Hb Disorders Research Articles

Hemoglobin SD-Punjab with Stroke and Moyamoya Syndrome and Genotype-Phenotype Correlation: A Report of Two Siblings

Background: Hemoglobin (Hb) SD-Punjab is a rare Hb disorder which may present at any age ranging from infancy to adulthood with variable clinical severity. There are very few reports of HbSD-Punjab with stroke, leading to moyamoya syndrome. We report two siblings with HbSD-Punjab, their diagnosis, genotype–phenotype correlation, and management. Clinical Description: Two siblings, ages 6 and 4 years, presented together to the emergency for the first time with different manifestations. The elder child had fever and acute onset right-sided weakness with anemia and splenomegaly, whereas the younger sister presented with fever and decreased appetite with anemia and splenomegaly. Management and Outcome: Both siblings showed severe anemia with the second sibling showing low platelet counts also. Magnetic resonance imaging of the brain of the elder sister showed chronic infarcts in the left frontal region in addition to acute infarction in the left cerebral hemisphere with angiography suggestive of moyamoya disease. Both siblings had blood pictures suggestive of hemolytic anemia and Hb electrophoresis showed co-inheritance of HbSD-Punjab. Both siblings responded to supportive treatment and blood transfusions and were started on oral hydroxyurea. At the genotype level, a combination of factors was noted with normal α-gene copies known to increase severity and heterozygosity for Xmn1 polymorphism explaining the higher HbF levels which decrease severity. Conclusion: Knowledge of the varied clinical presentation of hemolytic disorders and early detection is key to prevent more severe sequelae in such disorders.

Point-of-Care Diagnostic Test for Beta-Thalassemia.

Hemoglobin (Hb) disorders are among the most common monogenic diseases affecting nearly 7% of the world population. Among various Hb disorders, approximately 1.5% of the world population carries β-thalassemia (β-Thal), affecting 40,000 newborns every year. Early screening and a timely diagnosis are essential for β-thalassemia patients for the prevention and management of later clinical complications. However, in Africa, Southern Europe, the Middle East, and Southeast Asia, where β-thalassemia is most prevalent, the diagnosis and screening for β-thalassemia are still challenging due to the cost and logistical burden of laboratory diagnostic tests. Here, we present Gazelle, which is a paper-based microchip electrophoresis platform that enables the first point-of-care diagnostic test for β-thalassemia. We evaluated the accuracy of Gazelle for the β-Thal screening across 372 subjects in the age range of 4-63 years at Apple Diagnostics lab in Mumbai, India. Additionally, 30 blood samples were prepared to mimic β-Thal intermediate and β-Thal major samples. Gazelle-detected levels of Hb A, Hb F, and Hb A2 demonstrated high levels of correlation with the results reported through laboratory gold standard high-performance liquid chromatography (HPLC), yielding a Pearson correlation coefficient = 0.99. This ability to obtain rapid and accurate results suggests that Gazelle may be suitable for the large-scale screening and diagnosis of β-Thal.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Evaluation of Couples at Risk for Hemoglobin Disorders in Italy: The Results of an 18-Month Retrospective Observational Single-Center Study

