Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis.

The crosstalk between hematopoietic lineages is important for developmental hematopoiesis. However, the role of primitive red blood cells (RBCs) in the formation of definitive hematopoietic stem and progenitor cells (HSPCs) is largely unknown. Primitive RBC deficiencies in mammals always lead to early embryonic lethality, but zebrafish lines with RBC deficiencies can survive to larval stage. By taking advantage of a zebrafish model, we find that the survival of nascent HSPCs is impaired in alas2- or alad-deficient embryos with aberrant heme biosynthesis in RBCs. Heme-deficient primitive RBCs induce ferroptosis of HSPCs through the disruption of iron homeostasis. Mechanistically, heme-deficient primitive RBCs cause blood iron-overload via Slc40a1, and an HSPC iron sensor, Tfr1b, mediates excessive iron absorption. Thus, iron-induced oxidative stress stimulates the lipid peroxidation, which directly leads to HSPC ferroptosis. Anti-ferroptotic treatments efficiently reverse HSPC defects in alas2 or alad mutants. HSPC transplantation assay reveals that the attenuated erythroid reconstitution efficiency may result from the ferroptosis of erythrocyte-biased HSPCs. Together, these results illustrate that heme-deficient primitive RBCs are detrimental to HSPC production and may provide potential implications for iron dysregulation-induced hematological malignancies.

The amino acid sensor GCN2 controls red blood cell clearance and iron metabolism through regulation of liver macrophages.

GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that controls messenger RNA translation in response to amino acid availability and ribosome stalling. Here, we show that GCN2 controls erythrocyte clearance and iron recycling during stress. Our data highlight the importance of liver macrophages as the primary cell type mediating these effects. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout (GCN2-/-) mice displayed resistance to anemia compared with wild-type (GCN2+/+) mice. GCN2-/- liver macrophages exhibited defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2-/- cells demonstrated that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating red blood cell clearance and iron recycling.

Yet more evidence that non-aqueous myelin lipids can be directly imaged with ultrashort echo time (UTE) MRI on a clinical 3T scanner: a lyophilized red blood cell membrane lipid study.

Direct imaging of semi-solid lipids, such as myelin, is of great interest as a noninvasive biomarker of neurodegenerative diseases. Yet, the short T2 relaxation times of semi-solid lipid protons hamper direct detection through conventional magnetic resonance imaging (MRI) pulse sequences. In this study, we examined whether a three-dimensional ultrashort echo time (3D UTE) sequence can directly acquire signals from membrane lipids. Membrane lipids from red blood cells (RBC) were collected from commercially available blood as a general model of the myelin lipid bilayer and subjected to D2O exchange and freeze-drying for complete water removal. Sufficiently high MR signals were detected with the 3D UTE sequence, which showed an ultrashort T2* of ∼77-271 µs and a short T1 of ∼189 ms for semi-solid RBC membrane lipids. These measurements can guide designing UTE-based sequences for direct in vivo imaging of membrane lipids.

Red blood cells function as reservoirs of tumor DNA.

Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.

Leveraging deep learning for detecting red blood cell morphological changes in blood films from children with severe malaria anaemia.

In sub-Saharan Africa, acute-onset severe malaria anaemia (SMA) is a critical challenge, particularly affecting children under five. The acute drop in haematocrit in SMA is thought to be driven by an increased phagocytotic pathological process in the spleen, leading to the presence of distinct red blood cells (RBCs) with altered morphological characteristics. We hypothesized that these RBCs could be detected systematically and at scale in peripheral blood films (PBFs) by harnessing the capabilities of deep learning models. Assessment of PBFs by a microscopist does not scale for this task and is subject to variability. Here we introduce a deep learning model, leveraging a weakly supervised Multiple Instance Learning framework, to Identify SMA (MILISMA) through the presence of morphologically changed RBCs. MILISMA achieved a classification accuracy of 83% (receiver operating characteristic area under the curve [AUC] of 87%; precision-recall AUC of 76%). More importantly, MILISMA's capabilities extend to identifying statistically significant morphological distinctions (p < 0.01) in RBCs descriptors. Our findings are enriched by visual analyses, which underscore the unique morphological features of SMA-affected RBCs when compared to non-SMA cells. This model aided detection and characterization of RBC alterations could enhance the understanding of SMA's pathology and refine SMA diagnostic and prognostic evaluation processes at scale.

Survey-based assessment of hematopoietic stem cell graft manipulation (minimal) by cell therapy labs supporting hematopoietic stem cell transplants in India.

