Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

Genomic characterisation of clinically significant blood group variants in Aboriginal Australians.

BACKGROUND: Hematological disorders are often treated with blood transfusions. Many blood group antigens and variants are population-specific, and for patients with rare blood types, extensive donor screening is required to find suitable matches for transfusion. There is a scarcity of knowledge regarding blood group variants in Aboriginal Australian populations, despite a higher need for transfusion due to the higher prevalence of renal diseases and anaemia. MATERIALS AND METHODS: In this study, we applied next-generation sequencing and analysis to 245 samples obtained from Aboriginal Australians from South-East Queensland, to predict antigen phenotypes for 36 blood group systems. RESULTS: We report potential weak antigens in blood group systems RH, FY and JR that have potential clinical implications in transfusion and pregnancy settings. These include partial DIII type 4, weak D type 33, and Del RHD (IVS2-2delA). The rare Rh phenotypes D+ C+ E+ c- e+ and D+ C+ E+ c+ e- were also detected. DISCUSSION: The comprehensive analyses of blood group genetic variant profiles identified in this study will provide insight and an opportunity to improve Aboriginal health by aiding in the identification of appropriate blood products for population-specific transfusion needs.

A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

BACKGROUND: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. RESULTS: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. DISCUSSION: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. CONCLUSION: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.

Recurrent pregnancy loss in a patient with anti-Rh17.

BACKGROUND: Rh is one of the most important blood group systems in transfusion medicine. The two homologous genes RHD and RHCE are located on chromosome 1p36.11 and encode for RhD and RhCE proteins, respectively. Complex genetic polymorphisms result in a variety of antigenic expression of D, C, E, c, and e. Here, we describe a case of a young female with D-- who developed anti-Rh17 secondary to blood transfusion and had signs of haemolytic disease of the fetus and fetal death in five consecutive pregnancies. CASE DESCRIPTION: EDTA-whole blood samples were collected from the patient, husband and eight siblings for blood grouping, phenotyping, and red cell antibody screening. Extracted DNA was genotyped by SNP-microarray and massively parallel sequencing (MPS) with targeted blood group exome sequencing. Copy number variation analysis was performed to identify structural variants in the RHD and RHCE. Routine phenotyping showed all family members were D+. The patient's red blood cells were C-E-c-e-, Rh17- and Rh46- and had anti-Rh17 and anti-e antibodies. MPS showed the patient carried a wildtype RHD sequence and homozygous for RHCE (1)-D (2-9)-CE (10) hybrid gene predicted to express a D-- phenotype. CONCLUSIONS: Our patient had a rare D-- phenotype and confirmed to have RHCE/RHD hybrid gene with replacement of 2-9 exons of RHCE by RHD sequences. Unfortunately, our patient developed anti-Rh17 and anti-e antibodies due to blood transfusion and suffered fetal demise in her very first pregnancy. The adverse outcomes could have been prevented by active prenatal management.

Feasibility for non-invasive prenatal fetal blood group and platelet genotyping by massively parallel sequencing: A single test system for multiple atypical red cell, platelet and quality control markers.

Non-invasive prenatal tests (NIPT) to predict fetal red cell or platelet antigen status for alloimmunised women are provided for select antigens. This study reports on massively parallel sequencing (MPS) using a red cell and platelet probe panel targeting multiple nucleotide variants, plus individual identification single nucleotide polymorphisms (IISNPs). Maternal blood samples were provided from 33 alloimmunised cases, including seven with two red cell antibodies. Cell-free and genomic DNA was sequenced using targeted MPS and bioinformatically analysed using low-frequency variant detection. The resulting maternal genomic DNA allele frequency was subtracted from the cell-free DNA counterpart. Outcomes were matched against validated phenotyping/genotyping methods, where available. A 2.5% subtractive allele frequency threshold was set after comparing MPS predictions for K, RhC/c, RhE/e and Fya /Fyb against expected outcomes. This threshold was used for subsequent predictions, including HPA-15a, Jka /Jkb , Kpa /Kpb and Lua . MPS outcomes were 97.2% concordant with validated methods; one RhC case was discordantly negative and lacked IISNPs. IISNPs were informative for 30/33 cases as controls. NIPT MPS is feasible for fetal blood group genotyping and covers multiple blood groups and control targets in a single test. Noting caution for the Rh system, this has the potential to provide a personalised service for alloimmunised women.

