Broadband dielectric spectroscopy of blood above 50 GHz is scarce due to hardware limitations, affordability, and complexity. This study explores the use of a rectangular waveguide as a probe for analysing human blood in the 55 to 95 GHz range, at temperatures from 23 degrees C to 36 degrees C, and Haemoglobin levels between 0 and 16 g/dL, focusing on practical aspects such as sample preparation and the setup's mechanical, thermal, and calibration requirements. The research highlights the effectiveness of the Short-Open-Load calibration method, evidenced by precise measurements of saline solutions. Furthermore, analysis of 84 human blood samples indicates that while Haemoglobin levels introduce some uncertainty in dielectric constant measurements, dielectric loss readings remain consistent across temperature and Haemoglobin variations, with a low uncertainty of under 2.65%, indicating the potential use of this parameter in future millimetre-wave techniques for non-invasive testing of blood analytes.

BACKGROUND:Transfusion-related acute lung injury (TRALI) remains a major contributor to transfusion-associated mortality. While the pathogenesis of TRALI remains unclear, there is evidence of a role for blood components. We therefore investigated the potential effects of fresh frozen plasma (FFP), cryoprecipitate, and extracellular vesicles (EVs) derived from these blood components, on the viability of human lung microvascular endothelial cells (HLMVECs) in vitro. METHODS:EVs were isolated from FFP and cryoprecipitate using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. The potential effects of these blood components and their EVs on HLMVEC viability (determined by trypan blue exclusion) were examined in the presence and absence of neutrophils, either with or without prior treatment of HLMVECs with LPS. RESULTS:EVs isolated from FFP and cryoprecipitate displayed morphological and biochemical properties conforming to latest international criteria. While FFP, cryoprecipitate, and EVs derived from FFP, each reduced HLMVEC viability, no effect was observed for EVs derived from cryoprecipitate. CONCLUSION:Our findings demonstrate clear differences in the effects of FFP, cryoprecipitate, and their respective EVs on HLMVEC viability in vitro. Examination of the mechanisms underlying these differences may lead to an improved understanding of the factors that promote development of TRALI.

Transfusion-related acute lung injury (TRALI) is a potentially life-threatening adverse transfusion reaction. Many severe cases of TRALI relate to transfusion of antibodies against human neutrophil antigen 3 (HNA-3). Characterization of the HNA-3 antigen on choline transporter-like protein 2 (CTL2) led to the discovery that human anti-HNA-3a alloantibodies show some cross-reactivity with mouse CTL2. This suggested that human anti-HNA-3a antibodies may react with CTL2 from other species. Although small rodent models are crucial for scientific research, their clinical relevance may be limited due to differences in physiology and, as a result, findings obtained from murine models may not be directly applicable to clinical settings. Sheep have previously been used to model non-antibody mediated TRALI and were therefore assessed for their potential to model for anti-HNA-3a mediated TRALI. Sequences for CTL2 and the gene that encodes it ( SLC44A2 ) from humans, mice, and sheep were compared using publically available databases. Binding experiments were performed with human sera containing an antiHNA-3a antibody and human, mouse and sheep cells. Although analysis showed that sheep SLC44A2 and CTL2 shared high homology with both human and mouse counterparts (88-89% gene homology, 95-96% protein homology), including the Arg154 residue responsible the HNA-3a variant, no significant anti-HNA3a alloantibody binding was observed with sheep cells. Therefore, this study suggests that sheep would not be a suitable large animal in which to model TRALI mediated by human anti-HNA-3a antibodies.

Extracellular vesicles (EVs) are a heterogeneous collection of particles that play a crucial role in cell-to-cell communication, primarily due to their ability to transport molecules, such as proteins. Thus, profiling EV-associated proteins offers insight into their biological effects. EVs can be isolated from various biological fluids, including donor blood components such as cryoprecipitate and fresh frozen plasma (FFP). In this study, we conducted a proteomic analysis of five single donor units of cryoprecipitate, FFP, and EVs derived from these blood components using a quantitative mass spectrometry approach. EVs were successfully isolated from both cryoprecipitate and FFP based on community guidelines. We identified and quantified approximately 360 proteins across all sample groups. Principal component analysis and heatmaps revealed that both cryoprecipitate and FFP are similar. Similarly, EVs derived from cryoprecipitate and FFP are comparable. However, they differ between the originating fluids and their derived EVs. Using the R-package MS-DAP, differentially expressed proteins (DEPs) were identified. The DEPs for all comparisons, when submitted for gene enrichment analysis, are involved in the complement and coagulation pathways. The protein profile generated from this study will have important clinical implications in increasing our knowledge of the proteins that are associated with EVs derived from blood components.

