Significant Hemoglobinopathies Research Articles

Provisional diagnosis of clinically significant hemoglobinopathies and decision on suitability for marriage as part of the premarital screening program in Iraq: Iraqi Society of Hematology Guidelines

Provisional diagnosis of clinically significant hemoglobinopathies and decision on suitability for marriage as part of the premarital screening program in Iraq: Iraqi Society of Hematology Guidelines

Significant haemoglobinopathies: A guideline for screening and diagnosis: A British Society for Haematology Guideline: A British Society for Haematology Guideline.

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the National Health Service (NHS) Sickle Cell and Thalassaemia Screening Programme. Newborn screening and, when necessary, follow-up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening Programme. All babies under 1 year of age arriving in the United Kingdom should be offered screening for sickle cell disease (SCD). Preoperative screening for SCD should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with a sickle solubility test must always be followed by definitive analysis. Laboratories performing antenatal screening should utilise methods that are capable of detecting significant variants and are capable of quantitating haemoglobins A2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available for antenatal patients within three working days from sample receipt.

Study the Hemoglobin Electrophoresis Patterns in two Populations from Salah - addin Province - Iraq

The early identification of some clinically significant hemoglobinopathies and precise differentiation of hemoglobin variants are important to provide early comprehensive medical care to prevent some serious complications, assess prognosis and offer genetic counseling.
 This study examined the prevalence of α 2 - gene deletion in neonates in Samarra and Tikrit, Iraq. Screening study was carried out by examining fresh blood samples obtained from excised umbilical cord of 42 neonates born in neonates clinics in Samarra hospital and Tikrit hospital from October 2012 to March 2013. Hemoglobin electrophoresis were performed using Hellabio hemoglobin electrophoresis kit (He 10) and Hellabioscan system for detection of Hb bands and value % of fractions.
 The results showed 40 cases out of 42 (95.24%) have normal hemoglobin components. 2 cases out of 42 (4.76%) there was no visible band of HbA2 in hemoglobin electrophoresis pattern. Considering the missing of HbA2 band the two cases may have the deletion of δ gene so each one of them may has the genotype δ0/δ0.

Haemoglobinopathies

Haemoglobinopathies are inherited disorders of the globin component of haemoglobin. Genetic mutations lead to an altered structure of haemoglobin, as in sickle cell disease, or reduced quantity of haemoglobin, as in thalassaemia. Significant haemoglobinopathies are usually detected shortly after birth. Milder forms can present later in life, with anaemia or other complications, which is why it is important for GPs to have an understanding of haemoglobinopathies. The purpose of this article is to provide an overview of the two most significant clinical haemoglobinopathies, sickle cell disease and thalassaemia, and to focus on their primary care management.

Establishing a Sickle Cell Disease Registry in Africa: Experience From the Sickle Pan-African Research Consortium, Kumasi-Ghana.

Sickle cell disease (SCD) is the most common clinically significant hemoglobinopathy, characterized by painful episodes, anemia, high risk of infection, and other acute and chronic complications. In Africa, where the disease is most prevalent, large longitudinal data on patients and their outcomes are lacking. This article describes the experiences of the Kumasi Center for SCD at the Komfo Anokye Teaching Hospital (KCSCD-KATH), a Sickle Pan-African Research Consortium (SPARCO) site and a SickleInAfrica Consortium member, in establishing a SCD registry for the evaluation of the outcomes of patients. It also provides a report of a preliminary analysis of the data. The process of developing the registry database involved comprehensive review of the center’s SCD patient medical records, incorporating data elements developed by the SickleInAfrica Consortium and obtaining ethical clearance from the local Institutional Review Board. From December 2017 to March 2020, 3,148 SCD patients were enrolled into the SCD registry. Enrollment was during the SCD outpatient clinic visits or through home visits. A significant proportion of the patients was from the newborn screening cohort (50.3%) and was males (52.9%). SCD-SS, SCD-SC, and Sβ +thalassemia were seen in 67.2, 32.5, and 0.3% patients, respectively. The majority of the patients were in a steady state at enrollment; however, some were enrolled after discharge for an acute illness admission. The top two clinical diagnoses for SCD-SS patients were sickle cell painful events and acute anemia secondary to hyperhemolysis with incidence rates of 141.86 per 10,000 person months of observation (PMO) and 32.74 per 10,000 PMO, respectively. In SCD-SC patients, the top two diagnoses were sickle cell painful events and avascular necrosis with incidence rates of 203.09 per 10,000 PMO and 21.19 per 10,000 PMO, respectively. The SPARCO Kumasi site has developed skills and infrastructure to design, manage, and analyze data in the SCD registry. The newborn screening program and alternative recruitment methods such as radio announcement and home visits for defaulting patients were the key steps taken in enrolling patients into the registry. The registry will provide longitudinal data that will help improve knowledge of SCD in Ghana and Africa through research.