Genetic Characterization of Blood Group Antigens for Polynesian Heritage Norfolk Island Residents.

Improvements in blood group genotyping methods have allowed large scale population-based blood group genetics studies, facilitating the discovery of rare blood group antigens. Norfolk Island, an external and isolated territory of Australia, is one example of an underrepresented segment of the broader Australian population. Our study utilized whole genome sequencing data to characterize 43 blood group systems in 108 Norfolk Island residents. Blood group genotypes and phenotypes across the 43 systems were predicted using RBCeq. Predicted frequencies were compared to data available from the 1000G project. Additional copy number variation analysis was performed, investigating deletions outside of RHCE, RHD, and MNS systems. Examination of the ABO blood group system predicted a higher distribution of group A1 (45.37%) compared to group O (35.19%) in residents of the Norfolk Island group, similar to the distribution within European populations (42.94% and 38.97%, respectively). Examination of the Kidd blood group system demonstrated an increased prevalence of variants encoding the weakened Kidd phenotype at a combined prevalence of 12.04%, which is higher than that of the European population (5.96%) but lower than other populations in 1000G. Copy number variation analysis showed deletions within the Chido/Rodgers and ABO blood group systems. This study is the first step towards understanding blood group genotype and antigen distribution on Norfolk Island.

Investigation of the effect of pre-analytical factors on particle concentration and size in cryoprecipitate using nanoparticle tracking analysis.

BACKGROUND: Cryoprecipitate is used primarily to replenish fibrinogen levels in patients. Little is known about the presence of micro- or nano-sized particles in cryoprecipitate. Therefore, we aimed to quantify these particles and investigate some pre-analytical considerations. MATERIALS AND METHODS: Particle concentration and size distribution were determined in 10 cryoprecipitate units by nanoparticle tracking analysis (NTA). The effects of freeze-thawing cryoprecipitate and 0.45 µm filtration with either regenerated cellulose (RC) or polytetrafluoroethylene (PTFE) filters before sample analysis were examined. RESULTS: Neither the size nor concentration of particles were affected by two freeze/thaw cycles. PTFE filtration, but not RC filtration, significantly reduced particle mean and mode size compared to RC filtration and mode size compared to unfiltered cryoprecipitate. The 10 cryoprecipitate units had an average particle concentration of 2.50 × 1011 ± 1.10 × 1011 particles/mL, a mean particle size of 133.8 ± 7.5 nm and a mode particle size of 107.9 ± 11.1 nm. CONCLUSION: This study demonstrated that preanalytical filtration of cryoprecipitate units using RC filters was suitable for NTA. An additional freeze/thaw cycle did not impact NTA parameters, suggesting that aliquoting cryoprecipitate units prior to laboratory investigations is suitable for downstream analyses.

Next generation sequencing to identify iron status and individualise blood donors' experience.

BACKGROUND: Young adults form the majority of first-time blood donors to Australian Red Cross Lifeblood. However, these donors pose unique challenges for donor safety. Young blood donors, who are still undergoing neurological and physical development, have been found to have lower iron stores, and have higher risks of iron deficiency anaemia when compared to older adults and non-donors. Identifying young donors with higher iron stores may improve donor health and experience, increase donor retention, and reduce the burden on product donation. In addition, these measures could be used to individualise donation frequency. MATERIALS AND METHODS: Stored DNA samples from young male donors (18-25 years; No.=47) were sequenced using a custom panel of genes identified in the literature to be associated with iron homeostasis. The custom sequencing panel used in this study identified and reported variants to human genome version 19 (Hg19). RESULTS: 82 gene variants were analysed. Only one of which, rs8177181, was found to have a statistically significant (p<0.05) association with plasma ferritin level. Heterozygous alleles of this Transferrin gene variant, rs8177181T>A, significantly predicted a positive effect on ferritin levels (p=0.03). DISCUSSION: This study identified gene variants involved in iron homeostasis using a custom sequencing panel and analysed their association with ferritin levels in a young male blood donor population. Additional studies of factors associated with iron deficiency in blood donors are required if a goal of personalised blood donation protocols is to be achieved.