Background: Hemoglobinopathies are genetic disorders caused by mutations in the genes encoding globin chains. These conditions are the most common monogenic disorders worldwide, resulting in significant morbidity and mortality among those affected by the most severe forms. Antenatal evaluation for thalassemia and sickle cell disease (SCD) enables couples to know their reproductive risk, thus allowing an informed reproductive choice. However, in certain areas, couples do not often receive the appropriate counseling at the appropriate time. Aim: The aim of this study is to describe the characteristics of couples referred to our tertiary referral center in Milan, Italy, for evaluation of hemoglobinopathy risk and their management. Methods: in our Institution, a diagnostic-therapeutic pathway involving the Hemoglobinopathies unit, the Medical Genetic unit, and the central laboratory has been implemented. Subjects referred to the Institution, and their partner, underwent the following laboratory tests: complete blood count, hemolysis and iron parameters, vitamin pattern, and hemoglobin (Hb) pattern by high-performance liquid chromatography (HPLC). The couples were then evaluated by a hemoglobinopathy expert, and those at risk for Hb disorders were offered a molecular analysis. Couples were then referred to the geneticist to receive the results of the molecular analysis, discuss the genetic risk for the fetus and, in case of high risk, discuss possible reproductive choices (i.e., prenatal diagnosis, pre-implantation diagnosis). We retrospectively collected and analyzed couples' data referred to our center from the 1 st of July 2021 to the 31 st of December 2022. Results: Three hundred and fifty-one couples were evaluated for hemoglobinopathies over 18 months. The median age was 34 years, with a tendency for younger age in female partners; 67% (234/351) of the women were already pregnant at the time of evaluation (median gestational age 18 weeks, range 7-37), while 33% (117/351) were referred for preconception counseling. Individuals from more than 48 different countries were screened, and the most represented countries of origin included: Italy (50%), Nigeria (6%), Bangladesh (4%), and Ivory Coast (4%), underpinning a vastly diverse genomic background. The referral was triggered by either evidence of microcytic anemia (58%) or HPLC anomalies (42%), including Hb variants, HbA2 anomalies, or elevated HbF in at least one partner. 55% of the referrals did not require additional testing. The remaining 45% (158/351) underwent advanced molecular tests to determine the nature of the Hb defect, guided by the results of the first-level panel. Among these, 33 couples (12% of the total) were found to be at risk for transmission of severe clinical forms of β-thalassemia, sickle cell anemia, or compound β-disorder heterozygosity (HbS/β-thal, HbC/HbS). One additional couple (0.4%) was at risk for HbH disease. In addition, 4 couples (1.5%) were found to be at risk for thalassemia intermedia combining a β-thalassemia mutation and α-gene triplication. Of the above, all 34 couples underwent invasive fetal evaluation: 24 women underwent chorionic villous sampling, while 10 received amniocentesis. A diagnosis of thalassemia in the fetus was reported in 6/34 (18%) cases and 3 (9%) of SCD. Interestingly, during the 18 months of observation, among the evaluated adult subjects, we found 4 cases of non-transfusion dependent thalassemia (NTDT) and 1 of HbS/β-thal in otherwise healthy subjects. All 4 cases of NTDT resulted from α-gene triplication paired with a β-thalassemia heterozygous defect. Furthermore, α triplication was found alone in 4 subjects without additional Hb gene defects. Overall, α triplication was detected in 9 out of 154 (5.8%) individuals who underwent HBA gene analysis. Conclusion: The median gestational age at presentation was high, leading to a limited time for couples to receive the appropriate counseling to make an informed reproductive choice. Increasing awareness across all involved specialists remains the mainstay for an effective prevention program to promptly identify at-risk couples and offer counseling at the right time. In addition, the relatively high incidence of α triplication in our population of asymptomatic and hematologically normal subjects and the new diagnosis of 4 NTDT warrants further studies to confirm the prevalence of α triplication in the general population.

Rapid Assessment of Hemoglobin-Oxygen Dissociation

Introduction: Oxygen (O 2) delivery to tissues depends on the hemoglobin (Hb)-O 2 affinity and dissociation of O 2 from Hb in red blood cells (RBCs). Mutations that impact α and β-globin subunits of Hb often alter the Hb-O2 affinity of Hb and Hb-O 2 dissociation. A point mutation in the β-globin gene yields sickle hemoglobin (HbSS) which results in sickle cell disease (SCD). Recently, there has been an upsurge in improving SCD pathophysiology through modifying HbSS O 2 affinity. The ability to alter cellular phosphate such as 2,3-DPG that influences Hb-O 2 affinity is also under investigation. In this context, measuring Hb-O 2 affinity has gained additional significance as a clinical and laboratory parameter. Hb-O 2 affinity is determined by measuring Hb-O 2 saturation (SO 2, %) vs. partial pressure of oxygen (pO 2, mmHg). The Hb-O 2 affinity is commonly represented by the p50 value, which is the pO 2 value at 50% SO 2. p50 measurement is underutilized in clinical practice mainly due to cost, complexity, and limited throughput of existing instruments. Herein, we present a rapid assay that can detect Hb-O 2 affinity disorders, determine cellular oxygen availability, and examine the effectiveness of existing and novel therapies for SCD patients. Methods: In this assay, we optically measure the Hb-O 2 dissociation by induction of physiologic acidic pH (6.9). We exploited the acidic pH environment to expedite chemically induced partial deoxygenation of Hb (deoxy-Hb) during which the conformational changes in the tertiary structure of Hb are manifested as significant peak shifts in the optical absorption spectra. Hb absorption spectra in the 350-700 nm wavelength range primarily reflect the conformation of heme-heme interactions, which are sensitive indicators of changes in the Hb-O 2 affinity. We validated the assay by comparing it with the reference standard Hemox analyzer using blood from healthy individuals (HbAA) and from patients with SCD (HbSS) i.e., low Hb-O 2 affinity. Venous blood samples were collected in EDTA-coated tubes from subjects with HbSS (n=44), HbAS (n=14), and HbAA (n=17) following an IRB-approved study protocol. 75 µl of whole blood diluted 100-fold with pH 6.9 phosphate buffer was incubated for 2 minutes at room air to attain full oxygenation. The mixture of blood and the buffer was then deoxygenated with 0.053M of sodium metabisulphite (Na 2S 2O 5) for 3 minutes to achieve partial deoxy-Hb. We then obtained 350-450 nm spectra of deoxy-Hb using a spectroscopy microplate reader with a 2 nm resolution. We recorded each sample's Soret band shift in 4 minutes using the 414 nm Hb fully oxygenated absorption peak as the reference. Data were reported as SEM. Results: Hb exhibits three characteristic light absorption peaks. The Soret peak is located at 414 nm under full oxygenation at pO 2 of 162 mmHg, and upon full deoxy-Hb at pO 2 of 5 mmHg, the peak shifts to 430 nm ( Figure 1A). We initially tested different concentrations of Na 2S 2O 5 and the time of deoxygenation to achieve partial deoxy-Hb. At 75 mmHg we analyzed the Soret peak which was used as an optical marker of oxygen affinity, an equivalent to the p50 value. The peak shift significantly correlated to the p50 value measured by the Hemox device (PCC=0.81, p=0.004, n=28) ( Figure 1A). We also observed significant differences between HbAA (n=17), HbAS(n=14), SCD samples on various treatments (transfusion (Tx) (n=8), and Hydroxyurea (n=15) and SCD treatment-naïve patients' samples (n=20). HbAA vs. HbAS; p=0.01, HbAS vs. HbSS+Tx; p=0.02, HbSS+Tx vs. HbSS+Hu; p=0.04, HbAA vs. HbSS treatment naïve; p=0.000, HbSS treatment Naive vs. HbAS; p=0.001 ( Figure 1B). Additionally, the peak shift for HbSS correlated with the percentage of HbSS (PCC = 0.87, p = 0.000, n=20, data not shown). Discussion: We demonstrate fast, effective differentiation of abnormal Hb-O 2 affinity and characterize the changes in the HbSS-O 2 affinity in response to treatments by using chemically induced partial deoxygenation of Hb at a physiologic acidic pH.The ubiquity of optical absorption spectrophotometers positions this assay as an accessible, equitable, and accurate Hb-O 2 dissociation measurement resource for research and clinical use. The assay will facilitate the diagnosis and prognosis of red blood cells and Hb disorders requiring minimal technical expertise and laboratory infrastructure and assist with testing the effect of new targeted and genome-editing-based therapies for SCD.