Novel therapies in β‐thalassaemia

Beta-thalassaemia is one of the most significant haemoglobinopathies worldwide resulting in the synthesis of little or no β-globin chains. Without treatment, β-thalassaemia major is lethal within the first decade of life due to the complex pathophysiology, which leads to wide clinical manifestations. Current clinical management for these patients depends on repeated transfusions followed by iron-chelating therapy. Several novel approaches to correct the resulting α/β-globin chain imbalance, treat ineffective erythropoiesis and improve iron overload are currently being developed. Up to now, the only curative treatment for β-thalassemia is haematopoietic stem-cell transplantation, but this is a risky and costly procedure. Gene therapy, gene editing and base editing are emerging as a powerful approach to treat this disease. In β-thalassaemia, gene therapy involves the insertion of a vector containing the normal β-globin or γ-globin gene into haematopoietic stem cells to permanently produce normal red blood cells. Gene editing and base editing involves the use of zinc finger nucleases, transcription activator-like nucleases and clustered regularly interspaced short palindromic repeats/Cas9 to either correct the causative mutation or else insert a single nucleotide variant that will increase foetal haemoglobin. In this review, we will examine the current management strategies used to treat β-thalassaemia and focus on the novel therapies targeting ineffective erythropoiesis, improving iron overload and correction of the globin chain imbalance.

736 Comparing neonatal outcomes among patients undergoing intravenous glucose tolerance testing for diagnosis of gestational diabetes

To assess neonatal outcomes associated with abnormal intravenous glucose tolerance test (IV GTT) results in a cohort of patients unable to tolerate the standard oral glucose tolerance test for diagnosis of gestational diabetes (GDM). Patients who could not tolerate an oral glucose load, were > 24 weeks gestation, had a viable singleton or multifetal gestation, and spoke English or Spanish were enrolled prospectively between February 2019 and February 2020. Patients with pre-existing diabetes mellitus (DM) or clinically significant hemoglobinopathies were excluded. Enrolled patients underwent an IV GTT and glucose monitoring was initiated if the IV GTT resulted in a glucose metabolism rate below trimester-specific normative values. The primary outcome of interest was large for gestational age (LGA) infants. The study was powered to detect a 25% difference in LGA infants between patients with an abnormal IV GTT result compared to those with a normal IV GTT result. Of the 120 patients consented, 115 completed glucose testing and 13 (11.3%) had an abnormal IV GTT. Age, race/ethnicity, parity, family history of DM, insurance status, gestational age at testing, and body mass index did not differ between those with abnormal compared to normal IV GTT results. Participants with an abnormal IV GTT result were more likely to have multifetal pregnancies (15.4% vs 0%, p=0.01). Infants delivered to patients with an abnormal IV GTT result were more likely to be LGA, have lower mean neonatal blood glucose nadirs, and require treatment with supplemental glucose (Table). Participants with an abnormal IV GTT result were more likely to have an LGA neonate and clinically significant hypoglycemia. The IV GTT is a reasonable diagnostic test for GDM-related morbidities when an oral glucose load cannot be tolerated.

Study to identify the role of high performance liquid chromatography in detecting haemoglobinopathies in antenatal patients

