Cellulose Acetate Electrophoresis Research Articles

Reverse Phase-high-performance Liquid Chromatography (RP-HPLC) Analysis of Globin Chains from Human Erythroid Cells

β-hemoglobinopathies are severe genetic disorders characterized either by the abnormal synthesis of the adult β-globin chains of the hemoglobin (Hb) tetramer (βS-globin chains) in sickle cell disease (SCD) or by the reduced β-globin production in β-thalassemia. The identification and quantification of globin chains are crucial for the diagnosis of these diseases and for testing new therapeutic approaches aimed at correcting the β-hemoglobinopathy phenotype. Conventional techniques to detect the different Hb molecules include cellulose-acetate electrophoresis (CEA), capillary electrophoresis (CE), isoelectric focusing (IEF), and cation-exchange-HPLC (CE-HPLC). However, these methods cannot distinguish the different globin chains and precisely determine their relative expression. We have set up a high-resolution and reproducible reverse phase-HPLC (RP-HPLC) to detect and identify the globin chains composing the hemoglobin tetramers based on their different hydrophobic properties. RP-HPLC mobile phases are composed of acetonitrile (ACN) that creates a hydrophobic environment and trifluoroacetic acid (TFA), which breaks the heme group within the Hb tetramers releasing individual globin chains. Hb-containing lysates are loaded onto the AerisTM 3.6-µm WIDEPORE C4 200 Å LC Column and a gradient of increasing hydrophobicity of the mobile phase over time allows globin chain separation. The relative amount of globin chains is measured at a wavelength (λ) of 220 nm. This protocol is designed for evaluating globin chains in (i) red blood cells (RBCs) obtained from human peripheral blood, (ii) RBCs in vitro differentiated from hematopoietic stem/progenitor cells (HSPCs), and (iii) burst-forming unit-erythroid (BFU-E), i.e., erythroid progenitors obtained in vitro from human peripheral blood or in vitro cultured HSPCs. This technique allows to precisely identify the different globin chains and obtain a relative quantification. RP-HPLC can be used to confirm the diagnosis of β-hemoglobinopathies, to evaluate the disease severity and validate novel approaches for the treatment of these diseases.

SERUM PROTEIN EVALUATION OF PIT BULL, BULLMASTIFF AND CANE CORSO BREEDS OF DOMESTIC DOG (CANIS LUPUS FAMILIARIS)

The diversity of proteins, their metabolism and protein functions are attributed to a wide range of responses in cells, organs and tissues of animals. The study evaluated specific serum proteins from male and female gender of 3 dog breeds (Pit bull, Bullmastiff and Cane corso). Cellulose acetate electrophoresis technique was used to ascertain the concentrations (g/dL) of the individual serum proteins and compared to the respective normal reference values for domestic dog. Results obtained indicate that the Pit bull (male and female) and the Bullmastiff (female) had serum albumin concentrations that were higher than the normal reference range. An increase above normal in serum globulin (α1-globulin, α2-globulin and β2-globulin) concentrations was observed in the female Cane corso breed, as other dog gender and breed fell within reference range. A comparison based on serum albumin/globulin (A/G) ratio of the dog breeds revealed a normal A/G concentration except for the female Cane corso (0.36 g/dL) which was lower than normal, and the female Pit bull (1.19 g/dL) which was found to be higher. Such abnormal decrease and or increase in these respective serum protein concentrations could be attributed to prolong dehydration due to the dogs regular activity and low fluid (water) replacement and or incidences of mild to acute inflammatory response/disease due to consumption of a certain diet type over time.