BACKGROUND AIMS: Hematopoietic stem cell transplants (HSCTs) are increasingly being offered to patients in India for various conditions. The Indian Stem Cell Transplant Registry shows that a total of 2533 transplants were done in India in 2019. METHODS: An epidemiological descriptive cross-sectional survey (55 questions) of centers providing HSCT in India was planned to analyze variations in policies and practices regarding HSCT graft manipulation (i.e., plasma reduction, red blood cell [RBC] depletion and cryopreservation). A total of 63 of 102 centers responded to the survey (response rate, 61.7%), mostly from the northern part of India (27 of 63 [42.8%]). RESULTS: The majority of responding centers reported performing >50 HSCTs annually (n = 24 [38%]), and 92% (58 of 63) performed stem cell collections from a pediatric donor/patient (age <18 years). A total of 56 of 63 responding centers indicated that they did product manipulations involving cryopreservation (n = 45), plasma reduction (n = 42) and RBC depletion (n = 28). Cryopreservation was primarily done by blood centers (27 of 45 [60%]), with dimethyl sulfoxide (DMSO) being the primary constituent, used most commonly at a concentration of 5-10% (28 of 45 centers). Dump freezing was most commonly used (27 of 45) with a -80°C deep freezer. A 7-aminoactinomycin D based viability assessment was also most commonly used (30 of 45). Thawing of the product was done mainly at the bedside (30 of 45) using a wet-type thawer (36 of 45), and washing of DMSO was done by a few centers (seven of 45). Plasma reduction and RBC depletion were primarily done for ABO incompatibility at blood centers. CONCLUSIONS: This survey demonstrates the lack of standardization and uniformity in the minimal manipulation of hematopoietic stem cell grafts in centers supporting HSCT in India. This work also highlights the need for more studies and country-specific recommendations to establish best practices.

Expired blood transfusion and mortality outcomes in combat trauma patients.

BACKGROUND: Expired blood can be transfused if clinically indicated but outcome data do not exist. We hypothesized that modestly outdated blood can effectively support a hemorrhaging patient until surgical control is achieved. This study assessed whether expired blood was associated with mortality in combat trauma patients. STUDY DESIGN AND METHODS: A retrospective analysis of Armed Services Blood Program and Department of Defense Trauma Registry databases evaluated combat casualty records (2001-2023). The intervention of interest was transfusion of at least one unit of whole blood (WB), red blood cells (RBC), or platelets within one week past expiration. The outcome of interest was mortality at discharge. A control cohort that only received in-date blood was matched to the treatment cohort for logistic regression analysis. RESULTS: One hundred patients received expired RBCs (86), WB (11), and platelets (3). Mortality at discharge was 11.6% for expired RBC recipients and 13.4% for the control cohort (p = .97). After adjustment for injury severity, expired RBCs were not associated with mortality (OR = 0.40 [95% CI, 0.14-1.16]; p = .09). Of 10 patients who received the most expired RBCs by volume or storage duration, two were deceased at discharge. All 14 expired WB and platelet recipients were alive at discharge, but sample sizes were underpowered for regression analysis. DISCUSSION: Transfusion of modestly outdated RBCs was not associated with mortality in combat trauma patients. Expired WB and platelet recipients did well, but sample sizes were too small to draw significant conclusions. Expired blood should be further investigated for possible use in extenuating circumstances.

Patterns of red blood cell utilization: Harnessing electronic health records data from the Information Standard for Blood and Transplant (ISBT) 128 system within the Biologics Effectiveness and Safety (BEST) initiative.

BACKGROUND: Current hemovigilance methods generally rely on survey data or administrative claims data utilizing billing and revenue codes, each of which has limitations. We used electronic health records (EHR) linked to blood bank data to comprehensively characterize red blood cell (RBC) utilization patterns and trends in three healthcare systems participating in the U.S. Food and Drug Administration Center for Biologics Evaluation and Research Biologics Effectiveness and Safety (BEST) initiative. METHODS: We used Information Standard for Blood and Transplant (ISBT) 128 codes linked to EHR from three healthcare systems data sources to identify and quantify RBC-transfused individuals, RBC transfusion episodes, transfused RBC units, and processing methods per year during 2012-2018. RESULTS: There were 577,822 RBC units transfused among 112,705 patients comprising 345,373 transfusion episodes between 2012 and 2018. Utilization in terms of RBC units and patients increased slightly in one and decreased slightly in the other two healthcare facilities. About 90% of RBC-transfused patients had 1 (~46%) or 2-5 (~42%)transfusion episodes in 2018. Among the small proportion of patients with ≥12 transfusion episodes per year, approximately 60% of episodes included only one RBC unit. All facilities used leukocyte-reduced RBCs during the study period whereas irradiated RBC utilization patterns differed across facilities. DISCUSSION: ISBT 128 codes and EHRs were used to observe patterns of RBC transfusion and modification methods at the unit level and patient level in three healthcare systems participating in the BEST initiative. This study shows that the ISBT 128 coding system in an EHR environment provides a feasible source for hemovigilance activities.