Arboviruses pose a significant global public health threat, with Ross River virus (RRV), Barmah Forest virus (BFV), and dengue virus (DENV) being among the most common and clinically significant in Australia. Some arboviruses, including those prevalent in Australia, have been reported to cause transfusion-transmitted infections. This study examined the spatiotemporal variation of these arboviruses and their potential impact on blood donation numbers across Australia. Using data from the Australian Department of Health on eight arboviruses from 2002 to 2017, we retrospectively assessed the distribution and clustering of incidence rates in space and time using Geographic Information System mapping and space–time scan statistics. Regression models were used to investigate how weather variables, their lag months, space, and time affect case and blood donation counts. The predictors’ importance varied with the spatial scale of analysis. Key predictors were average rainfall, minimum temperature, daily temperature variation, and relative humidity. Blood donation number was significantly associated with the incidence rate of all viruses and its interaction with local transmission of DENV, overall. This study, the first to cover eight clinically relevant arboviruses at a fine geographical level in Australia, identifies regions at risk for transmission and provides valuable insights for public health intervention.

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 anemia. 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.

The Rh blood group is one of the most polymorphic and clinically significant blood groups. When the maternal Rh blood group is discordant with that of the fetus, maternal alloantibodies can be produced, resulting in a risk of haemolytic disease of the fetus and newborn (HDFN) in future pregnancies. While red blood cell screening can prevent this, the panels are designed for highly prevalent antigens. A novel low-frequency Rh blood group mutation, CETW (c.486C>G), has been identified. This report aimed to use recombinant protein technology to confirm that alloantibodies in donor serum, associated with HDFN, bind to the CETW variant.

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.

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.

Background: Blood donor history questionnaires are used world-wide to detect blood supply and donor safety risks. Donors are less likely to return to donate blood following deferral, therefore removing questions with limited impact on safety would likely improve donor satisfaction and donation rates. Early human immunodeficiency virus (HIV) infection can manifest as acute retroviral syndrome (ARS). This study aimed to evaluate the impact of questioning and deferring blood donors for self -reported symptoms of ARS on Australian blood transfusion safety. Methods: A retrospective analysis of Australian Red Cross Lifeblood databases for the 17 -year period, 2000-2016, was undertaken. Voluntary Australian blood donors who reported rash and lymphadenopathy on the donor questionnaire (DQ) were determined at risk of ARS and thus HIV. The proportion of donors deferred for possible ARS, the proportion who later returned, and donor return time for ARS and other 12 -month deferrals was assessed. HIV status on subsequent donation in ARS declaring donors and proportion of HIV -positive donors with ARS -like symptoms was also reviewed. Results: Of donors who declared possible ARS, 65.56% [95% confidence interval (CI): 55.74-75.37%] were deferred and only 22.34% (95% CI: 17.40-27.29%) of deferred donors later returned. Compared to other 12 -month deferral categories, donors deferred for possible ARS were less likely to return. No donor who declared possible ARS and subsequently donated tested HIV -positive. Moreover, no donors who tested HIV -positive reported both rash and lymphadenopathy in combination. Conclusions: The ARS question does not effectively differentiate HIV -positive from HIV -negative donors. The continued questioning of donors about ARS, through a self -reported combination of rash and lymphadenopathy, resulted in loss of donors who were unlikely to pose a threat to transfusion safety.

Blood donors can lose up to 200–250 mg of iron per whole blood donation, significantly increasing their risk of iron deficiency and anaemia. Recovery of donor iron stores following whole blood donation varies considerably. Using genomic DNA from blood donors, we have reported preliminary data for two molecular typing strategies to profile genetic variants associated with iron metabolism and storage.1,2 These studies on small numbers of donors using next generation sequencing (NGS) for a panel of genes associated with iron homeostasis2,3 and TaqMan arrays for 10 selected single-nucleotide polymorphisms (SNP)1 established proof-of-principle. Future work of this nature must consider the advantages of each tool. NGS, is more expensive and time consuming for both testing and data analysis, however, is required for identification of variants of significance in donor iron recovery. TaqMan is a fast, convenient, and less expensive tool to rapidly identify a donor’s variant profile for individual management. A two-stage approach would benefit future studies. First, a large-scale survey using sequencing techniques to identify genetic variants associated with recovery of post-donation iron stores. Second, rapid testing with a less expensive tool such as TaqMan suitable for rapid testing and individual tailoring of blood donation frequency. References 1. Ji Y, Flower R, Hyland C, et al. Genetic factors associated with iron storage in Australian blood donors. Blood Transfus 2018; 16: 123–9. 2. Jacko G, Sivakaanthan A, Obeysekera M, et al. Next generation sequencing to identify iron status and individualise blood donors’ experience. Blood Transfus 2023; (in press). 3. McDonald CJ, Ostini L, Wallace DF, et al. Next-generation sequencing: application of a novel platform to analyze atypical iron disorders. J Hepatol 2015; 63: 1288–93.