Hemoglobin E Prevalence among People Residing in Malaria Areas

Background: Malaria can infect erythrocytes and hence cause different pathogenesis episodes leading to death mostly in pregnant women and children under the age of 5 years. The selective pressure of these parasites leads to the production of new human genetic diseases. The most prevalent genetic alterations in the human genome are thalassemia and hemoglobinopathies (Hb E, Hb S), which are recognized throughout the world, including Saudi Arabia. Methods: From May 2018 to August 2019, 13972 Saudi citizens from King Fahd Central Hospital and premarital facilities in the Saudi Arabian province of Jazan participated in this study. This study aims to compare the prevalence of Hb E and other hemoglobinopathies in positive versus negative cases of malaria. So, CBC, malaria test, Hb-electrophoresis, and molecular study were investigated. Result: For thalassemias and Hb disorders, 36% with abnormal Hb (47% of them) carried Hb S in their blood, 37% with á-thalassemia, 11% for â-thalassemia and 4% of Hb E. Significant variations in CBC parameters were observed in Hb E patients. There was significant decrease in MCV, MCH and MCHC and slightly increase in WBCs, RBCs, RDW and PLT as compared to controls.

Types of Inherited Hemoglobin Disorders Among the Patients Attending Hematology Outdoor of a Tertiary Care Hospital in Bangladesh

Background: Inherited Haemoglobin (Hb) disorders are the most frequent genetic hereditary disorders with an increasing global health burden, especially in low- and middle-income countries. This disorder is mostly caused by a point mutation on a globin gene resulting mostly in single amino acid substitutions and consequently defective globin chain production or reduced production of normal haemoglobin. Despite the limited study of this country regarding this issue, the number of individuals suffering from inherited Hb disorders is not negligible. Thus, the study was designed to evaluate the types of inherited haemoglobin disorders among the patients attending the haematology outdoor of Dhaka Medical College Hospital (DMCH). Methods: This hospital-based cross-sectional study was conducted at the Department of Haematology in DMCH, for a period of 6 months (June 2022 to November 2022. People attending the Haematology outpatient department and diagnosed with a case of inherited Hb disorder were approached for inclusion. Written informed consent was taken from each subject. Ethical issues were ensured properly. For the study, a total of 100 patients were interviewed for less than 30 minutes of duration in each case. Relevant investigations such as, Hemoglobin (Hb), Red Cell Count (RCC), MCV, MCH, MCHC, RDW, Reticulocyte, Serum ferritin were done in the Haematology lab of DMCH, and data were collected in a semi-structured questionnaire. Collected Data were analyzed by SPSS 21. Results: Out of 100 inherited Hb disorder subjects, the majority (60 or 60%) were from the age group of 14 to 24 years. The mean age of patients was 38.78 ± 6.09 with range 15-51 years. The male-female ratio was 1:1. The majority (58%) came from rural areas and 42% came from urban areas. Mean value of Hb was 10.54; subsequently, MCV (63.25), MCH (22.18), S ferritin (140.43). The majority of patients had their onset of clinical presentation of illness for 5 years. Among total subjects, two-thirds of the patients (66 or 66%) had Hb E Disease followed by 14% Beta thalassaemia trait, 14% Hb E trait, and 6% b thalassaemia major. No association was noted between the type of thalassaemia with age & sex of the subjects. Conclusion: Irrespective of age and sex variations, Hb E disease is the most frequent inherited haemoglobin disorder among the patient attending the haematology outdoor of a tertiary care hospital. J MEDICINE 2023; 24(2): 96-105

The gene spectrum of thalassemia in Yangjiang of western Guangdong Province.