Introduction: Haemoglobinopathy is a major genetic public health problems in India, responsible for significant morbidity and mortality. Individuals with trait (carriers) are healthy and unaware of their carrier status unless specifically screened. If a couple carry a clinically significant haemoglobinopathy trait there is a 1 in 4 chance with each pregnancy that their child will inherit a major haemoglobinopathy. The most effective approach to reduce the burden of the society is to reduce the incidence by implementation of a carrier screening programme. Material and Methods: The present study was carried out in a tertiary care hospital for a period of 2 years. 500 Antenatal patients attending Obstetric-out patient department for routine check-up were included in the study. Screening of patient was done by Solubility test, Sickling test and Alkali denaturation test. Finally a very standardized method to detect HbA2 and HbF was done by using an automated High Performance Liquid Chromatography. Results: Out of a total of 500 pregnant women 4% were having haemoglobinopathies, whereas 96% had normal haemoglobin. Out of 20 cases, maximum number of cases i.e. 2.2% was of ? –Thalassemia trait, followed by Sickle cell trait cases i.e. 1.2%. Other haemoglobinopathies are also found i.e. sickle cell disease 0.2% ?? Thalassemia case 1.2% and double heterozygous 0.2%.Out of a total of 20 positive pregnant women only partners of 8 women were screened and out of them 37.5% were found positive. Conclusion: Detection of carrier status using HPLC during pregnancy along with couple screening provides prospective parents with the option of testing the fetus for hemoglobinopathy. Keywords: Antenatal, Double heterozygous, Haemoglobinopathies, High Performance Liquid Chromatography, ? –Thalassemia trait.

Antenatal haemoglobinopathy screening - Experiences of a large Australian Centre.

Antenatal screening is vital to identifying couples at risk of having children with a clinically significant haemoglobinopathy. In Australia, immigration is increasing carrier incidence. 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. 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. 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.

Advances in genome editing: the technology of choice for precise and efficient β-thalassemia treatment.

Beta (β)-thalassemia is one of the most significant hemoglobinopathy worldwide. The high prevalence of the β-thalassemia carriers aggravates the disease burden for patients and national economies in the developing world. The survival of β-thalassemia patients solely relies on repeated transfusions, which eventually results into multi-organ damage. The fetal γ-globin genes are ordinarily silenced at birth and replaced by the adult β-globin genes. However, mutations that cause lifelong persistence of fetal γ-globin, ameliorate the debilitating effects of β-globin mutations. Therefore, therapeutically reactivating the fetal γ-globin gene is a prime focus of researchers. CRISPR/Cas9 is the most common approach to correct disease causative mutations or to enhance or disrupt the expression of proteins to mitigate the effects of the disease. CRISPR/cas9 and prime gene editing to correct mutations in hematopoietic stem cells of β-thalassemia patients has been considered a novel therapeutic approach for effective hemoglobin production. However, genome-editing technologies, along with all advantages, have shown some disadvantages due to either random insertions or deletions at the target site of edition or non-specific targeting in genome. Therefore, the focus of this review is to compare pros and cons of these editing technologies and to elaborate the retrospective scope of gene therapy for β-thalassemia patients.

The association of HBB-related significant hemoglobinopathies and low fetal fraction on noninvasive prenatal screening for fetal aneuploidy

Objectives HBB-related significant hemoglobinopathies have been anecdotally associated with low fetal fraction on noninvasive prenatal screening (NIPS). We sought to compare the difference in fetal fraction using NIPS in women with HBB-related significant hemoglobinopathies (HSH) and women with normal hemoglobin. Study design This is a retrospective case-control study. Cases were women with a diagnosis of HSH using NIPS from a commercial laboratory. The comparison group was women with hemoglobin AA from a tertiary care center database. We tested for differences in median fetal fraction using quantile regression analysis, adjusting for maternal body weight and gestational age. Results This study includes 35 women with clinically significant HSH and a comparison group of 636 women with hemoglobin AA. Adjusting for gestational age and body weight, the median fetal fraction was 4.1 point lower in the HSH than in the comparison group (β − 4.1; 95% −5.7 to −2.5, p < .05). The rate of no-calls due to low fetal fraction was significantly higher in the clinically significant HSH group than in the comparison group [HSH: n = 9/35, 25.7% versus comparison: n = 32/636, 5.0% (p < .001)]. Conclusion Women with HSH were more likely to have a lower fetal fraction and ultimately a five-fold higher no-call rate. What’s already known about this topic? Low fetal fraction is one of the most common causes of no-call result in noninvasive prenatal screening High maternal weight, early gestational age and fetal aneuploidies are associated with low fetal fraction What does this study add? HBB-related significant hemoglobinopathies are associated with low fetal fraction Reduction in fetal fraction due to HBB-related significant hemoglobinopathies may also result in higher no-call rate

Compound Heterozygote of Hb S (HBB: c.20A>T)/Hb Westdale (HBB: c.380_396delTGCAGGCTGCCTATCAG): Report of Four Cases from Odisha State, India