Evaluation of haemoglobin variants of Red Sokoto goats and Yankasa sheep in Beji, Niger State, Nigeria: Effect on body weight and reproductive parameters

Blood was sampled from 76 Red Sokoto (RS) goats and 76 Yankasa Sheep (YS) to evaluate Haemoglobin (Hb) genotypes and their effect on body weight and some reproductive parameters of the animals. The blood samples were analyzed by cellulose acetate electrophoresis. The electrophoretic test revealed the presence of three co-dominant alleles (A, B and C) which produced four haemoglobin genotypes (AA, AB, BB andBC) in RS goats, and two codominantalleles (AandB) which gave rise to two haemoglobin genotypes (AA and AB) in YS. The frequencies of alleles A, B and C were 0.32, 0.61 and 0.07 in RS goats; and 0.945 and 0.055 for alleles A and B in YS. The estimated heterozygosities were 0.406 and 0.118 in RS goats and YS while the estimated inbreeding coefficients were 0.009 and 0.008 in the RS goats and YS, respectively. The observed and expected genotype frequencies deviated significantly from the expected Hardy-Weinberg equilibrium (P 0.05;chi-square=7.82for RS goats, P 0.01;chi-square = 13.13 for YS).There were significant (P<0.05) differences observed in the body weight of male YS as a result of the Hb type; individuals with Hb AB were observed to have higher body weight (44.00 kg) compared to those with Hb AA (22.83 kg).No significant (P>0.05) difference was observed however in the body weight of RS goats. Male YS with Hb AB had longer (P 0.05) scrotum (26.00 kg) and wider scrotal circumference (33.00 kg) compared to those with Hb AA (18.83kg;25.67kg,respectively). No significant (P>0.05) difference was however observed in there productive parameters of RS goats. It was concluded that polymorphism exist at the Hb locus in the YS and RS goats sampled in Beji and this had an influence on body weight and the reproductive parameters evaluated particularly in the sheep. More heterozygotes were also observed in the population studied there by leading to alow rate of in breeding coefficient in the animals.

Blood protein polymorphism and differentiation of selected indigenous cattle breeds in Nigeria

Genetic variation is the basis of effective improvement in farm animals. Population differentiation is used for objective choice of parental genotypes that constitutes new hybrids in crossbreeding. In Nigeria, population characteristics of selected indigenous cattle breeds have not been fully documented. Therefore, blood protein electrophoretic patterns of selected indigenous cattle breeds in Nigeria were assessed. Blood samples (5mL) were taken underneath the tail by venipuncture from 40 cattle randomly selected from each of the five breeds. The samples were subjected to cellulose acetate electrophoresis to determine the genetic variants of haemoglobin (Hb), carbonic anhydrase (CA) and transferrin (Tf) following standard procedure. Data were analysed using descriptive statistics, cluster analysis and Euclidean genetic distance. Allele frequencies ranged between 0.10 (Hb ) and 0.90 (Hb ), 0.11 (CA ) and 0.89 (CA ) and 0.02 (Tf ) and 0.49 (Tf ) across the breed.Two main clusters from the dendrogram were observed for each of Hb, CA and Tf. Euclidean genetic distance at the blood protein polymorphism level between WF and SG, WF and RB, WF and BK, WF and Muturu were 29, 30, 80 and 93, respectively. Genetic variants of transferring were largest within breed which indicated potential for selection

Diversity of West African dwarf goat in southwestern Nigeria based on allozyme Markers

Effective conservation, rational management and inadequate information on genetic diversity are the major challenges in livestock production. Genetic diversity has been used to reveal the extent of differentiation within livestock species. However, information on the use of allozymes in genetic diversity of the West African Dwarf (WAD) goat is insufficient. Therefore, genetic diversity of the WAD goat populations in southwestern Nigeria was investigated in this study. Three protein loci markers were used. Blood (5 mL each) samples were randomly collected from20, 20, 40 and 60 goats from Ondo ,Oyo, Ogun andOsun States respectively. The samples were subjected to cellulose acetate electrophoresis to determine the genetic variants at Haemoglobin (Hb), Carbonic Anhydrase (CA) and Transferrin (Tf) loci. Another set of blood (5 mL) from 20 different individual animals were randomly obtained from each of Ondo, Oyo, Ogun, and Osun States. Allele frequency, observed heterozygosity (H ), Polymorphic Information Content (PIC), F-statistic (F F and F ), gene flow (Nm), gene diversity (D), number of alleles per loci (A ), effective number of allele (AE) Mean Number of Allele (MNA) were generated from the data obtained. Data were analysed using Hardy-Weinberg equilibrium (HWE) at α0.05. The allele frequency ranged between 0.11 (Hb ) and 0.58 (Hb ), 0.17 (CA ) and 0.44 (CA ) and 0.08 (Tf ) to 0.60 (Tf ). Deviation from HWE was not significant in all populations except at Tf locus (0.00). The H ranged from 0.43 to 0.62 andNm and D ranged between 3.68 and 32.40 and 0.34 to 0.50 respectively. TheMNAwas 0.67 butA ranged from 1.52 to 2.00. The allozymes revealed some level of genetic diversity and a genetic differentiation indicative of the amount of genetic differences among individualswithin theWestAfricanDwarf goat population.