Background: Thalassemia presents a higher incidence in southern China. The objective of this study is to analyze the genotype distribution of thalassemia in Yangjiang, a western city of Guangdong Province in China. Methods: The genotypes of suspected cases with thalassemia were tested by PCR and reverse dot blot (RDB). Unidentified rare thalassemia genotypes of the samples were further ascertained by PCR and direct DNA sequencing. Results: Among 22467 suspected cases with thalassemia, 7658 cases were found with thalassemia genotypes using our PCR-RDB kit. Among these 7658 cases, 5313 cases were found with α-thalassemia (α-thal) alone, --SEA/αα was the most common genotype, accounting for 61.75% of α-thal genotypes, and the following mutations were found: α3.7/αα, -α4.2/αα, αCSα/αα, αWSα/αα, and αQSα/αα. A total of 2032 cases were found with β-thalassemia (β-thal) alone. βCD41-42/βN, βIVS-II-654/βN, and β-28/βN accounted for 80.9% of all β-thal genotypes, and the following genotypes were found: βCD17/βN, βCD71-72/βN, and βE/βN. Compound heterozygotes of β-thal and β-thalassemia homozygotes were identified in 11 and five cases, respectively, in this study. α-thal combined with β-thal was identified in 313 cases, showing 57 genotype combinations of the coincidence of both Hb disorders; one extreme patient had a genotype of --SEA/αWSα and βCD41-42/β-28. In addition, four rare α-mutations (--THAI, HKαα, Hb Q-Thailand, and CD31 AGG>AAG) and six rare β-mutations (CD39 CAG>TAG, IVS-Ⅱ-2 (-T), -90(C>T), Chinese Gγ+(Aγδβ)0, CD104 (-G), and CD19 A>G) were also found in this study population. Conclusion: This study provided detailed genotypes of thalassemia in Yangjiang of western Guangdong Province in China and reflected the complexity of genotypes in this high-prevalence region, and this would be valuable for diagnosis and counseling for thalassemia in this area.

Prevalence and spectrum of haemoglobinopathies in females of reproductive age group- A first tertiary care center experience in Punjab, North India.

Thalassemia and hemoglobinopathies are a group of inherited conditions characterized by abnormalities in the synthesis or structure of hemoglobin (Hb). According to estimates, approximately 7% of the world population is a carrier of Hb disorders, leading to high morbidity and mortality. To reduce the burden of these highly prevalent monogenic disorders, detecting them in the carrier stage is crucial to prevent disease progression. We aimed to estimate the prevalence and spectrum of hemoglobinopathies in females in the reproductive (20-40 years) age group. It was a retrospective observational study carried out for 2.5 years (from January 2018 till June 2020). All the females in the age group of 20-40 years age whose blood samples were received in the department for High-Performance Liquid Chromatography (HPLC) were included. The cases with abnormal HPLC findings were analyzed for hematological parameters including hemoglobin, RBC count, and RBC indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), & red cell distribution width - coefficient of variation (RDW-CV)]. Statistical package for social science (SPSS) statistics 21 version for Microsoft Windows (Chicago, USA) was used for statistical analysis of data. The data were described in terms of range, mean ± standard deviation (SD), frequencies (number of cases), and relative frequencies (percentage) as appropriate. During the study period, 72.2% of the females were affected with β-thalassemia trait, followed by HbD Punjab trait (17.8%), HbQ India trait (2.9%), β-thalassemia major (1.8%), and two cases (1.2%) each of HbS trait, HbD Iran trait, and compound heterozygous of HbD Punjab and β-thalassaemia, whereas HbE trait, compound heterozygous of HbQ and β-thalassemia, compound heterozygous of HbJ-variant and β-thalassemia had one case each (0.6%). Preventive strategies are cost-effective and include population screening, premarital screening, screening of spouses, genetic counseling, and prenatal diagnosis. Educating the carrier females about the potential risk and various screening methods may help in controlling the disease.

Molecular prevalence of HBB-associated hemoglobinopathy among reproductive-age adults and the prenatal diagnosis in Jiangxi Province, southern central China.