Hemolytic disease of the fetus and newborn caused by anti-sD antibody in a GP.Mur/Mur Thai mother and review of the prevalence of sD in Thai blood donors.

BACKGROUND: Low-prevalence antigen sD (MNS23) is encoded by GYPB c.173C > G. Hemolytic disease of the fetus and newborn (HDFN) due to anti-sD is rare. A mother delivered a newborn whose red blood cells (RBCs) were DAT-positive and was later diagnosed with HDFN. Serum from the mother was incompatible with the father's RBCs and was used to screen 184 Thai blood donors. This study aimed to investigate the cause of HDFN in a Thai family and determine the prevalence of sD in Thai blood donors. MATERIALS AND METHODS: Three family members and four blood donors were investigated in the study. Massively Parallel Sequencing (MPS) was used for genotyping. Standard hemagglutination techniques were used in titration studies, phenotyping, and enzyme/chemical studies. Anti-s, anti-Mia , anti-JENU, and anti-sD reagents were used in serological investigations. RESULTS: The mother was GYP*Mur/Mur. The father and the four donors were GYPB*s/sD predicting S - s + sD +. The baby was GYP*Mur/sD and his RBCs were Mia +, s + w with anti-s (P3BER) and JENU+w . RBCs from two GYPB*sD -positive blood donors reacted with anti-sD (Dreyer). Proteolytic enzyme α-chymotrypsin-treated sD + cells did not react with anti-sD (Wat) produced by the GP.Mur/Mur mother but reacted with the original anti-sD (Dreyer). DISCUSSION: This is the first report of HDFN due to anti-sD in the Asian population. The genotype frequency for GYPB*sD in a selected Thai blood donor population is 2.2% (4/184). Anti-sD should be considered in mothers with Southeast Asian or East Asian background when antibody identification is unresolved in pregnancies affected by HDFN.

The genomic landscape of blood groups in Indigenous Australians in remote communities.

BACKGROUND: Red blood cell (RBC) membrane-associated blood group systems are clinically significant. Alloimmunisation is a persistent risk associated with blood transfusion owing to the antigen polymorphisms among these RBC-associated blood groups. Next-generation sequencing (NGS) offers an opportunity to characterize the blood group variant profile of a given individual. Australia comprises a large multiethnic population where most blood donors are Caucasian and blood group variants remain poorly studied among Indigenous Australians. In this study, we focused on the Tiwi Islanders, who have lived in relative isolation for thousands of years. METHODS AND MATERIALS: We predicted the blood group phenotype profiles in the Tiwi (457) and 1000 Genomes Phase 3 (1KGP3-2504) cohort individuals using RBCeq (https://www.rbceq.org/). The predicted phenotype prevalence was compared with the previous literature report. RESULTS: We report, for the first time, comprehensive blood group profiles corresponding to the 35 known blood group systems among the Indigenous Tiwi islander population and identify possible novel antigen variants therein. Our results demonstrate that the genetic makeup of the Tiwi participants is distinct from that of other populations, with a low prevalence of LU (Au[a-b+]) and ABO (A2) and D+C+c+E+e- phenotype, an absence of Diego blood group variants, and a unique RHD (DIII type4) variant. CONCLUSION: Our results may contribute to the development of a database of predicted phenotype donors among the Tiwi population and aid in improving transfusion safety for the ~2800 Tiwi people and the ~800,000 other Indigenous Australians throughout the nation.

Using whole-genome sequencing to characterize clinically significant blood groups among healthy older Australians.