Hemoglobin C/Korle-Bu Identified In A Patient With A Previous Diagnosis Of Hemoglobin S/C Disease

Abstract Casestudy Hemoglobin Korle-Bu (Hb-KB) is an uncommon Hb variant that can be mistaken for Hb-S on electrophoretic screening. While Hb-KB alone has no clinical manifestations, there are only limited case studies describing KB in combination with other Hb variants. Here we report a rare case of Hb-C/KB misdiagnosed and managed as Hb-S/C disease for over 20 years. Results A 21-year-old African American woman with presumed Hb-S/C disease presented with generalized abdominal pain, nausea, and vomiting. In 1999, Hb electrophoresis showed 59% of abnormal hemoglobin presumed to be HbS and 41% HbC+A2; agar/acetate gel analysis was not employed and the hemoglobinopathy remained incompletely characterized. She had a history of back, chest, abdomen, and extremity pains requiring multiple hospital admissions, with treatments including dilaudid, oxycontin, oxycodone, and hydroxyurea. Her vital signs were normal and her examination was only significant for abdominal tenderness. Imaging studies did not show any evidence vascular occlusion, avascular necrosis, or end-organ dysfunction. RBC indices were remarkable for mild borderline anemia, microcytosis, decreased MCH/elevated MCHC, and borderline elevated RDW. Peripheral smear showed microcytic red cells with anisocytosis, scattered target cells, and a notable absence of sickled cells and Hb-C-crystals. The diagnosis of Hb-S/C disease was then revisited. HPLC showed abnormal hemoglobins in the Hb-D window at 55.1% and in the Hb-C window at 40.6%, with 3.5% Hb-A2 and no normal Hb-A. Gel electrophoresis with cellulose acetate followed by citrate agar suggested Hb-C in combination with either D, G, or Korle-Bu. Definitive diagnosis was obtained by beta globin gene sequencing that demonstrated one copy Hb-C (19G>A, Glu7Lys) and one copy Hb- Korle-Bu (220G>A, Asp74Asn). Given the absence of Hb-S/C disease, her gastrointestinal distress and pain episodes were re-evaluated. Conclusion Hb-S and Hb-Korle-Bu migrate similarly in cellulose acetate electrophoresis but can be distinguished on citrate agar. Challenging beta chain variants can now be readily differentiated by complete gene sequencing. This case study emphasizes the importance of distinguishing Hb-KB from clinically-significant Hb-S.

Production of sialic acid rich glycopeptide from bovine κ-casein glycomacropeptide by hydrolyzing with papain

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide having many biological activities. The study described in this research communication was undertaken to determine whether sialic acid rich glycopeptide can be produced from GMP by proteinase treatment. A sample of GMP was hydrolyzed with papain, and the obtained hydrolysate was chromatographed on a column of diethylaminoethyl-Sephacel to obtain a glycopeptide fraction (GPF). This product accounted for average 48.1% dry weight of GMP or 81.1% total recovered sialic acid from GMP. The content of sialic acid (expressed as % dry weight) was 1.7 times higher in GPF (22.6) than in unhydrolyzed GMP (13.4). Major differences in amino acid composition between GPF and GMP were found in the contents (mol%) of: lysine (<1 and 4.5, respectively), serine (20.3 and 10.3, approximately twice higher in GPF), asparagine/aspartic acid and isoleucine. The contents of the last two amino acids were approximately twice lower in GPF. On gel filtration chromatography with Sephacryl S-100, GMP was eluted as a single peak with elution volume similar to that of dimeric β-lactoglobulin (36.6 kDa) whereas GPF was eluted in two peaks both with elution volumes greater than that of α-lactalbumin (14.2 kDa). These peak fractions containing high (fraction I) and low (fraction II) molecular size glycopeptides gave different sialic acid to peptide ratio, which was 1.7 times higher in fraction I than in fraction II. Results of size exclusion HPLC on Superdex-75 were consistent with those of gel filtlation chromatography. On cellulose acetate electrophoresis, the mobility of GPF relative to that of GMP as 1.0 was found to average 1.2, suggesting a higher negative charge density in GPF than in GMP. It was concluded that papain digestion of GMP is an efficient method to produce glycopeptide with high sialic acid content.