We report four cases of compound heterozygotes for Hb S (HBB: c.20A>T) and a rare β0-thalassemia (β0-thal) mutation, Hb Westdale (HBB: c.380_396delTGCAGGCTGCCTATCAG), characterized by a 17 bp deletion between codons 126 to 131 in exon 3 of the β-globin gene of human hemoglobin (Hb) confirmed by direct β-globin gene sequencing. All four cases were from four unrelated families belonging to the Agharia caste, an endogamous ethnic community of the Sundargarh and Jharsuguda districts of Odisha State, India. Detailed observations indicated that all four cases of Hb S/Hb Westdale were clinically severe. On family screening, six family members were found to be heterozygous for Hb Westdale and were asymptomatic. Deletional α-thalassemia (α-thal) and XmnI polymorphism were studied for all the Hb Westdale cases. The Hb S/Hb Westdale cases had an early median age at onset of symptoms and presentation, more requirement of blood transfusions, splenomegaly and hepatomegaly and were found to be clinically more severe when compared with the Hb S-β-thal with IVS-I-5 (G>C) (HBB: c.92 + 5G>C) cases. Overall, the findings indicate that this rare and hitherto unreported compound heterozygosity of Hb S/Hb Westdale is a clinically significant hemoglobinopathy and its finding in a large endogamous community of Odisha State, India will have important implication in the epidemiology and understanding of the clinical spectrum of sickle cell disease in Indian context and prenatal diagnosis.

Detection of hemoglobinopathies in patients of anaemia using high performance liquid chromatography (HPLC) - a hospital based prospective study

Introduction: In India, the gene frequency of hemoglobinopathies is 4.2%, with a population over 1 billion and over 12000 infants born each year have a clinically significant hemoglobinopathies. In various parts of India, the prevalence of β-Thalassemia is different. β- Thalassemia has a high prevalence in some communities, such as Sindhi, Luvana, Tribes, and Rajputs. There is no such study done in and around Karimnagar district of Telangana state, therefore a preliminary hospital-based study was carried out. Materials &amp; Methods: In this cross-sectional hospital-based study, target group adopted was anaemic patients (&lt;11gm/dl). Haemoglobin and Red Blood Cell indices were measured on automated– three part differential cell counter. All these samples were analysed for haemoglobin disorders by BIORAD ‘VARIANT’ HPLC machine. Results: Among the cases, Karimnagar (62), Adilabad (143) and Medak (15) districts amounting for 220 cases in which 36 (16.36%) were having Hemoglobinopathies of which Karimnagar had 12 cases, Adilabad 23 cases and Medak had one case. The present study also revealed the prevalence of hemoglobinopathies according to caste. Mala caste had a higher frequency 12/36 (33.3%) followed by Munnur Kapu 6/36 (16.6%) and Christians 5/36(13.8%). This might be due to higher population of mala community as compared to others reported to the hospital. Conclusion: The data regarding prevalence and distribution can be useful in prevention and management of various hemoglobinopathies which may play a vital role in the hospital blood bank as well as in the formulation of transfusion policies.

Detection of Hemoglobin (Hb) Variants By HPLC Screening in Cord Blood Units (CBU) Donated to the National Cord Blood Program (NCBP)