RHD-negative red cells may be avoided for patients with ambiguous serologic typing for the RHD antigen.

BACKGROUND AND OBJECTIVES: Serologic typing with monoclonal anti-D is mandatory for RHD antigen determination before transfusion, but due to aberrant (weak or partial) variants of RHD, results may be ambiguous and molecular RHD-typing is required. Before that, RHD-negative (RHD -) red blood cells concentrates (RBCs) shall be transfused to avoid anti-D formation, which probably leads to wastage of RHD - RBCs. STUDY DESIGN AND METHODS: All patients with ambiguous results in serologic RHD-typing and molecular RHD-typing were assessed retrospectively. The proportions of patients at risk for anti-D formation and the proportion of RHD - RBCs transfused unnecessarily were evaluated for the following transfusion strategies: (1) RHD-positive (RHD + )RBCs for all patients, (2) RHD + RBCs for patients with at least 2+ reaction with anti-D, (3) RHD + RBCs for patients with C and/or E in their RHCE-phenotype, (4) RHD + RBCs for patients with C and/or E and at least 2+ reaction, and (5) RHD - RBCs for all patients. RESULTS: A total of 112 patients were included. Most had weak D type 1-3 and a minority had other, rare RHD variants. The risk of anti-D formation was 4.5%, 2.9%, 1.8%, 1.0%, and 0% for strategies 1-5, respectively. The proportion of RHD - RBCs transfused unnecessarily was 0%, 49.5%, 0.9%, 50.5%, and 95.5%. CONCLUSION: Transfusing patients with a C and/or E in their RHCE-phenotype with RHD + RBCs resulted in a very low risk of immunization while avoiding wastage of RHD - RBCs. Therefore, this strategy should be used for some patients with ambiguous results in serologic RHD-typing and pending results of molecular RHD-typing.

Post-transfusion biotin-labeled red blood cell survival studies in pediatric sickle cell disease with antibodies of uncertain significance.

BACKGROUND: Red blood cell (RBC) antibodies are common in multiply transfused patients with sickle cell disease (SCD). Unlike RBC alloantibodies, the potential of autoantibodies to cause post-transfusion hemolysis may be uncertain. Biotin-labeling provides a direct measurement of red cell survival (RCS) over time, thus can be used to assess the clinical significance of RBC antibodies. Antibodies to biotinylated RBC (B-RBC) occasionally are detected after exposure, which may impact B-RBC survival in subsequent RCS studies. STUDY DESIGN AND METHODS: Pediatric patients with SCD receiving monthly chronic transfusions underwent RCS studies, receiving aliquots of allogeneic RBC labeled at distinct densities of biotin (2-18 µg/mL). B-RBC survival was followed for 4 months post-transfusion, and B-RBC antibody screening for 6 months. Patients with warm autoantibodies (WAA) or B-RBC antibodies are reported here. RESULTS: RBC antibodies were detected during RCS in four patients: one with WAA, one with WAA followed by B-RBC-specific antibodies, and two with transient B-RBC antibodies within the first 5 weeks of exposure. B-RBC half-lives (T50) ranged 37.6-61.7 days (mean 47.8 days). There was no evidence of increased hemolysis or accelerated B-RBC clearance in the presence of WAA or B-RBC antibodies. DISCUSSION: Biotinylation of allogenic RBC can be used to assess the possible effects of RBC antibodies on transfusion survival in individual cases, particularly when it is uncertain if the detected antibodies may result in hemolysis. In the cases presented here, neither WAA nor B-RBC antibodies were associated with significant shortening of B-RBC survival in individuals with SCD.

Radiofrequency identification tracking system (RFID) significantly improves blood bank inventory management and decreases staff work effort.