There have been no comprehensive studies of a full range of blood group polymorphisms within the Australian population. This problem is compounded by the absence of any databases carrying genomic information on chronically transfused patients and low frequency blood group antigens in Australia. Here, we use RBCeq, a web server-based blood group genotyping software, to identify unique blood group variants among Australians and compare the variation detected vs global data. Whole-genome sequencing data were analyzed for 2796 healthy older Australians from the Medical Genome Reference Bank and compared with data from 1000 Genomes phase 3 (1KGP3) databases comprising 661 African, 347 American, 503 European, 504 East Asian, and 489 South Asian participants. There were 661 rare variants detected in this Australian sample population, including 9 variants that had clinical associations. Notably, we identified 80 variants that were computationally predicted to be novel and deleterious. No clinically significant rare or novel variants were found associated with the genetically complex ABO blood group system. For the Rh blood group system, 2 novel and 15 rare variants were found. Our detailed blood group profiling results provide a starting point for the creation of an Australian blood group variant database.

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.

RBCeq: A robust and scalable algorithm for accurate genetic blood typing.

BACKGROUND: While blood transfusion is an essential cornerstone of hematological care, patients requiring repetitive transfusion remain at persistent risk of alloimmunization due to the diversity of human blood group polymorphisms. Despite the promise, user friendly methods to accurately identify blood types from next-generation sequencing data are currently lacking. To address this unmet need, we have developed RBCeq, a novel genetic blood typing algorithm to accurately identify 36 blood group systems. METHODS: RBCeq can predict complex blood groups such as RH, and ABO that require identification of small indels and copy number variants. RBCeq also reports clinically significant, rare, and novel variants with potential clinical relevance that may lead to the identification of novel blood group alleles. FINDINGS: The RBCeq algorithm demonstrated 99·07% concordance when validated on 402 samples which included 29 antigens with serology and 9 antigens with SNP-array validation in 14 blood group systems and 59 antigens validation on manual predicted phenotype from variant call files. We have also developed a user-friendly web server that generates detailed blood typing reports with advanced visualization (https://www.rbceq.org/). INTERPRETATION: RBCeq will assist blood banks and immunohematology laboratories by overcoming existing methodological limitations like scalability, reproducibility, and accuracy when genotyping and phenotyping in multi-ethnic populations. This Amazon Web Services (AWS) cloud based platform has the potential to reduce pre-transfusion testing time and to increase sample processing throughput, ultimately improving quality of patient care. FUNDING: This work was supported in part by Advance Queensland Research Fellowship, MRFF Genomics Health Futures Mission (76,757), and the Australian Red Cross LifeBlood. The Australian governments fund the Australian Red Cross Lifeblood for the provision of blood, blood products and services to the Australian community.

Past and future epidemic potential of chikungunya virus in Australia.

BACKGROUND: Australia is theoretically at risk of epidemic chikungunya virus (CHIKV) activity as the principal vectors are present on the mainland Aedes aegypti) and some islands of the Torres Strait (Ae. aegypti and Ae. albopictus). Both vectors are highly invasive and adapted to urban environments with a capacity to expand their distributions into south-east Queensland and other states in Australia. We sought to estimate the epidemic potential of CHIKV, which is not currently endemic in Australia, by considering exclusively transmission by the established vector in Australia, Ae. aegypti, due to the historical relevance and anthropophilic nature of the vector. METHODOLOGY/PRINCIPAL FINDINGS: We estimated the historical (1995-2019) epidemic potential of CHIKV in eleven Australian locations, including the Torres Strait, using a basic reproduction number equation. We found that the main urban centres of Northern Australia could sustain an epidemic of CHIKV. We then estimated future trends in epidemic potential for the main centres for the years 2020 to 2029. We also conducted uncertainty and sensitivity analyses on the variables comprising the basic reproduction number and found high sensitivity to mosquito population size, human population size, impact of vector control and human infectious period. CONCLUSIONS/SIGNIFICANCE: By estimating the epidemic potential for CHIKV transmission on mainland Australia and the Torres Strait, we identified key areas of focus for controlling vector populations and reducing human exposure. As the epidemic potential of the virus is estimated to rise towards 2029, a greater focus on control and prevention measures should be implemented in at-risk locations.

Low Genetic Diversity of Hepatitis B Virus Surface Gene amongst Australian Blood Donors.