BackgroundPatient outcomes are influenced by many confounding factors peri-operatively, including the type of surgery, anaesthesia, transfusion, and immune competence. We have previously demonstrated (in-vitro) that compared to allogeneic blood transfusion (ABT), intraoperative cell salvage (ICS) improves immune competence. The peri-operative immune response is complex. Altered or impaired immune responses may predispose patients to develop adverse outcomes (i.e., post-operative wound infection, pneumonia, urinary tract infection etc.) Surgical patients may develop infection, even without the confirmed presence of a definite microbiological pathogen. With all these factors in mind it is important to consider changes in immune cell numbers (and sub-populations) and functional capacity during peri-operative transfusion. MethodsIn this TRIMICS-Cell (Transfusion Related Immune Modulation and Intraoperative Cell Salvage-Cell numbers) study (n = 17, October 2018-November 2019) we prioritized and analysed peri-operative changes in the number and proportions of immune cell populations and sub-populations (B cells (CD20(+)), NK (natural killer) cells (CD56(+)), monocytes (CD14(+)), T cells (total CD3(+) and sub-populations: T helper cells (CD4(+)), cytotoxic T cells (CD8(+)), effector T cells (CD4(+) CD127(+)), activated effector T cells (CD4(+) CD25(+) CD127(+)) and regulatory T cells (CD4(+) CD25(+) CD127(-))), plasmacytoid dendritic cells (pDC; Lineage(-), HLA-DR+, CD11c(-), CD123(+)), classical dendritic cell (cDC) (Lineage(-), HLA-DR+, CD11c(+)), and cDC activation (Lineage(-), HLA-DR+, CD11c(+)), co-stimulatory/adhesion molecules and pDC (CD9(+), CD38(+), CD80(+), CD83(+), CD86(+), CD123(+)). Firstly we analysed the whole cohort of study patients and secondly according to the relevant transfusion modality (i.e., three study groups: those who received no transfusion, received ICS only (ICS), or both ICS and allogeneic packed red blood cells (pRBC) (ICS & RBC)), during major orthopaedic surgery. ResultsFor the whole study cohort (all patients), changes in immune cell populations were significant: leucocytes and specifically neutrophils increased post-operatively, returning towards pre-operative numbers by 48h post-operatively (48h), and lymphocytes reduced post-operatively returning to pre-operative numbers by 48h. When considering transfusion modalities, there were no significant peri-operative changes in the no transfusion group for all immune cell populations studied (cell numbers and proportions (%)). Significant changes in cell population numbers (i.e., leucocytes, neutrophils and lymphocytes) were identified in both transfused groups (ICS and ICS & RBC). Considering all patients, changes in immune cell sub-populations (NK cells, monocytes, B cells, T cells and DCs) and functional characteristics (e.g., co-stimulation markers, adhesion, activation, and regulation) were significant peri-operatively and when considering transfusion modalities. Interestingly DC numbers and functional capacity were specifically altered following ICS compared to ICS & RBC and pDCs were relatively preserved post-operatively following ICS. ConclusionA transient peri-operative alteration with recovery towards pre-operative numbers by 48h post-surgery was demonstrated for many immune cell populations and sub-populations throughout. Immune cell sub-populations and functional characteristics were similar peri-operatively in those who received no transfusion but changed significantly following ICS and ICS & RBC. Interesting changes that require future study are a post-operative monocyte increase in the ICS & RBC group, changes in cDC considering transfusion modalities, and possibly preserved pDC numbers post-operatively following ICS. Future studies to assess changes in immune cell sub-populations, especially during peri-operative transfusion, while considering post-operative adverse outcomes, is recommended.

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.

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.

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.

•Blood donations dropped worldwide since the onset of the COVID-19 pandemic due to generalized fear, lockdowns, and the absence of existing pandemic plans.•Blood donations increased after earthquakes, mainly due to a surge in first-time donors.•Successful strategies to balance blood supply and demand during the COVID-19 pandemic included suspension of elective surgeries, public awareness campaigns, home visits, and donor transportation.•Risk modelling, seroprevalence, and haemovigilance should be coordinated in conjunction with public health surveillance to optimise responses to the threat of emerging infections.

The monocyte monolayer assay (MMA) is used to predict transfusion outcomes when antibodies to red blood cell (RBC) antigens are present and compatible antigen-negative blood isn't available. Typically, the monocytes used are peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers. This study aimed to determine the between-volunteer variability in monocyte reactivity in the MMA. PBMCs were isolated (Ficoll density gradient) from freshly collected whole blood from 28 individuals and incubated with RBCs sensitised with anti-D (1 hour, 37oC, 5% CO2; positive control). Monocytic index (MI%) was calculated after microscopic analysis of the proportion of monocytes with phagocytosed or adherent RBCs. Mean MI was 23.6% (SD 10.35); however, between-volunteer variation was wide (MI range: 1.6–45.9%). Based on a 20% MI as a cut off, 11 volunteers (39.3%) did not reach the positive threshold for the anti-D positive control, which would result in assay failure and inconclusive results that could not be used to inform transfusion decision making. These results indicate MMA outcomes are impacted by individual volunteer monocytes. Therefore, a curated pool of volunteers with demonstrated monocyte reactivity is required for a reliable MMA. Further investigation of monocyte characteristics that contribute to the variability of MMA results between individuals is warranted.