A new high-prevalence LW antigen detected by an antibody in an Indigenous Australian homozygous for LW*A c.309C>A variant.

BACKGROUND AND OBJECTIVES: The LW gene encodes the LW glycoprotein that carries the antigens of the LW blood group system. LW antigens are distinct from D antigen, however, they are phenotypically related and anti-LW antibodies are often mistaken as anti-D. An antibody was detected in an Australian patient of Aboriginal descent who consistently typed as LW(a+b-). This study aimed to describe the antibody recognizing a high-prevalence antigen on the LW glycoprotein. STUDY DESIGN AND METHODS: Samples from the patient and her four siblings were investigated. DNA was genotyped by single nucleotide polymorphism (SNP)-microarray and massively parallel sequencing (MPS) platforms. Red blood cells (RBCs) were phenotyped using standard haemagglutination techniques. Antibody investigations were performed using a panel of phenotyped RBCs from adults and cord blood cells. RESULTS: SNP-microarray and MPS genotyped all family members as LW*A/A, (c.299A), predicting LW(a+b-). In addition, a novel LW*A c.309C>A single nucleotide variant was detected in all family members. The patient and one of her siblings (M4) were LW c.309C>A homozygous. Antibody from the patient reacted positive to all reagent panel RBCs and cord blood cells but negative with RBCs from LW(a-b-), Rhnull and sibling M4. Antibody failed to react with RBCs treated with dithiothreitol. CONCLUSION: Antibody detected in the patient recognized a novel high-prevalence antigen, LWEM, in the LW blood group system. LWEM-negative patients who developed anti-LWEM can be safely transfused with D+ RBCs, however, D- is preferred. Accurate antibody identification can help better manage allocation of blood products especially when D- RBCs are in short supply.

ABO serology in a case of persistent weak A in a recipient following a group O-matched unrelated bone marrow transplant.

CONCLUSIONS: HLA-matched hematopoietic stem cell transplantation (HSCT) from red blood cell (RBC)-incompatible donors is not uncommon. The engraftment process following ABO-incompatible allogeneic HSCT results in the transition from patient blood group to donor blood group in the recipient. In contrast, most non-hematopoietic tissues retain expression of the patient's original blood group for life, and these antigens may adsorb from the plasma onto the donor-derived RBCs. Correct serologic interpretation of the ABO blood group during this engraftment process can be difficult. We present the serologic findings of a 15-year-old girl of Maori descent, who was diagnosed with acute myeloid leukemia and transplanted with an HLA-matched unrelated group O, D+ bone marrow. Despite engraftment, her RBCs showed persistence of weak A. This case report showcases the importance of awareness and correct serologic interpretation of weak persistence of recipient ABH substance on the patient's RBCs for clinical decision-making, blood component support, and patient wellbeing.

α-thalassemia trait caused by frameshift mutations in exon 2 of the α2-globin gene: HBA2:c.131delT and HBA2:c.143delA.

We describe two frameshift mutations associated with an α-thalassemia (α-thal) phenotype, identified in three unrelated individuals investigated for persistent microcytosis. The first mutation, HBA2:c.131delT, is located in codon 43, and the second, HBA2:c.143delA, is located in codon 47. Both are due to single base pair deletions that cause a frameshift and a premature termination codon (PTC) at positions 48/49. The presence of a PTC at this position has been documented to result in nonsense mediated mRNA decay that would account for the thalassemic phenotype.

A molecular tool to assess the pathological relevance of alpha-globin DNA variants.

AIM: While the phenotype for heterozygous beta-thalassaemia is straightforward, it is more difficult to confirm a causative relationship for mutations in the alpha-globin genes. The aim of this study was to generate an in vitro system to evaluate the pathological relevance of α-globin mutations. METHODS: The novel variant HBA1:c.301-3C>G was used as a model. In silico analysis predicted an aberrant acceptor splice site in the mutant sequence. Subsequent in vitro studies included generation of and transfection of an expression vector carrying the HBA1:c.301-3C>G mutation, RNA purification, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA sequencing. Immunofluorochemistry (IFC) with antibodies specific to the N- and or C- terminal of the α-globin protein was used in protein detection. RESULTS: In vitro molecular characterisation of this point mutation confirmed the preferential utilisation of a cryptic splice site at intron 2 of the pre-mRNA, resulting in a shift in the reading frame causing a premature termination codon (PTC) at codons 101/102 and generation of a truncated protein. CONCLUSION: We have described here a molecular tool to study mutations that affect α-globin pre-mRNA splicing and translation. We confirm in silico predictions of the consequences of the HBA1:c.301-3C>G mutation, proving aberrant RNA splicing and the production of a truncated α-globin protein.