β-Globin Gene Mutations in Pediatric Patients with β-Thalassemia in the Region of Çukurova, Turkey

β-Thalassemia (β-thal) is one of the most common genetic disorders in Turkey. In this study, we investigated the mutations and frequency of β-thal at the molecular level in pediatric β-thal patients in the Çukurova region. The β-thal mutations of 52 cases were analyzed. An automated blood cell counter was used for hematological data. Cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) methods were used for hemoglobin (Hb) typing. Amplification refractory mutation system (ARMS), restriction fragment length polymorphism (RFLP), gap-polymerase chain reaction (gap-PCR) and DNA sequencing analysis methods were used to determine genomic features. In this study, we found that 36 subjects carried homozygous mutations [IVS-I-110 (G>A) (HBB: c.93-21G>A) (58.3%), codon 8 (–AA) (HBB: c.25_26delAA) (5.6%), −30 (T>A) (HBB: c.-80T>A) (5.6%), IVS-I-6 (T>C) (HBB: c.92+6T>C) (5.6%) and IVS-II-1 (G>A) (HBB: c.315+1G>A) (5.6%)]. We found that 13 subjects carried compound heterozygosities for IVS-I-110/IVS-I-6 (15.4%) and IVS-I-110/frameshift codon (FSC) 44 (–C) (HBB: c.135delC) (15.4%). We observed that the Syrian subject also carried a compound heterozygosity for IVS-I-6/IVS-I-25 (–25 bp) (HBB: c.93_21del). We determined that the most frequently observed β-thal mutation in the Çukurova region, where various types of hemoglobinopathies have been observed, is the IVS-I-110 mutation. As the prevalence of the disease will affect the region where the immigrant population is dense, population screening and prenatal diagnosis (PND) should be increased and the public should be made aware of the consequences.

Studies of Genetic Distance, Gene and Genotypic Frequencies of Hemoglobin Types of West African Dwarf and Yankassa Sheep

Hemoglobin (Hb) alleles, genotype and their frequencies were studied in 50 West African Dwarf (WAD) and 50 Yankasa sheep. Blood samples were collected from the animals and analysed, hemoglobin (Hb) type were determined by cellulose acetate electrophoresis. The resultant data were subjected to statistical analyses. The gene frequencies estimated in each allele of the population and were tested using Hardy-Weinberg equilibrium. The genetic distance was thereafter estimated according to Bodmer and Cavalli-Sforza method. The frequencies of Hb(A) and Hb(B) in WAD sheep were 0.35 and 0.65 and genotype frequencies were HbAA (0.34), HbAB (0.00) and HbBB (0.66). The corresponding values in Yankassa sheep were 0.75 and 0.25, 0.70, 0.10 and 0.20, respectively. The distribution of the haemoglobin genotypes was tested using Chi-square analysis. The genetic distance at the haemoglobin locus between WAD and Yankassa sheep was estimated and was found to be 0.16, which shows that within the limit of these small populations sampled, there is a similarity of alleles at the locus. Sampling larger number animals in different breeds of sheep will allow for a better genetic studies and marker-assisted selections in sheep improvement programs. Keywords: Genetic Distance, hemoglobin, Sheep, WAD, Yankassa.