Variants in the small surface gene of hepatitis B virus (HBV), which codes for viral surface antigen (HBsAg), can affect the efficacy of HBsAg screening assays and can be associated with occult HBV infection (OBI). This study aimed to characterise the molecular diversity of the HBV small surface gene from HBV-reactive Australian blood donors. HBV isolates from 16 HBsAg-positive Australian blood donors' plasma were sequenced and genotyped by phylogenies of viral coding genes and/or whole genomes. An analysis of the genetic diversity of eight HBV small surface genes from our 16 samples was conducted and compared with HBV sequences from NCBI of 164 international (non-Australian) blood donors. Genotypes A-D were identified in our samples. The region of HBV small surface gene that contained the sequence encoding the 'a' determinant had a greater genetic diversity than the remaining part of the gene. No escape mutants or OBI-related variants were observed in our samples. Variant call analysis revealed two samples with a nucleotide deletion leading to truncation of polymerase and/or large/middle surface amino acid sequences. Overall, we found that HBV small surface gene sequences from Australian donors demonstrated a lower level of genetic diversity than those from non-Australian donor population included in the study.

Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display.

BACKGROUND: RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display. MATERIAL AND METHODS: Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count. RESULTS: Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity. DISCUSSION: BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.

Non-invasive prenatal testing for management of haemolytic disease of the fetus and newborn induced by maternal alloimmunisation.

Anti-D immunoglobulin prophylaxis reduces the risk of RhD negative women becoming alloimmunised to the RhD antigen and is a major preventative strategy in reducing the burden of haemolytic disease of the fetus and newborn (HDFN). HDFN also arises from other maternal red cell antibodies, with the most clinically significant, after anti-D, being anti-K, anti-c and anti-E. Among the 39 human blood group systems advanced genomic technologies are still revealing novel or rare antigens involved in maternal alloimmunisation. Where clinically significant maternal antibodies are detected in pregnancy, non-invasive prenatal testing (NIPT) of cell-free fetal DNA provides a safe way to assess the fetal blood group antigen status. This provides information as to the risk for HDFN and thus guides management strategies. In many countries, NIPT fetal RHD genotyping as a diagnostic test using real-time PCR has already been integrated into routine clinical care for the management of women with allo-anti-D to assess the risk for HDFN. In addition, screening programs have been established to provide antenatal assessment of the fetal RHD genotype in non-alloimmunised RhD negative pregnant women to target anti-D prophylaxis to those predicted to be carrying an RhD positive baby. Both diagnostic and screening assays exhibit high accuracy (over 99 %). NIPT fetal genotyping for atypical (other than RhD) blood group antigens presents more challenges as most arise from a single nucleotide variant. Recent studies show potential for genomic and digital technologies to provide a personalised medicine approach with NIPT to assess fetal blood group status for women with other (non-D) red cell antibodies to manage the risk for HDFN.

Frequency of Mia (MNS7) and Classification of Mia-Positive Hybrid Glycophorins in an Australian Blood Donor Population.

BACKGROUND: MNS blood group system genes GYPA and GYPB share a high degree of sequence homology and gene structure. Homologous exchanges between GYPA and GYPB form hybrid genes encoding hybrid glycophorins GP(A-B-A) and GP(B-A-B). Over 20 hybrid glycophorins have been characterised. Each has a distinct phenotype defined by the profile of antigens expressed including Mia. Seven hybrid glycophorins carry Mia and have been reported in Caucasian and Asian population groups. In Australia, the population is diverse; however, the prevalence of hybrid glycophorins in the population has never been determined. The aims of this study were to determine the frequency of Mia and to classify Mia-positive hybrid glycophorins in an Australian blood donor population. METHOD: Blood samples from 5,098 Australian blood donors were randomly selected and screened for Mia using anti-Mia monoclonal antibody (CBC-172) by standard haemagglutination technique. Mia-positive red blood cells (RBCs) were further characterised using a panel of phenotyping reagents. Genotyping by high-resolution melting analysis and DNA sequencing were used to confirm serology. RESULT: RBCs from 11/5,098 samples were Mia-positive, representing a frequency of 0.22%. Serological and molecular typing identified four types of Mia-positive hybrid glycophorins: GP.Hut (n = 2), GP.Vw (n = 3), GP.Mur (n = 5), and 1 GP.Bun (n = 1). GP.Mur was the most common. CONCLUSION: This is the first comprehensive study on the frequency of Mia and types of hybrid glycophorins present in an Australian blood donor population. The demographics of Australia are diverse and ever-changing. Knowing the blood group profile in a population is essential to manage transfusion needs.