Background and aims: Hemoglobinopathy associated with the HBB gene, with its two general subtypes as thalassemia and abnormal hemoglobin (Hb) variants, is one of the most prevalent hereditary Hb disorders worldwide. Herein we aimed to elucidate the prevalence of ß-thalassemia and abnormal hemoglobin variants and the prenatal diagnosis of the HBB gene in Jiangxi Province, southern central China. Methods: Hematological indices and capillary Hb electrophoresis were conducted for 136,149 subjects who were admitted to Jiangxi Maternal and Child Health Hospital and requested for hemoglobinopathy investigation. Routine α- and ß-globin genotyping were performed by gap-polymerase chain reaction (Gap-PCR) and reverse dot-blot (RDB) hybridization for the 11,549 individuals suspected to be thalassemia carriers. For participants whose genotypes could not explain their hematological indices, further Sanger sequencing and Gap-PCR were conducted for the detection of rare or novel variants in related globin genes. Prenatal diagnosis was performed for 77 pregnant couples both carrying ß-thalassemia trait at appropriate gestational ages. Results: Among the 11,549 subjects, 2,548 individuals were identified with HBB-associated hemoglobinopathy based on molecular analysis. A total of 2,358 subjects were identified as ß-thalassemia heterozygous carriers and nine cases were diagnosed as compound heterozygous ß-thalassemia. Additionally, 125 cases were detected with composite α- and ß-thalassemia and the remaining 56 individuals with abnormal Hb variants in the HBB. A total of 35 types of variants were identified in the HBB gene, including 26 types of ß-thalassemia and nine types of abnormal Hb variants. Four novel variants were firstly reported, including one variant in HBA2 and three variants in HBB. Overall, 77 prenatal samples underwent ß-thalassemia molecular diagnosis; 20 fetuses were identified with normal ß-thalassemia genotypes, 30 fetuses as ß-thalassemia heterozygotes, 11 as homozygotes, and 16 as compound heterozygotes in HBB. Conclusion: We have demonstrated a relatively high prevalence rate at 1.872% of ß-hemoglobinopathies including common and rare ß-thalassemia as well as abnormal Hb variants among large child-bearing population in the Jiangxi area of southern central China for the first time. Our data presents that prenatal diagnosis is an effective way to prevent and control birth defects of ß-thalassemia.

Epidemiological and molecular study of hemoglobinopathies in Mauritanian patients.

BackgroundHemoglobinopathies, inherited disorders of hemoglobin (Hb), are the most common hereditary monogenic diseases of the red cell in the world. Few studies have been conducted on hemoglobinopathies in Mauritania. Therefore, the aim of this work is to establish the molecular and epidemiological basis of hemoglobinopathies in a cohort of Mauritanian patients and to determine the haplotype of the β‐globin gene cluster in sickle cell subjects.MethodsThe molecular screening of Hb disorders in 40 Mauritanian patients was done by a polymerase‐restriction fragment length polymorphism (RFLP) for the sickle cell disease (SCD) mutation, a PCR/sequencing method for β‐thalassemia mutations, and by the multiplex polymerase chain reaction method for the α‐thalassemia. The exploration of eight polymorphic sites (SNPs) within the β‐globin gene cluster was conducted by PCR/RFLP method, to identify the HbS haplotypes from the sickle cell subjects.ResultsThe epidemiological study of our patients showed a high incidence in the Senegal River area (52.5%) and a high ethnic prevalence for the Heratin (47.5%) and the Pular (35%). Molecular study allowed us to identify eight different mutations in our sample analyzed. They are respectively: HbS (HBB:c.20A>T) (68.75%), Cd44 ‐C (HBB:c.135delC) (8.75%), −29A>G (HBB:c.‐79A>G) (4.8%), −α‐3.7 (g.34164_37967del3804) (3.75%), IVS‐II‐849A>G (HBB:c.316‐2A>G) (2.25%) and Cd24T>A (HBB:c.75T>A), Hb Siirt (HBB:c.83C>G) and HbC (HBB:c.19G>A) each with (1.25%). Six different haplotypes are being explored among the SCD subjects with the Senegal haplotype as the most prevalent (66.7%), followed by Benin (10%), Arab‐Indians (6.7%), Bantu (3.3%), and two atypical haplotypes.ConclusionOur findings enrich the epidemiological data in our population and could contribute to the establishment of a strategy of prevention and management through screening, genetic counseling, and prenatal diagnosis of Hemoglobinopathies in the Mauritanian population.