Hematological Profiles in Hemoglobinopathy Patients in South Western Nepal

INTRODUCTION: Homozygous inheritance of the hemoglobin results in sickle cell anemia (SCA), heterozygous inheritance results in sickle cell trait (SCT). Sickle cell anemia and β-thalasemia (β-TT) have been a major health threat for the tharu living in the South-Western Terai of Nepal. This study is carried out to apply and optimise the phenotypic method and haematological profile to characterise the SCT, SCA and β-TT from suspected tharu community dwellers.&#x0D; MATERIAL AND METHODS: We enrolled 100 suspected cases of hemoglobinopathies of 12-14 years children and ≥15 years adults tharu community dwellers from Dang, Kapilvastu, Nepalgunj, Rupandehi and Nawalparasi of South-Western Nepal from May 2018 to November 2018. Five mililitre of blood was collected in EDTA vial and transported to the laboratory maintaining cold chain. The hematological profile was recorded after investigations. The hemolysate from blood samples were subjected to phenotypic testing by adopting cellulose acetate electrophoresis at pH 8.6.&#x0D; RESULTS: Our result showed the commonest hemoglobinopathy was SCT (38%) followed by β-TT (21%) and SCA (5%). Males were more affected with SCA (60%) while that of females were most affected with β-TT (57.1%). Only 44.7% females were affected with SCT while 42.9% males were affected with β-TT. The significant difference in mean was observed in Hb level (p=0.0001), RBC (p=0.004), MCHC (p=0.015) and RDW (p=0.028) whereas the non-significant difference in mean was observed for glucose 6 phosphate dehydrogenase (G6PD) level (p=0.063) in hemoglobinopathy patients.&#x0D; CONCLUSION: Most cases develop severe type of anemia as shown by change in hematological parameters. This information could advocate for timely counselling before constellation of associated condition appeared in hemoglobinopathy patients.

Blood Donors' Age, Haemoglobin Type, G6PD Status, and Blood Group Impact Storability of CPDA-1 Banked Whole Blood: A Repeated-Measure Cohort Study in Cape Coast, Ghana.

Background The high prevalence of haemoglobin variants and glucose 6-phosphate dehydrogenase disorder (G6PDd) in sub-Saharan Africa means that substantial proportions of donor blood units carry these red cell abnormalities. Aim This study investigated the impact that inherited haemoglobin variants and/or G6PD status have on whole blood banked at 4–6°C for 35 days. Method This repeated-measure cohort study was undertaken on 103 donor blood units collected into blood bag containing CPDA-1 anticoagulant. On days 0, 7, 14, 21, and 35, full blood count, osmotic-induced haemolysis, and plasma K+ levels were estimated. Also, on day 0, G6PD status, haemoglobin variants, % foetal haemoglobin, and blood group of donor units were determined using methaemoglobin reductase, cellulose acetate electrophoresis, modified Bekte alkali denaturation assay, and slide haemagglutination test, respectively. Result Overall, although plasma K+ levels increased during storage, donor units from individuals ≥20 years, G6PD normal, Hb AC, or blood group B had comparatively higher percentage change in plasma K+ during storage. Osmotically induced haemolysis of donor units was significantly decreased in Hb AC (compared with Hb A or AS) donor units on days 7, 14, 21, and 35 (p < 0.0001 in each case). G6PDd donor units had comparatively reduced osmotic-induced lysis compared with G6PD normal units, reaching a statistical significance on day 35 (p = 0.043). Also, Hb AC units had comparatively nonstatistically higher plasma K+ at all time points (compared with Hb A or AS). Furthermore, whereas donor units from individuals ≥20 years showed significantly higher median free haemoglobin on day 21 (compared to donor <20 years), when donor units were stratified per Hb variants, only Hb AS units had median free haemoglobin below the 0.8% threshold after 35 days' storage. Conclusion Age of donor, blood group, Hb AC variant, and G6PD status may be important considerations in the storability of whole blood.

Use of an automated pyrosequencing technique for confirmation of sickle cell disease.

BackgroundThe diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sβ0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.ObjectivesTo develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB geneMethodsWe developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.ResultsWe identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified as Sβ0 thalassemia and 81 as Sβ+ thalassemia. The most frequent beta thalassemia mutations of Sβ0 and Sβ+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.DiscussionThe PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common β+ and β0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD cohort in Brazil.