Purpose. To evaluate the frequency and types of Hb abnormalities in CBU collected by the NCBP at the New York Blood Center.Background and Methods. Established in 1992, NCBP has collected and tested over 76,000 ethnically diverse CBU and manages an inventory of over 60,000 clinical grade CB products, including the first FDA-licensed (HEMACORD®). NCBP has released over 5,600 CBU for transplantation worldwide. To prevent transplants from donors affected with clinically significant hemoglobinopathy (HP), current Good Tissue Practices (cGTP) require Hb screening of all CBU before they are accepted to the public CB inventories. Clinically significant Hb disorders are caused by either structurally abnormal Hb variants (e.g., Hb S, Hb C, Hb E) or by decreased or absent production of α- or β-globin chains (thalassemia). Evaluation of NCBP CBU for possible HP includes: a) detailed family history for HP; b) complete blood count (CBC) and RBC indices, particularly mean corpuscular volume (MCV); c) screening using High-Performance Liquid Chromatography (HPLC) and d) confirmatory diagnostic testing using molecular methods (Hemoglobinopathy Reference Laboratory, Oakland, CA). To date, 76,312 CBU have been screened at the NCBP with the Bio-Rad Variant HPLC system using the Sickle Cell Short Program, a 3-min assay, specifically designed and FDA approved to provide a qualitative result for the presence of Hbs A, F, S, C, D and/or E in the neonate.Results.The HPLC chromatographic patterns were normal (Hb FA, where F is fetal and A is adult Hb) in 73,163 CBU (95.87%), and revealed an abnormal Hb in the remaining 3,149 CBU (4.13%), the majority of which were not clinically significant.The most frequent abnormal Hb phenotypes were, sickle cell trait (FAS): 1,755 CBU (57%), Hb E trait (FAE): 562 (18%) and Hb C trait (FAC): 506 (17%). Among the 3,149 CBU with abnormal Hb, 87 carried two genes for Hb disorders of clinical relevance, of which 54 were homozygous for sickle hemoglobin (Hb FS), 6 for Hb C (Hb FC) and 19 were double heterozygotes (Hb FSC). The HPLC results of all CBU with Hb traits and homozygous patterns were confirmed on repeat testing. In addition, all homozygous HPLC results were confirmed by molecular testing.Thalassemia and other abnormalities. Hb Barts (γ4) elutes "Fast" in the HPLC chromatogram. Its normally low concentration in CB increases when γ-chains replace non-functional or deleted α-globin chains in α-thalassemia (α-thal). Testing of all 76,312 NCBP CBU showed levels of Hb Barts from 0.5% to 34% of total Hb: 69.3% of the samples had Hb Barts level below 2.5%, 29.9% between 2.6 and 6.0% and 0.8% greater than 6.1%.Accordingly, for presumptive identification of α- or β-thalassemia, HPLC results must be interpreted together with red cell indices and family history. Of 788 CBU samples submitted for molecular (DNA) analyses as "possible thalassemia carriers", one was confirmed as Hb H disease (three α-globin genes deleted), 270 had α-thal trait (two α-globin gene deletions) and 517 had one or no α-globin gene deletion. The 270 CBU with α-thal trait had Hb Barts levels between 4.0% (above 6% in most cases) and 18.9% of total Hb. Further, 258 (95.6%) of the CBU with α-thal trait had MCV below 105 fL (range: 79.9 - 104.8 fL); only 12 cases (4.4%) had MCV >105 fL, all of which had levels of Hb Barts > 5%. The Hb H disease case had Fast Hb 34.2% (by HPLC) and MCV was 76.1 fL.β-thalassemia (β-thal): 4 CBU had β-thal major and in those, only Hb F (no Hb A) was seen in the HPLC result. Among 26 CBU found to have β-thal trait by the reference lab’s DNA analysis, 16 (61.5%) were tested because of positive family history and 10 (38.5%) because of MCV <105 fL (seven CBU had both, low MCV and family history of thalassemia). Another CBU with a still-uncharacterized β-globin gene mutation had MCV <105 fL.HPLC patterns disclosing "Unknown Hbs" as "traits" in 5 CBU, were confirmed by molecular testing.Conclusions.HPLC is a simple to perform, accurate, comprehensive, inexpensive and fast screening method for HP in cord blood.All homozygous Hb detected by HPLC were confirmed by the reference lab. This is particularly relevant for sickle cell disease, as it is the most common HP in ethnic minority donors.Screening with HPLC, when used in combination with CBC and family history, is a useful tool to identify possible α- and β-thalassemia carriers in CBU.Molecular DNA testing is diagnostic in confirming and identifying globin gene deletions and mutations. DisclosuresNo relevant conflicts of interest to declare.

Genetic Testing for the Diagnosis of Hemoglobinopathies Has Very High Clinical Utility