Molecular and cellular characterization of a new α-thalassemia mutation (HBA2:c.94A>C) generating an alternative splice site and a premature stop codon.

The identification of α-thalassemia (α-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the α2-globin gene, causing a mild α-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of α-thal.

Hb Lynwood [α107(G14) (-T) (α2) HBA2:c.323delT)] in conjunction with the α(3.7) deletion produces a moderately severe α-thalassemia phenotype.

We describe a novel frameshift mutation associated with an α-thalassemia (α-thal) phenotype in a patient of Sudanese origin investigated for persistent microcytosis. In addition to the α(3.7) deletion, a novel mutation on the α2 gene was detected: HBA2:c.323delT. This mutation causes a frameshift at codon 107 of the α2 gene. The result is a disturbed amino acid sequence for the following 24 amino acids, and a premature termination codon at position 132.

Hb East Timor [ß80(EF4)Asn→His, AAC>CAC (HBB c.241A>C)], a variant hemoglobin associated with normal hematology.

Routine hemoglobin (Hb) analyses identified a new ß-globin variant in a family from East Timor. The red cell indices were within normal limits for all affected family members. The variant is due to a missense mutation at amino acid codon 80 (AAC>CAC) which results in the substitution of histidine for asparagine.

Moderate exposure to allogeneic blood products is not associated with reduced long-term survival after surgery for coronary artery disease.

BACKGROUND: It has been suggested that blood transfusion has an adverse effect on long-term health, mainly through immune modulation and tumor promotion. To further assess this concern, the authors have performed a prospective observational study with the hypothesis that after taking perioperative risk factors relevant to long-term survival into account, patients undergoing coronary artery surgery who receive a perioperative allogeneic blood transfusion have worse long-term survival than those who do not. METHODS: The health outcomes of 1,841 consecutive subjects who had isolated nonemergency first-time coronary artery surgery and who survived more than 60 days after surgery were determined by record linkage. The association between length of survival, blood products transfused, and risk factors for long-term survival at entry to the study were determined by Cox proportional hazards regression. RESULTS: A total of 1,062 subjects were transfused. Of these, 266 subjects died during a mean follow-up of 8.1 yr. Of subjects who were transfused, 27% had a new malignant condition recorded on the death certificate, compared with 43% who were not transfused. Older age, cerebrovascular disease, use of a mammary graft, chronic pulmonary disease, renal dysfunction, reduced left ventricular function, and preoperative anemia were predictive of reduced long-term survival. There was no association between transfusion of blood products and long-term survival. CONCLUSIONS: Patients who have undergone coronary artery surgery and who have received moderate amounts of blood as part of responsible and conservative management should be reassured that they are unlikely to experience a reduction in long-term survival.

Postpartum anti-D: can we safely reduce the dose?

OBJECTIVE: To assess the potential for dose-reduction of prophylactic anti-D postpartum. DESIGN: Retrospective audit of fetomaternal haemorrhage (FMH) quantitation by flow cytometry. PARTICIPANTS AND SETTING: 5148 consecutive Rhesus D-negative women aged 15-45 years who had FMH estimation by flow cytometry at a central laboratory in Western Australia in the 65 months between 1 August 1999 and 31 January 2005. MAIN OUTCOME MEASURES: Quantitation of FMH volume for adequate prophylactic anti-D administration in a timely fashion. RESULTS: 90.4% (4651/5148) of the women had an FMH volume of 1.0 mL or less of Rh D-positive red cells, and 98.5% (5072/5148) had a volume of less than 2.5 mL. Only 0.4% of cases had an FMH volume of 6.0 mL or greater (range, 6.0-92.4 mL). CONCLUSIONS: This large retrospective audit shows that a currently available dose of 250 IU (50 mg) of anti-D would have been sufficient for 98.5% of the 5148 Rh D-negative women. On the basis of this evidence, a reduction in the recommended routine postpartum dose of anti-D from 625 IU to 250 IU when flow cytometric quantitation for FMH is available should be considered. Adopting such a strategy would ensure the ongoing provision of a valuable human blood product currently in limited supply.