Prevalence of Sickle Cell Disease and Other Haemoglobin Variants in Calabar, Cross River State, Nigeria

Background: Sickle cell disease (SCD) is the commonest genetic disorder worldwide with a global prevalence of 20-25 million. About 12-15 million affected persons are in Sub-Sahara Africa with Nigeria bearing the highest burden of people living with sickle cell disease. SCD is a disease characterized as an autosomal, recessive, heterogeneous, and a monogenetic disorder caused by an A-to-T point mutation in the β-globin gene responsible for the production of abnormal hemoglobin S (HbS), which polymerizes in the deoxygenated state and results in the sickling of erythrocytes. Haemoglobin variants are mutant forms of haemoglobin in a population usually occurring as a result of genetic changes in specific genes, or globins that causes change on alterations in the amino acid. They could affect the structure, behavior, the production rate and the stability of the specific gene. Well-known haemoglobin variants such as sick-cell anaemia are responsible for diseases and are considered haemoglobinopathies. Other variants cause no detectable pathology and are thus considered as non-pathological variants.&#x0D; Aim: The study is aimed at evaluating the burden of sickle cell disease and other haemoglobin variants in Calabar, South-South Nigeria.&#x0D; Methods: This is a retrospective study done at the haematology laboratory of University of Calabar Teaching Hospital, Calabar. Cellulose acetate electrophoresis at alkaline pH was used for the evaluation of haemoglobinopathies. The data were entered into Microsoft Excel 2016 spreadsheet and analysed with the IBM SPSS Version 22. Data were summarized into percentage of different phenotypes.&#x0D; Results: Results of the total 3648 haemoglobin electrophoresis recorded, 1368 (37.50%) were male while the remaining 2280 (62.5%) females given a male to female ratio of 1:1.7. Five haemoglobin phenotypes were identified as HbAA, HbAS, HbAC, HbSC and HbSS. The overall average values of their prevalence were HbAA 64.78%, HbAS 32.62%, HbSS 2.14%, HbAC 0.33%, HbSC 0.14%. Thus, the prevalence of SCD (Prevalence of HbSS+HbSC) was 2.28%. The highest proportion of SCD was observed in 2011 with least in 2016 and 2017 respectively.&#x0D; Conclusion: The prevalence of SCD and other haemoglobin variants in Calabar is similar to that of the national prevalence rate. There is need for continuous enlightenment and premarital counselling on the pattern of inheritance of SCD most especially with the increased burden of sickle traits in the environment has reported in this study.

International Multi-Site Clinical Validation of Point-of-Care Microchip Electrophoresis Test for Hemoglobin Variant Identification