BACKGROUND: Before implementation of the radio frequency identification (RFID) system, there was a high loss rate of 4.0%-4.3% of red blood cell (RBC) units every year expiring on the shelf in our transfusion service laboratory. We introduced RFID technology to improve inventory management and the burden of work on the staff. The goal of this study was to evaluate the impact of RFID technology on the inventory management of RBC units and the staff workload in a transfusion service laboratory. STUDY DESIGN AND METHODS: Using an RFID system involves encoding RBC units with an RFID tag capturing information such as donor identification number, product code, blood type, expiration date, product volume, and negative antigen(s). Tag information is collected through retrofitted storage shelves linked to the RFID server. The study analyzed RBC usage by unit and by volume (mL) and staff work effort to carry out inventory management tasks before and after the implementation of the RFID system. RESULTS: Implementation of the RFID technology reduced the loss, or discard, of RBC units to less than 1% annually (a statistically significant change, p < .001). The RFID computer dashboard provides a constant visual update of the inventory, allowing technologists to have accurate product counts and reducing their work burden. DISCUSSION: Implementation of RFID technology substantially reduced RBC product loss, improved inventory management, and lessened staff work burden.

Quality of life and cost-effectiveness of convalescent plasma compared to standard care for hospitalized COVID-19 patients in the CONCOR-1 trial.

BACKGROUND: The CONvalescent Plasma for Hospitalized Adults With COVID-19 Respiratory Illness (CONCOR-1) trial was a multicenter randomized controlled trial assessing convalescent plasma in hospitalized COVID-19 patients. This study evaluates the cost-effectiveness of convalescent plasma and its impact on quality-of-life to provide insight into its potential as an alternative treatment in resource-constrained settings. METHODS: Individual patient data on health outcomes and resource utilization from the CONCOR-1 trial were used to conduct the analysis from the Canadian public payer's perspective with a time horizon of 30 days post-randomization. Baseline and 30-day EQ-5D-5L were measured to calculate quality-adjusted survival. All costs are presented in 2021 Canadian dollars. The base case assessed the EQ-5D-5L scores of hospitalized inpatients reporting at both timepoints, and a utility score of 0 was assigned for patients who died within 30 days. Costs for all patients enrolled were used. The sensitivity analysis utilizes EQ-5D-5L scores from the same population but only uses costs from this population. RESULTS: 940 patients were randomized: 627 received CCP and 313 received standard care. The total costs were $28,716 (standard deviation, $25,380) and $24,258 ($22,939) for the convalescent plasma and standard care arms respectively. EQ-5D-5L scores were 0.61 in both arms (p = .85) at baseline. At 30 days, EQ-5D-5L scores were 0.63 and 0.64 for patients in the convalescent plasma and standard care arms, respectively (p = .46). The incremental cost was $4458 and the incremental quality-adjusted life day was -0.078. DISCUSSION: Convalescent plasma was less effective and more costly than standard care in treating hospitalized COVID-19.

Preoperative hemoglobin thresholds for increased risk of "medical" complications in fast-track total hip and knee arthroplasty, a secondary analysis of a machine-learning algorithm.

BACKGROUND: There is increasing evidence that gender-specific hemoglobin thresholds may not be ideal in the surgical population. Thus, preoperative anemia defined as a hemoglobin of <13.0 g/dL is a well-established risk factor in elective surgery. However, few studies have investigated the specific influence of preoperative hemoglobin within a machine-learning model using data from an optimized fast-track surgical setup. STUDY DESIGN AND METHODS: A secondary analysis on the specific influence of preoperative hemoglobin level on a machine-learning model developed for identifying patients at increased risk of a length of stay (LOS) of >4 day or readmissions due to medical complications in fast-track total hip and knee arthroplasty within a well-defined fast-track protocol. To evaluate the effect of hemoglobin on the model we calculated SHaply Additive Explanation (SHAP) values for the 3913 patients from our previous test-dataset and stratified by gender and total hip and knee arthroplasty, respectively. RESULTS: The study period ran from January 2017 to August 2017. Median LOS was 1 day and mean preoperative Hb was 15.5 g/dL (SD:1.5), lower in women (14.9 vs. 16.2 g/dL) and with 30.5% of women versus 12.0% of men having a Hb of <13.0 g/dL. There was a steep increase in SHAP value with a preoperative Hb < 14.8 g/dL, and irrespective of gender age and procedure type. DISCUSSION: A machine-learning model found a hemoglobin threshold of <14.8 g/dL for increased risk of impaired recovery, regardless of gender or age, supporting reevaluation of preoperative anemia thresholds in the elective surgical setting.

Iatrogenic blood loss from phlebotomy during adult extracorporeal membrane oxygenation: A retrospective cohort study.