RISK FACTORS FOR THE ANEMIA IN PREGNANT WOMEN: A LITERATURE REVIEW

Background: Anemia in pregnancy is still a global public health problem and is the largest contributor to morbidity and mortality. Some studies report that risk factors for anemia vary from place to place, so more in-person observations are needed regarding anemia risk factors.
 Objectives: This study aimed to determine the risk factors of anemia in pregnant women. Methods of using literature review studies.
 Design: This study design is a literature review to search and review article from database and the theory which is descriptive.
 Data Sources: Search for scientific articles using the Mendeley application by entering the keyword "Risk Factors, Anemia, Pregnancy" with Published year 2018-2021, English and Indonesian, full text, National journal has ISSN and open access.
 Review Methods: The literature review method using narrative review based on inclusion criteria, namely articles discussing anemia in pregnant women, published in 20158-202, International and National Publications, having ISSN National Journals, Articles using English, original articles, full text and open access. While the Exception Criteria for Articles other than English, type of research literature review and RCT, articles are then collected and a journal summary is made containing the name of the researcher, the year the journal was published, the title of the study, the method and a summary of the results or findings. The summary of research journals is entered into a table according to the format, and 10 articles are eligible.
 Results: From the 10 selected articles, it can be concluded that the risk factors for anemia, especially in developing countries, are multifactorial, including micro-iron deficiency, folate, and vitamins A and B12 as well as anemia due to parasitic infections such as malaria and hookworms or chronic infections such as TB, HIV, parasites. intestines, infection of working mothers (farmers), contaminated drinking water sources, consumption of coffee/tea and diet. Meanwhile, other factors in Indonesia are unfavorable geographical location, level of education, lack of public awareness of health, socio-economic situation, parity (number of children), and chronic energy shortages (KEK).
 Conclusion: Anemia is still a global health problem and still has to be addressed, especially in developing countries. Therefore, efforts are needed to better understand the main causes of anemia, including iron deficiency as well as other nutritional deficiencies, diseases, and Hb disorders related to anemia so that appropriate action can be given. Biochemical measurement of micronutrients (especially iron and Vitamin A), inflammatory signs in addition to hematological index when assessing anemia clinically are also urgently needed.

Spectrum of Genetic Mutation in Beta Globin Gene in Various Type of Thalassaemia in Bangladesh

Background: Hb-E/Beta thalassaemia is a congenital haemoglobin disorder which is a compound heterozygous state consists of qualitative disorder like Hb E variant & quantitative Hb disorder caused by genetic mutation of Beta chain. Objective: The aim of the study was to identify the beta gene mutation in Hb E/Beta thalassaemia. Method: A total of 32 diagnosed Hb E/Beta thalassaemia patients were included in this cross-sectional study from May 2019 to July 2020. Genetic analysis was done by sanger sequencing. Results: In this observational study, we found 13 different types of Beta gene mutations. Heterozygous for IVS 1-5(G>C) mutation was most frequent (53.1%). Conclusion: Genetic mutation is the confirmatory diagnosis for thalassaemia as well as one of the main factors for clinical expression. Mutation pattern also varies according to the geographical distribution. So, this study shows the frequently found mutation in Bangladesh and should carry out routinely to point out phenotypic expression.

Thalassaemia (part 1)

The thalassaemias are a group of inherited blood disorders characterised by decreased or absent globin chain synthesis. Classification of thalassaemia is based on the type of globin chain that is deficient. There are four globin chain subtypes, viz. α, β, γ and δ; deficiencies of these are designated as α-, β-, γ- or δ-thalassaemia, respectively. As genetic defects or deletions may affect one or more globin genes, a variety of clinical phenotypes may be encountered. Heterozygotes (thalassaemia carriers) enjoy partial protection against malaria, with consequential survival advantage. This explains the unexpectedly high gene frequencies found in malaria-endemic areas. South Africa is not a malaria-endemic area, and therefore has a predictably low prevalence of thalassaemia and other inherited Hb disorders. However, because of migration, these conditions are increasingly encountered in countries not endemic to malaria. In this issue (part 1 of the 2-part CME series), discussion is centred around epidemiology, pathophysiology, clinical features and diagnosis. Management is discussed in the next issue (part 2).

Surrogate Markers of Subclinical Atherosclerosis and Its Associated Factors in Patients with β-Thalassemia Major

β-Thalassemia major (β-TM) is a severe genetic hemoglobin (Hb) disorder with cardiovascular complications such as atherosclerosis due to transfusion-dependent iron overload. We aimed to determine the associated factors with surrogate markers of subclinical atherosclerosis in these patients. Sixty subjects with β-TM referred to the Thalassemia Clinic of the Iranian Blood Transfusion Organization (IBTO) were included in our study. The blood samples were collected for laboratory measurements. The carotid intima-media thickness (CIMT), was measured by ultrasonography, and ankle-brachial index (ABI) was calculated. The multivariate linear analysis was performed to determine the appropriate indicators of subclinical atherosclerosis in β-TM. There was no significant difference in baseline characteristics between the study groups. In multivariate linear analysis, age and systolic blood pressure (SBP) were negatively associated with inverse-transformed CIMT [unstandardized β coefficient (B): −0.024, 95% confidence interval (95% CI): −0.032- −0.010, p < 0.001; B: −0.009, 95% CI: −0.017- −0.001, p 0.031, respectively]. There was also a significant correlation between the serum level of high-density lipoprotein cholesterol (HDL-C) and insulin with higher ABI, after adjustment for confounding variables (B: 0.003; 95% CI: 0.000–0.005; p = 0.030, and B: 0.004, 95% CI: 0.000–0.009, p = 0.037, respectively). Our results show that advancing age and increased SBP, HDL-C and insulin, associated with higher CIMT or ABI, are appropriate indicators of subclinical atherosclerosis in β-TM patients.