Introduction: Nearly 24% of the world's population carry hemoglobin (Hb) gene variants, with the large majority of affected births occurring in low-income countries. The most prevalent structural Hb variants are the recessive β-globin gene mutations, βS or S, βC or C, and βE or E1. Hb S mutation is prevalent in sub-Saharan Africa and in Central India. Hb C is common in West Africa, and Hb E is common in Southeast Asia and in India. Homozygotes or compound heterozygotes with βS (e.g., Hb SS or SC) have sickle cell disease (SCD), a chronic sickling disorder associated with pain, chronic multi-organ damage, and high mortality. While Hb EE causes only a mild microcytic anemia, Hb E in combination with β-thalassemia can lead to transfusion dependent thalassemia. Though carriers are typically asymptomatic, they may pass the mutations to their offspring. Screening is needed so that these disorders can be diagnosed early and managed in a timely manner2. For example, in low-income countries, due to lack of nationwide screening and comprehensive care programs, up to 80% of babies born with SCD are undiagnosed and less than half of them survive beyond 5 years of age2. The unmet need for affordable, portable, accurate point-of-care tests to facilitate decentralized hemoglobin testing in resource-constrained countries is well-recognized 2,3. Here, we present international multi-site clinical validation results and high diagnostic accuracy of the 'HemeChip' (Fig. 1), an affordable, 10-minute point-of-care microchip electrophoresis test for identifying common Hb variants S, C, and E. Methods: Institutional Review Board approvals were obtained at each study site, and blood samples were collected as part of the standard clinical care. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. 315 children (6 weeks to 5 years of age) were tested in Kano, Nigeria. Study participants were enrolled at three hospitals, Amino Kano Teaching Hospital, Murtala Mohammed Specialist Hospital, and Hasiya Bayero Pediatric Hospital. 124 subjects (7 weeks to 63 years old) were included in the study at Siriraj Thalassemia Center in Bangkok, Thailand. 298 subjects (8 months to 65 years old) were tested at a referral testing facility of ICMR-National Institute of Research in Tribal Health, located at Late Baliram Kashayap Memorial Medical College, Jagdalpur, Chhattisgarh, India. Blood samples were tested with both HemeChip and the standard reference methods, high performance liquid chromatography or cellulose acetate electrophoresis. Reference test results were not available to the HemeChip users. Similarly, HemeChip test results were not available to the users of the standard reference tests. Clinical validation studies presented here were performed with a fully functional, portable HemeChip prototype developed at Case Western Reserve University (Fig. 1A). A commercial product has been developed based on this technology by Hemex Health Inc. under the product name, GazelleTM(Fig. 1B). Results and Discussion: Among the total 768 tests performed with HemeChip in all test sites, 732 were valid tests, as defined by the Standards for Reporting Diagnostic Accuracy (STARD)4. HemeChip correctly identified all subjects with Hb SS, Hb SC, Hb AS, Hb AE, and Hb EE with 100% accuracy (Table 1). Nine subjects with normal Hb (Hb AA) were identified as HbSS in Nigeria. No subjects with disease were identified as normal or trait by HemeChip. Three subjects with compound heterozygous Hb Sβ-thalassemia (2 subjects with Hb Sβ+-thalassemia, 1 subject with Hb Sβ0-thalassemia) were identified as Hb SS. Sensitivity was 100% for all Hb types tested. Specificity was 98.7% for Hb SS versus other Hb types, and 100% for all other Hb types tested. HemeChip displayed an overall diagnostic accuracy of 98.4% in comparison to standard reference methods for the Hb variants tested in all clinical testing sites (Table 1). HemeChip is a versatile point-of-care system that enables affordable, accurate, decentralized hemoglobin testing in resource-limited settings.

Hemoglobin Phenotypes in Nigeria: Data From a National Reference Laboratory

Abstract On a global scale, 5% to 7% of the population carries an abnormal hemoglobin. With a sickle hemoglobin (HbS) carrier prevalence of 25% to 40%, Nigeria bears the greatest burden of sickle cell disorder worldwide. Until recent times, detection of other clinically significant hemoglobin variants associated with HbS has been unavailable, resulting in missed/wrong diagnosis, weak national data, and suboptimal control of hemoglobin disorders. This study aimed to review hemoglobin assays over a 2-year period at a national reference laboratory in Nigeria, in order to describe the prevalent hemoglobin phenotypes for health planning. The study was a retrospective, laboratory data-based survey. Sociodemographics and hemoglobin assay results of 6,851 specimens between January 2016 and December 2017 were analyzed. Study protocol was approved by the Institutional Review Board. Participants aged less than 1 year and who had recent transfusions and a history of hydroxyurea therapy were excluded. At the hemoglobin laboratory, specimens were analyzed using cation exchange high-performance liquid chromatography (CE-HPLC) (Bio Rad D10, California). When indicated, further analysis of suspected abnormal hemoglobins was confirmed using a second method, capillary electrophoresis (SEBIA Capiflex II, France). The distribution of the identified hemoglobin phenotypes was determined as a proportion of the total number of participants. The mean (SD) age of the participants was 31.8 (14.9) years. The most frequent hemoglobin phenotypes were AA (59.4%), AS (23.3%), and SS (13.3%). Hemoglobin phenotypes AC (2.1%), SC (0.9%), AGPhiladelphia (0.2%), and CC (0.2%) were less frequent. Least occurring phenotypes were ADIbadan, AE, AGPhiladelphia, AOArab, DD, and hereditary persistence of fetal hemoglobin (HPFH). In the Nigerian context, accurate diagnosis of hemoglobin variants using quantitative, high-resolution hemoglobin assays, compared to zone (cellulose acetate) electrophoresis at alkaline pH will provide data for health planning, better access to genetic counseling, informed reproductive health choices, and secondary prevention of hemoglobinopathies.