BACKGROUND: Adult extracorporeal membrane oxygenation (ECMO) patients are at high risk for allogeneic blood transfusion. Few studies have characterized iatrogenic blood loss from phlebotomy in adult ECMO patients. We hypothesized that iatrogenic phlebotomy would be a significant source of blood loss during ECMO. METHODS: Adults who had their entire ECMO run at our medical center between 2020 and 2022 were included. Average daily phlebotomy volume and total phlebotomy volume during ECMO were estimated based on the total number of laboratory tests that were processed. In addition, the crude and adjusted association between total phlebotomy volume during ECMO and RBC transfusion during ECMO was evaluated using linear regression and Loess curve analysis. RESULTS: A total of 161 patients who underwent 162 ECMO runs were included. Of the 162 ECMO runs, 88 (54.3%) were veno-arterial and 74 (45.7%) were veno-venous ECMO. Median duration of ECMO was 5 days [25th, 75th percentile = 2, 11]. Median daily phlebotomy volume was 130 mLs [25th, 75th percentile = 94, 170] and median total phlebotomy volume during ECMO was 579 mLs [25th, 75th percentile = 238, 1314]. There was a significant crude and adjusted association between total phlebotomy volume and RBC transfusion during ECMO (beta coefficient = 0.0023 and 0.0024 respectively, both p < .001) based on linear regression analysis. DISCUSSION: Phlebotomy for laboratory testing is a significant source of blood loss during ECMO in adults. Comprehensive patient blood management for adult ECMO patients should include strategies to reduce laboratory testing and/or phlebotomy volume during ECMO.

Lack of RBC transfusion independence by Day 30 following allogeneic hematopoietic stem cell transplant strongly predicts inferior survival and high non-relapse mortality in acute myeloid leukemia patients.

BACKGROUND: Studies have suggested that acute myeloid leukemia (AML) patients with incomplete hematologic recovery undergoing allogeneic stem cell transplantation (allo-HSCT) had inferior overall survival (OS). STUDY DESIGN AND METHODS: This single-center, retrospective study of AML patients evaluated the relationship between red blood cell (RBC) and platelet (PLT) transfusion requirements during the first 30 days and long-term outcomes after allo-HSCT through multivariate analyses. RESULTS: A total of 692 AML patients received peripheral blood stem cells (89.2%), marrow (5.6%), or umbilical cord (5.2%) from matched related (37.4%), unrelated (49.1%), or haploidentical (8.2%) donors in 2011-2017. Transfusion requirements during the first 30 days for RBC (89.5% transfused, median 3, range 1-18 units) or PLT (98.2% transfused, median 6, range 1-144 units) were variable. By Day 30, 56.7% (95% confidence interval [CI]: 52.8-60.3%) and 86.1% (95% CI: 83.2-88.5%) had achieved RBC and PLT transfusion independence, respectively. Median follow-up among survivors (n = 307) was 7.1 years (range: 2.7-11.8). Lack of RBC transfusion independence by Day 30 was strongly and independently associated with worse 5-year OS (39.2% vs. 59.6%, adjusted hazard ratio [HR] 1.83, 95% CI: 1.49-2.25), leukemia-free survival (35.8% vs. 55.5%, HR = 1.75, 95% CI: 1.43-2.14), and NRM (29.7% vs. 13.7%, HR = 2.05, 95% CI: 1.45-2.89) (p < .001). There was no difference in relapse rates among patients who achieved or did not achieve RBC (p = .34) or PLT (p = .64) transfusion independence. CONCLUSION: Prolonged RBC dependence predicted worse survival and NRM rates, but not increased relapse. Posttransplant surveillance of such patients should be adjusted with more attention to non-relapse complications.

Compound heterozygosity induces a rare Gerbich-negative phenotype in an immunized blood donor.

BACKGROUND: Antibodies to Gerbich blood group antigens are exceedingly rare and can cause moderate transfusion reactions. Several deletional variants of the GE-gene, that harbors long sequence repeats, enable alloimmunization and formation of naturally occurring antibodies. SUBJECT AND METHODS: A female blood donor and soldier of the German Army without history of pregnancy or transfusion showed an antibody reactive with all test cells except for GE:-2-3 RBC. Thus, anti-Ge2 was suspected. Molecular analysis including fragment length specific PCR, Sanger sequencing and NGS should reveal the molecular background of the deficiency. Segregation of the variant alleles should be demonstrated by family analysis. RESULTS: Compound heterozygosity for GYPC exon 2 (GE*01.-02) and exon 3 (GE*01.-03) deletion was detected in the donor and her sister. The mother had one exon 3 amplicon of reduced length, while the father heterozygously exhibited a truncated GYPC exon 2. NGS clearly demonstrated reduced coverages within the deletional fragments within each family member. The donor and her sister showed the complete absence of a 640 bp fragment. DISCUSSION AND CONCLUSION: Rare GE deletion variants can induce naturally occurring anti-Ge2 in Caucasians. Because of an enhanced risk of injury as soldier autologous RBC of the donor were cryopreserved. The donor and her sibling can give blood for each other because of identical ABO, Rh, and K antigen blood types.