▪Background: The hemoglobinopathies are a genetically complex group of blood disorders that includes sickle cell disease (SCD), thalassemia, and other structural and/or functional hemoglobin (Hb) variants. For decades, the mainstay of diagnostic testing for hemoglobinopathies has been Hb separation techniques (e.g., electrophoresis, chromatography), sometimes paired with functional assays of Hb (e.g., solubility, instability, oxygen affinity). Such protein-based testing cannot detect or adequately differentiate a number of clinically significant hemoglobinopathies. Nevertheless, most clinicians continue to rely primarily on protein-based diagnostic methods despite the availability of genetic testing. The reasons for this are not fully understood, but include concerns about the cost of genetic testing and the perception that protein-based methods are usually sufficient for clinical care.Objective: To determine the clinical utility of genetic testing for the diagnosis of hemoglobinopathies by quantifying how often a suspected clinical diagnosis is changed, clarified or excluded by genetic testing.Methods: We reviewed the results of 500 sequential orders for clinical genetic testing for known or suspected hemoglobinopathies that were performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013 and 5/2015. This comprehensive genetic testing service is a collaboration between the divisions of Hematology and Human Genetics. For this analysis we reviewed orders for copy number variation analysis (CNV) of the α-globin gene cluster, sequencing of the α-globin genes (HBA1, HBA2), CNV of the β-globin gene cluster, and/or sequencing of the β-globin genes (HBB). An order for testing could include any combination of these 4 tests. We compared the stated indications and suspected diagnoses for testing provided by the ordering physician with the final results of the genetic testing. For each order, the results of this comparison (suspected diagnosis vs. genetic diagnosis) were classified into 1 of 4 mutually exclusive groups: (1) suspected diagnosis confirmed, (2) suspected diagnosis excluded, (3) suspected diagnosis clarified, or (4) new or unexpected diagnosis. The category "suspected diagnosis clarified" includes cases where the overall suspected diagnosis was confirmed, but additional clinically meaningful genetic data was also uncovered (e.g., homozygous sickle cell anemia was confirmed, but co-inherited α-globin gene deletions were also identified).Results: 500 serial genetic testing panels were performed on 475 unique individuals (7 specimens were from an embryo or fetus). These 500 panels included 1-4 individual tests: HBB sequencing in 423, α-globin CNV in 475, β-globin CNV in 345, and HBA1/2 sequencing in 164. The final diagnosis was a specific genotype of SCD (± α-thalassemia and/or gene-deletion HPFH) in 234; thalassemia (α, β, or δβ) in 139; Hb S trait (± α-thalassemia) in 18; Hb C trait or disease (± α-thalassemia) in 13; Hb E trait or disease (± α-thalassemia) in 9; another named or novel Hb variant in 22; and normal Hb in 65. Overall, the suspected diagnosis was confirmed in 246 (49.2%), clarified in 156 (31.2%), excluded in 77 (15.4%), and in 21 (4.2%) a new or unsuspected diagnosis was made. For patients with a suspected diagnosis of SCD, the diagnosis was mostly confirmed (66.7%) and never excluded by genetic testing, but it was clarified in 31.6%, and an unsuspected genotype of SCD was identified in 1.8%. For patients with a suspected diagnosis other than SCD, the diagnosis was confirmed in 34%, clarified in 30.6%, excluded in 29.1%, and an unsuspected genotype was identified in 6.4%. Not considering the clinical utility of the "suspected diagnosis clarified" category, a suspected diagnosis was excluded or determined to be a new or unsuspected diagnosis in 98/500 (19.6%) of all cases. Additionally, follow-up genetic testing was recommended for 31/500 (6.2%).Conclusion: In half (50.8%) of all cases, genetic testing provided new or additional diagnostic information that was not apparent on protein-based diagnostic methods. Genetic testing excluded or substantially changed a suspected diagnosis in one-quarter (19.6%) of all cases. Therefore, genetic testing for hemoglobinopathies has very high clinical utility, and we propose that it should be considered a standard of care for all patients with known or suspected hemoglobinopathies. DisclosuresQuinn:Amgen: Research Funding; Eli Lilly: Research Funding; MAST Therapeutics: Research Funding; Glycomimetics: Research Funding; Silverlake Research: Consultancy. Begtrup:GeneDx: Employment.

Graft Failure after Hematopoietic Cell Transplantation for Hemoglobin Disorders: Successful Application of a Rescue Regimen