Study of prevalence of haemoglobin disorders in sindhi community with reference to HPLC

Introduction: Inherited haemoglobin disorders are major health problems in certain communities in different geographical areas in India. These disorders pose a huge burden on affected families physically, financially & psychologically. This study is carried out to find the Prevalence of these disorders in Sindhi community in Aurangabad city in state of Maharashtra, India. Materials and Methods: The present cross-sectional study was undertaken among 338, apparently healthy Sindhi individuals from community in Aurangabad, Maharashtra from July 2014 to March 2017. All the samples were tested in automated hematology cell counter and then for HPLC on Bio-Rad variant classic. Various CBC parameters and Hemoglobin patterns were derived on all samples. Result: The overall prevalence of Hemoglobinopathy was 60/338 (17.75%), comprised of 42 (12.42%) cases of BTT, 3 (0.88%) cases of BTM, 3 (0.88%) cases of SCT, 11 (3.25%) cases of Hb Q-India & One (0.29%) case of Hb Q-India+BTT. Conclusion: Out of 338 samples, BTT and Hb Q-India were found to be more common Hb disorders in Sindhi community. Screening of community and counselling regarding the nature of these disorders are very important measures to reduce the burden of disease. Keywords: Hemoglobin disorders, Prevalence, HPLC, β-thalassemia trait, Hb Q-India, Maharashtra, India.

Computer Vision and Deep Learning Assisted Microchip Electrophoresis for Integrated Anemia and Sickle Cell Disease Screening

Introduction: Anemia affects a third of the world's population with the heaviest burden borne by women and children. Anemia leads to preventable impaired development in children, as well as high morbidity and early mortality among sufferers. Inherited hemoglobin (Hb) disorders, such as sickle cell disease (SCD), are associated with chronic hemolytic anemia causing high morbidity and mortality. Anemia and SCD are inherently associated and are both prevalent in the same regions of the world including sub-Saharan Africa, India, and south-east Asia. Anemia and SCD-related complications can be mitigated by screening, early diagnosis followed by timely intervention. Anemia treatment depends on the accurate characterization of the cause, such as inherited Hb disorders. Meanwhile, Hb disorders or SCD treatments, such as hydroxyurea therapy, requires close monitoring of blood Hb level and the patient's anemia status over time. As a result, it is crucially important to perform integrated detection and monitoring of blood Hb level, anemia status, and Hb variants, especially in areas where anemia and inherited Hb disorders are the most prevalent. Blood Hb level (in g/dL) is used as the main indicator of anemia, while the presence of Hb variants (e.g., sickle Hb or HbS) in blood is the primary indicator of an inherited disorder. The current clinical standards for anemia testing and Hb variant identification are complete blood count (CBC) and High-Performance Liquid Chromatography (HPLC), respectively. State-of-the-art laboratory infrastructure and trained personnel are required for these laboratory tests. However, these resources are typically scarce in low- and middle-income countries, where anemia and Hb disorders are the most prevalent. As a result, there is a dire need for high accuracy portable point-of-care (POC) devices to perform integrated anemia and Hb variant tests with affordable cost and high throughput. Methods: In 2019, the World Health Organization (WHO) listed Hb electrophoresis as an essential in vitro diagnostic (IVD) technology for diagnosing SCD and sickle cell trait. We have leveraged the common Hb electrophoresis method and developed a POC microchip electrophoresis test, Hemoglobin Variant/Anemia (HbVA). This technology is being commercialized under the product name "Gazelle" by Hemex Health Inc. for Hb variant identification with integrated anemia detection (Fig. 1A&amp;B). We hypothesized that computer vision and deep learning will enhance the accuracy and reproducibility of blood Hb level prediction and anemia detection in cellulose acetate based Hb electrophoresis, which is a clinical standard test for Hb variant screening and diagnosis worldwide (Fig. 1C). To test this hypothesis, we integrated, for the first time, a new, computer vision and artificial neural network (ANN) based deep learning imaging and data analysis algorithm, to Hb electrophoresis. Here, we show the feasibility of this new, computer vision and deep learning enabled diagnostic approach via testing of 46 subjects, including individuals with anemia and homozygous (HbSS) or heterozygous (HbSC or Sβ-thalassemia) SCD. Results and Discussion: HbVA computer vision tracked the electrophoresis process real-time and the deep learning neural network algorithm determined Hb levels which demonstrated significant correlation with a Pearson Correlation Coefficient of 0.95 compared to the results of reference standard CBC (Fig.1D). Furthermore, HbVA demonstrated high reproducibly with a mean absolute error of 0.55 g/dL and a bias of -0.10 g/dL (95% limits of agreement: 1.5 g/dL) according to Bland-Altman analysis (Fig. 1E). Anemia determination was achieved with 100% sensitivity and 92.3% specificity with a receiver operating characteristic area under the curve (AUC) of 0.99 (Fig. 1F). Within the same test, subjects with SCD were identified with 100% sensitivity and specificity (Fig. 1G). Overall, the results suggested that computer vision and deep learning methods can be used to extract new information from Hb electrophoresis, enabling, for the first time, reproducible, accurate, and integrated blood Hb level prediction, anemia detection, and Hb variant identification in a single affordable test at the POC. Disclosures An: Hemex Health, Inc.: Patents &amp; Royalties. Hasan:Hemex Health, Inc.: Patents &amp; Royalties. Ahuja:Genentech: Consultancy; Sanofi-Genzyme: Consultancy; XaTec Inc.: Consultancy; XaTec Inc.: Research Funding; XaTec Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Genentech: Honoraria; Sanofi-Genzyme: Honoraria. Little:GBT: Research Funding; Bluebird Bio: Research Funding; BioChip Labs: Patents &amp; Royalties: SCD Biochip (patent, no royalties); Hemex Health, Inc.: Patents &amp; Royalties: Microfluidic electropheresis (patent, no royalties); NHLBI: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees. Gurkan:Hemex Health, Inc.: Consultancy, Current Employment, Patents &amp; Royalties, Research Funding; BioChip Labs: Patents &amp; Royalties; Xatek Inc.: Patents &amp; Royalties; Dx Now Inc.: Patents &amp; Royalties.