Estimation of mosquito-borne and sexual transmission of Zika virus in Australia: Risks to blood transfusion safety.

BACKGROUND: Since 2015, Zika virus (ZIKV) outbreaks have occurred in the Americas and the Pacific involving mosquito-borne and sexual transmission. ZIKV has also emerged as a risk to global blood transfusion safety. Aedes aegypti, a mosquito well established in north and some parts of central and southern Queensland, Australia, transmits ZIKV. Aedes albopictus, another potential ZIKV vector, is a threat to mainland Australia. Since these conditions create the potential for local transmission in Australia and a possible uncertainty in the effectiveness of blood donor risk-mitigation programs, we investigated the possible impact of mosquito-borne and sexual transmission of ZIKV in Australia on local blood transfusion safety. METHODOLOGY/PRINCIPAL FINDINGS: We estimated 'best-' and 'worst-' case scenarios of monthly reproduction number (R0) for both transmission pathways of ZIKV from 1996-2015 in 11 urban or regional population centres, by varying epidemiological and entomological estimates. We then estimated the attack rate and subsequent number of infectious people to quantify the ZIKV transfusion-transmission risk using the European Up-Front Risk Assessment Tool. For all scenarios and with both vector species R0 was lower than one for ZIKV transmission. However, a higher risk of a sustained outbreak was estimated for Cairns, Rockhampton, Thursday Island, and theoretically in Darwin during the warmest months of the year. The yearly estimation of the risk of transmitting ZIKV infection by blood transfusion remained low through the study period for all locations, with the highest potential risk estimated in Darwin. CONCLUSIONS/SIGNIFICANCE: Given the increasing demand for plasma products in Australia, the current strategy of restricting donors returning from infectious disease outbreak regions to source plasma collection provides a simple and effective risk management approach. However, if local transmission was suspected in the main urban centres of Australia, potentially facilitated by the geographic range expansion of Ae. aegypti or Ae. albopictus, this mitigation strategy would need urgent review.

Soluble mediators in packed red blood cells augment lipopolysaccharide-induced monocyte interleukin-1ß production.

BACKGROUND AND OBJECTIVES: Soluble mediators in packed red-blood-cell (PRBC) units have been hypothesized as a mechanism associated with transfusion-related immune modulation. Soluble mediators including damage-associated molecular patterns (DAMPs) are known to activate inflammasomes. Inflammasome complexes maturate caspase-1 and interleukin (IL)-1ß. We assessed whether PRBC supernatants (SN) modulated IL-1ß driven inflammation and whether macrophage migration inhibitory factor (MIF) was a contributing factor. MATERIALS AND METHODS: Isolated monocytes were incubated with PRBC-SN in an in vitro transfusion model. Lipopolysaccharide (LPS) was added in parallel to model a bacterial infection. Separately, recombinant MIF was used in the model to assess its role in IL-1ß driven inflammation. IL-1ß and caspase-1 were quantified in the PRBC-SN and culture SN from the in vitro model. RESULTS: PRBC-SN alone did not induce IL-1ß production from monocytes. However, PRBC-SN alone increased caspase-1 production. LPS alone induced both IL-1ß and caspase-1 production. PRBC-SN augmented LPS-driven IL-1ß and caspase-1 production. Recombinant MIF did not modulate IL-1ß production in our model. CONCLUSIONS: Soluble mediators in PRBC modulate monocyte IL-1ß inflammation, which may be a contributing factor to adverse effects of transfusion associated with poor patient outcomes. While MIF was present in PRBC-SN, we found no evidence that MIF was responsible for IL-1ß associated immune modulation.