BackgroundAlthough patients with clinically significant hemoglobinopathies can be supported with blood transfusions and iron chelation therapy, the only curative option is allogeneic hematopoietic stem cell transplantation (HCT). Unfortunately, there is a risk of graft rejection as a consequence of donor sensitization and alloimmunization related to transfusion exposures and a hyperproliferative marrow niche. Typically, graft rejection after HCT for hemoglobinopathies is accompanied by autologous hematopoietic recovery, but there is also a risk of graft failure and marrow aplasia which can cause a fatal outcome. To accommodate individuals who lack a suitable sibling donor, transplant options are being expanded to include alternative sources of hematopoietic donors which carry higher risks of graft rejection, graft-versus-host disease and transplant-related mortality. Given the significant risk of graft failure after alternative donor HCT, it is important to develop a plan for stem cell rescue when these complications occur.Methods and ResultsWe reviewed 3 patients with hemoglobin disorders who were successfully rescued by alternative transplantation regimens following primary or secondary graft failures. Two patients with sickle cell disease and one with β-thalassemia major were treated at our institution with a reduced-intensity regimen. All 3 patients had graft rejection accompanied by marrow aplasia within 2 months after HCT. Patient 1 was successfully rescued with mobilized peripheral blood stem cells from the same HLA mismatched sibling donor after a minimal toxicity preparative regimen. Patients 2 and 3 were successfully rescued from HLA-haploidentical donors after a minimal toxicity preparative regimen that included in vivo T-cell depletion by post-grafting high dose cyclophosphamide. Refer to Table 1 for patient and transplant characteristics.ConclusionThere are 3 methods to rescue patients from graft rejection: donor lymphocyte infusions, the intensification of immunosuppression or a rescue allogeneic transplant. Here we report our local experience with graft failure and its successful management in 3 patients with hemoglobin disorders. Although graft rejection remains a major obstacle to successful transplantation for hemoglobinopathies, better donor selection and screening by lymphocyte cross matching, optimized conditioning regimens and improved supportive care should improve outcomes after alternate donor HCT. We speculate that it is also possible now to rescue from graft failure in most cases and that an allogeneic HCT rescue might be utilized for a curative outcome.Table 1Patient characteristics, rescue transplant regimen, engraftment, graft-versus-host-disease (GVHD), and duration of immune suppression post-rescue HCT. BM, Bone Marrow; UCB, Umbilical Cord Blood; ATG, Anti-thymocyte Globulin; HLA, Human Leukocyte Antigen; MMF, Mycophenolate Mofetil; TBI, Total Body IrradiationPatient 1Patient 2Patient 3Age, Gender14 yo Male3 ½ yo Male14 yo MaleDiagnosisHemoglobin SSβ-Thalassemia majorHemoglobin SSIndication for TransplantSubarachnoid Hemorrhage Veno-occlusive crisesAlternative to life-long transfusions and iron chelation therapyMultiple Cerebral infarcts Acute Chest Syndrome Veno-occlusive crisesTransplant #1 Conditioning RegimenBusulfan Thiotepa Cyclophosphamide Rabbit ATGBusulfan Thiotepa Fludarabine Rabbit ATGBusulfan Fludarabine Rabbit ATGTransplant #1 Cell SourceBM, 9/10 HLA mismatch at A locusSingle 6/6 HLA matched UCBBM, 10/10 HLA matched siblingGVHD Prophylaxis for Transplant #1Cyclosporine MMFCyclosporine MethotrexateType of Graft FailureSecondaryPrimarySecondaryRescue Transplant Conditioning RegimenFludarabine 30mg/m2 on days -4 through -2; TBI 400cGy on day 0Fludarabine 30 mg/m2 on days -6 through -2; TBI 400cGy on day 0; Cyclophosphamide 14.5 mg/kg/day on days -6, -5; and 50 mg/kg/day on days +3,+4Recue Transplant Cell SourceSame donor, Mobilized PBSCHaploidentical motherHaploidentical fatherRescue GVHD ProphylaxisTacrolimus MMFTime to Neutrophil Engraftment (days)141415Time to Platelet Engraftment (days)182522Day 28 Percent Donor Cells98%100%99%Acute GVHDNoneSkinNoneChronic GVHDNoneOral and Integument (resolved)Pericardial effusion (resolved)Duration of Post-Rescue Transplant Immune Suppression17 months37 months16 months DisclosuresNo relevant conflicts of interest to declare.

Antenatal haemoglobinopathy screening: Patterns within a large obstetric service. Working towards a standard of care.