δ-Globin Chain Variants Associated with Decreased Hb A2 Levels: A National Reference Laboratory Experience

High prevalence of hemoglobin (Hb) disorders mandates national programs for screening and genetic counseling in many countries. Increased Hb A2 levels are commonly associated with β-thalassemias, however, various disorders including alteration of δ chains may result in decreased production of Hb A2, thus hindering the diagnosis of β-thalassemias. The reported data reflect the experience of a large reference laboratory in the United States. In the current study, we have attempted to assess the prevalence and also tried to characterize the identified mutations in the HBD gene resulting in decreased Hb A2 levels. In our cohort, 1.6% of 6486 patients were found to have Hb A2 values of <1.9%. Bidirectional sequencing of the HBD gene demonstrated mutations in 20 cases (19.0% of the individuals with decreased Hb A2). In addition to the previously reported variants, one novel mutation (Hb A2-Utah or HBD: c.46T>C).

The Homozygous Hemoglobin EE Variant Is Associated with Poorer Riboflavin Status in Cambodian Women of Reproductive Age

Riboflavin is required for erythropoiesis, which is increased in people with hemoglobinopathies due to increased hemolysis and erythrocyte turnover. Dietary intake and status of riboflavin is poor in Cambodia, where hemoglobinopathies are common. We assessed the association between genetic hemoglobin disorders and riboflavin status in women of reproductive age in Cambodia. Venous blood samples from 515 Cambodian women of reproductive age, 18-45 y, were analyzed for biomarker status of riboflavin [erythrocyte glutathione reductase activation coefficient (EGRac)], genetic hemoglobin (Hb) disorders, and hematological indices. Linear regression analysis was used to estimate the association between EGRac with Hb, ferritin, and Hb genotypes. EGRac was log transformed in the analyses, and the regression coefficients represent the geometric mean differences. Genetic Hb disorders were present in 57% of the population, with the homozygous hemoglobin E variant (Hb EE) occurring in ∼10% of women (n=53). Deficient (EGRac ≥1.40) or marginal riboflavin status (EGRac ≥1.30 and <1.40) was observed in 92% (n=475) of women. The variant Hb EE genotype was associated with 18% (95% CI: 9%, 28%) higher geometric mean EGRac values than the normal Hb AA genotype (P<0.001). Although riboflavin biomarker deficiency or marginal status is widely prevalent in Cambodian women, lower riboflavin status was observed more frequently in women with the Hb EE genotype than in women with normal Hb AA. The relation between genetic Hb disorders and riboflavin warrants further investigation. This trial was registered at clinicaltrials.gov as NCT01593423 and NCT02481375.

Hb Rush (HBB: c.304G>C): A Rare Variant Hemoglobin Mimicking the Hb S (HBB: c.20A>T) Variant on High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a useful and rapid tool in the evaluation of hemoglobin (Hb) disorders that include thalassemia and various hemoglobinopathies. Most of the techniques or programs used in automated testing platforms are customized to identify the common variants seen in that particular region. At times, variant Hbs may be identified which are not commonly seen in the local population. This may cause diagnostic dilemma and may require further studies for definitive characterization. We present a patient with Hb Rush (HBB: c.304G>C), a rare unstable Hb variant eluting in the Hb S (HBB: c.20A>T) window on HPLC.