Association between Non-secretion of ABH Antigens and Sickle Cell Anaemia

Aim: To determine whether non-secretion of ABH blood group antigens was associated with Sickle Cell Anaemia.&#x0D; Materials and Methods: Haemaglutination inhibition test was carried out on saliva samples from 300 individuals; 100 of whom had haemoglobin (Hb) genotype AA, 100 HbAS, 50 HbAC and 50 HbSS. ABO blood grouping was carried out by standard methods and Haemoglobin genotype test was performed by cellulose acetate electrophoresis technique.&#x0D; Results: Eighteen percent (18%) of HbAA, 23% of HbAS, 18% of HbAC and 42% of HbSS individuals were non-secretors of ABH antigens (p = 0.007). Non-secretion of ABH substances was more associated with HbSS persons than HbAA (p = 0.002), HbAS (p = 0.016) and HbAC (p = 0.009) individuals.&#x0D; Conclusion: Non-secretion of ABH blood group substances is associated with Sickle Cell Anaemia.

Clinical Biomarkers for Diagnosis of Damages in Individuals with Long-Term Exposure to X-Rays.

To determine altered manifestation of plasma proteins in X-rays technicians who are regularly exposed to low doses of radiations over a long period during their job. Descriptive study. District Headquarters Hospital and Mufti Mahmood Memorial Teaching Hospital; from January 2017 to January 2018. The study enrolled 70 individuals consisting of 50 X-ray technicians working 8 to 12 hours/day for five days per week and 20 unexposed healthy controls. The serum protein expression pattern (concentrations of various serum proteins) was evaluated through cellulose acetate electrophoresis and serum antioxidant status was measured through ferric reducing ability of plasma (FRAP) assay. The antioxidant assay showed significantly low trolox-equivalent antioxidant capacity (TEAC) status and FRAP value in X-ray technicians as compared to controls (p<0.001). Analysis of serum protein demonstrated a significantly reduced concentrations of albumin (p<0.001) and elevated level of the Ɣ-globulins (p<0.001), while other globulins fractions like α1 and β remain unchanged. There was a strong negative correlation (p<0.001) according to Pearson coefficient (r=87%⁽⁻⁾) between albumin and Ɣ-globulins fraction. Whereas, a positive correlation (p<0.001) (r=46%⁽⁺⁾) between alpha 1 globulin and albumin fraction was observed. A correlation between other globulin fractions and albumin was found statistically significant (p<0.001). Elevated serum gamma globulins may be a potential protein biomarker for triage and detection of X-radiation induced damages.

HETEROGENEITY OF PROTEINS IN BIRDS’ EGG-WHITES

Little is known about the comparative heterogeneity of protein compounds among a wide variety of birds’ egg white, particularly, an analysis of their detailed, in-parallel protein composition. Hence this research is conducted mainly to evaluate the extent of variability among 42 types of birds’ egg white. To improve the perception of these biological fluids, the main phenotypic variations of egg whites were evaluated using the discontinuous denaturing polyacrylamide gel electrophoresis (SDS-PAGE), Gradient SDS-PAGE, Native-PAGE, cellulose acetate electrophoresis, and the reverse-phase high-performance liquid chromatography (RP-HPLC). The results showed that the Native-PAGE and SDS-PAGE produced better screening results than other methods in identifying protein. Although  Native-PAGE and SDS-PAGE did not show remarkable variability in terms of hydrophobicity, several electrophoretic differences of egg-white proteins were observed. Several unknown proteins in the egg white samples of different bird species were also identified through the electrophoretic experiments. Hence, it might be possible, as in the case of egg white samples, to provide a characterized assessment among birds by using only the available gel electrophoresis techniques. This study also provided a rapid snapshot of the initial identification of several unknown components of egg white proteins. Accordingly, this study constituted the first large-scale comparative proteomics investigation performed among the largely varying types of egg whites from commercial stores and bird keepers in the middle Euphrates areas in Iraq.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center

The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15years. Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. The study includes 31075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

Hematological and Molecular Findings in the First Case of Hb J-Norfolk [HBA2: c.173G>A (or HBA1] in an Indian Patient

We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.