Antenatal screening can predict clinically significant haemoglobinopathies, however in Australia, practices are not standardised and are evolving as the population becomes more ethnically diverse. This study describes antenatal screening practices in a large Australian laboratory/antenatal service. Data were collected retrospectively on consecutive antenatal haemoglobin electrophoresis over 16 months and correlated with obstetric data, obtained from the local obstetric database. 462 patients were included, with an average gestation of 25.8 weeks. 'Pregnancy' was the most common stated indication, with absent indication/clinical information in 8%. Gestational age was documented in 54%. In 15%, no contact details of the referrer were documented and partner screening was traceable in only 25 cases (5.4%). In 82% of cases, no abnormalities were detected. Beta thalassemia trait was the most common positive result. Only 52% of patients had recent iron studies. The mean haemoglobin was 111.6 g/L and mean cell volume was 80.5 fl at the time of testing. Ethnicity was documented on the request form in 3%. After Australasia, the most common ethnicity of patients was South East Asia and the Middle East. Referral patterns in our health service are diverse and reflect our changing population and care practices. Detailed guidelines are required and we propose a comprehensive algorithm for general use where selective screening is practiced within an Australasian population or one with similar demographics.

Mass spectrometric analysis of unknown haemoglobin fractions identified by high-performance liquid chromatography in an antenatal screening programme

AimsThe majority of haematology laboratories use automated high-performance liquid chromatography (HPLC) as a primary method in the screening of samples for haemoglobin variants and thalassaemia. HPLC detects adducted fractions, such...

Characterizing a Cohort of α-Thalassemia Couples Collected During Screening for Hemoglobinopathies: 14 Years of an Iranian Experience

Our study aimed to determine the number of couples with normal hemoglobin (Hb) electrophoresis and low-borderline hematological values, which may come up with a clinically critical status in their offspring. The number of couples at risk for severe α-thalassemia (α-thal) needed to be estimated before recommending genetic counseling and prenatal diagnosis (PND). During the past 14 years, from at least 7000 referrals, 754 couples were investigated for α-thal by direct mutation detection methods followed by reverse strip assay and α-globin gene sequencing for inconclusive cases. Detection of silent β-thalassemia (β-thal) mutations was done in suspected cases by complete β-globin gene sequencing. We were able to provide a molecular diagnosis in 87.3% (658/754) of couples. A total of 9.1% (60/658) may have a clinically significant hemoglobinopathy in their offspring. Significant conditions included hydrops fetalis (20.0%; 12/60), certain Hb H (β4) genotypes (78.3%; 47/60) and β-thal intermedia (β-TI) (1.7%; 1/60). The diagnostic flowchart for couples with microcytic hypochromic anemia in countries with a high prevalence of hemoglobinopathies should include α and β gene sequencing. As our results indicate, every nine out of 100 of these couples will face significant hemoglobinopathies and every two out of 100 can carry Hb Bart’s (γ4) hydrops fetalis in their pregnancies. For such cases, PND should be utilized to allow the carrier couples to decide whether or not to abort the fetus.

Testing for haemoglobinopathies in Johannesburg, South Africa: A 30-year review

Haemoglobinopathies are seen mostly in regions where malaria occurs or has occurred, but population migration has resulted in affected individuals being identified in many countries globally. The first molecular genetics services for diagnostic testing and prenatal diagnosis were established, both worldwide and in South Africa (SA), for haemoglobinopathies. To analyse the diagnostic service offered by the Division of Human Genetics, National Health Laboratory Service and University of the Witwatersrand, from 1983 to 2012. A retrospective file analysis (N=1 249) was performed for all individuals who had molecular genetic testing for α-thalassaemia, β-thalassaemia and sickle cell anaemia to examine indications for testing, population origins of patients and molecular genetics findings. The α-thalassaemia testing was requested predominantly to explain microcytic hypochromic haematological indices. Five α-globin deletions were identified, the most common being the -α3.7, in individuals of different ethnicities. For β-thalassaemia and sickle cell anaemia, most testing was performed for prenatal diagnosis purposes. For sickle cell anaemia, most prenatal tests were requested by African families. The β-thalassaemia families were mostly of Indian or Mediterranean origin. The most common mutation identified in all Indian groups was IVS1 nt5 (G>C) (c.92+5G>C) and in individuals from the Mediterranean, IVS1 nt110 (G>A) (c.93-21G>A). The molecular genetics service for haemoglobinopathies in SA is comprehensive and specific to the needs of local ethnic groups. Clinically significant haemoglobinopathies occur at significant frequencies in specific high-risk ethnic groups. Appropriate screening programmes should be initiated so that genetic counselling and reproductive options can be offered.