected HbAS in 11,846 (9.8%), HbAC in 4508 (3.7%) and other electrophoretic abnormalities in 1090 babies. Among 101 previously unconfirmed cases, 34/90 (38%) with HPLC evidence of a HbSS phenotype had other genotypes, and 7/11 (64%) with a HbCC phenotype had other genotypes. Syndromes from the interaction of β thalassaemia occurred in 112 babies (85 with HbS, 27 with HbC) and of genes for hereditary persistence of fetal haemoglobin (HPFH) in 18 (12 with HbS, 6 with HbC). Variants other than HbS and HbC occurred in 270 babies, 16 in combination with either HbS or HbC, and 254 as traits. Most variants are benign even when inherited with HbS, although HbO Arab, HbD Punjab, or Hb Lepore Washington, which occurred in 6 cases, may cause sickle cell disease. Conclusions: Genes for β thalassaemia and HPFH are common in western Jamaica and when associated with HbS may present diagnostic challenges in newborns, as HbF and HbA2 have not reached diagnostic levels. Family and DNA studies may be necessary for genotype confirmation. [ABSTRACT FROM AUTHOR]

Background: Antenatal screening is vital to identifying couples at risk of having children with a clinically significant haemoglobinopathy. In Australia, immigration is increasing carrier incidence. Methods: A retrospective analysis was performed of full blood count, high-performance liquid chromatography and haemoglobin electrophoresis of women and their partners who underwent antenatal haemoglobinopathy screening over three years at a major NSW laboratory. Genetic testing results were included where available. Results: One thousand six hundred and twenty-eight women and 729 male partners were screened at a median gestation of 14 weeks. 8.2% of women had a clinically significant result, with a median 16-day interval to partner testing. In 35% of couples screened simultaneously, the partner did not require testing. Genetic confirmatory testing was performed in 65% of high risk couples. Conclusion: There was a significant delay to antenatal haemoglobinopathy screening for mothers, limiting time for genetic diagnosis, prenatal diagnosis and management of affected pregnancies. Screening should be performed earlier. Simultaneous couple testing is not cost-effective. [ABSTRACT FROM AUTHOR]

Hemoglobinopathies are caused by point mutation in globin gene that results in structural variant of hemoglobin. While 7 % of world populations are carrier of hemoglobinopathies, the prevalence of the disease varies between 3 to 17 % across different population groups in India. In a diagnostic laboratory, alkaline gel electrophoresis and cation exchange-based HPLC (CE-HPLC) are most widely used techniques for characterization of hemoglobin variants. In the above methods, the differential surface charge of hemoglobin molecule in variants is exploited for their characterization. Sometime, co-migration of variants in gel electrophoresis and co-elution or elution with unknown retention time in automated CE-HPLC might lead to ambiguity in the analysis of hemoglobinopathies. Under such circumstances, it is necessary to use other analytical methods that provide unambiguous results. Mass spectrometry-based proteomics approach and DNA sequence analysis are examples of such alternative methods. In the present study, liquid chromatography coupled to mass spectrometry has been used for three commonly observed variants in India, e.g., HbE, HbQ India and HbD Punjab that appeared with inappropriate results in the conventional analysis. A customized hemoglobin variant database has been used in the mass spectrometry-based analysis of those three variants. Mass spectrometry-based proteomics approach was used to analyze above variant sample accurately. [ABSTRACT FROM AUTHOR]

The thalassemias are among the most common monogenic diseases worldwide, a national health burden in India. There are estimated 7500–12,000 babies born with β-thalassemia major (β-TM) every year in this country. Couples who are at-risk of having children with hemoglobin (Hb) disorders desired to have the option of avoiding the birth of an affected child by prenatal diagnosis (PND). Thus, the prenatal women are a highly important target group for carrier screening and preventing the birth of thalassemic children in the country. The present study was conducted among 20,883 pregnant women, irrespective of gravida and duration of pregnancy, from the prenatal clinic of Nilratan Sarkar (NRS) Medical College & Hospital, Kolkata, West Bengal, India, from February 2009 to November 2012. Thalassemia carrier status was assessed by high performance liquid chromatography (HPLC) along with red blood cell (RBC) indices. Husbands of all thalassemia carrier women were advised and persuaded to undergo screening for hemoglobinopathies. The couples were counseled to undergo PND if both of them were detected to be thalassemia carriers. The data were statistically analyzed to evaluate the efficacy of this procedure. [ABSTRACT FROM AUTHOR]

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1canalysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [β17(A14)Lys→Asn;HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1cmeasurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1canalysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing. [ABSTRACT FROM AUTHOR]

Objective: England has universal screening for sickle cell disease in newborns, with a policy of reporting beta thalassaemia major where it is detected. We report a case with a high gamma/beta ratio (20.167), but all other tandem mass spectrometry results within expected normal limits. The second test did not support the presumed diagnosis of beta thalassaemia major. As the two results were discrepant, further investigations were carried out. Methods and results: Sanger sequencing identified a novel mutation HBB:c.40 G > A (Hb Tower Hamlets) with concomitant beta plus (severe) thalassaemia due to the HBB:c.92 + 5 G > C mutation. Conclusions: This case highlights considerations for policy makers and implementers of new methods in screening programmes. For the English screening programme, the importance of adherence to the two-part testing strategy for newborn haemoglobinopathy screening is demonstrated. When discrepancies occur in results between the two tests, a thorough investigation is indicated. Communication, training, incident review and oversight are vital aspects of any screening programme. Protocols, risk assessments and guidance require constant monitoring and review to ensure that they remain fit for purpose. When new methods are introduced, stringent application of these principles is recommended. [ABSTRACT FROM AUTHOR]

The article highlights the increasing scope of sickle cell screening in Europe for the treatment of sickle cell disease in children around the world. Topics discussed include Introduction of Sickle haemoglobinopathies as common genetic disorder related to sickle cell diseases; It discusses the importance of newborn screening for the diagnoses of the disease; and treatment for the disease such as pneumococcal immunizations, caregiver education and preventive medical screening.