Red cell transfusion thresholds in outpatients with myelodysplastic syndromes: Results of a pilot randomized trial RBC-ENHANCE.

BACKGROUND: The optimal hemoglobin (Hb) threshold for red blood cell transfusions in adult patients with myelodysplastic syndromes (MDS) has not been defined. STUDY DESIGN AND METHODS: We conducted a pilot randomized multi-center study of two transfusion algorithms (liberal, to maintain Hb 110-120 g/L, transfuse 2 units if Hb < 105 g/L and 1 unit if Hb 105-110 g/L vs. restrictive, 85-105 g/L, transfuse 2 units when Hgb < 85 g/L). Primary objectives were 70% compliance in maintaining the q2 week hemoglobin within the targeted range and the achievement of a 15 g/L difference in pre-transfusion Hb. Secondary outcomes included measures of quality of life (QOL), iron studies and safety. RESULTS: Twenty-eight patients were randomized between February 2015-2020, 13 to the restrictive arm and 15 to the liberal arm in three tertiary care centers. The compliance was 66% and 45% and the mean pre-transfusion Hb thresholds were 86 (standard deviation [SD] 8) and 98 g/L (SD 10) in the restrictive and liberal arms, (mean difference 11.8 g/L, p < .0001), respectively. Patients in the liberal arm experienced a mean of 3.4 (SD 2.6) more transfusion visits and received a mean of 5.3 (SD 5.5) more units of blood during the 12-week study. Ferritin increased by 1043 (SD 1516) IU/L and 148 (SD 1319) IU/L in the liberal and restrictive arms, respectively. Selected QOL scores were superior pre-transfusion and more patients achieved clinically important improvements in the liberal arm compared with the restrictive arm for selected symptoms and function domains. CONCLUSION: The results establish that policies for transfusion support can be delivered in practice at multiple hospitals, but further research is required to understand the full clinical effects and safety of liberal transfusion policies in MDS outpatients.

Rethinking the transfusion pathway in myelodysplastic syndromes: Study protocol for a novel randomized feasibility n-of-1 trial of weekly-interval red cell transfusion in myelodysplastic syndromes.

BACKGROUND: Anemia in myelodysplastic syndromes (MDS) is associated with poorer health-related quality of life (HRQoL) and physical function, and is frequently treated with transfusions. The current common practice of transfusing multiple red blood cells (RBC) units every 2-4 weeks may result in peaks/troughs in hemoglobin (Hb) level, yet maintaining a stable Hb may better improve HRQoL. We describe a study protocol aiming to investigate the feasibility of weekly low-dose RBC transfusion in MDS patients, including assessing HRQoL and physical function outcomes. STUDY DESIGN AND METHODS: In this n-of-1 pilot study, patients receive two treatment arms, with randomly allocated treatment sequence: arm A (patient's usual transfusion schedule) and arm B (weekly transfusion, individualized per patient). To facilitate timely delivery of weekly transfusion, extended-matched RBCs are provided, with transfusion based upon the previous week's Hb/pre-transfusion testing results to eliminate delays of awaiting contemporaneous cross-matching. Primary outcome is the feasibility of delivering weekly transfusion. Secondary outcomes include HRQoL, functional activity measurements, RBC usage, and alloimmunization rates. A qualitative substudy explores patient and staff experiences. RESULTS: The trial is open in Australia, Netherlands, and UK. The first patient was recruited in 2020. Inter-country differences in providing RBCs are observed, including patient genotyping versus serological phenotyping to select compatible units. DISCUSSION: This pilot trial evaluates a novel personalized transfusion approach of weekly matched RBC transfusion and challenges the dogma of current routine pre-transfusion matching practice. Findings on study feasibility, HRQoL, and physical functional outcomes and the qualitative substudy will inform the design of a larger definitive trial powered for clinical outcomes.

In vivo genotoxicity assessment of N-(-9 acridinyl)-b-alanine hydrochloride (S-300) using a validated Pig-a mutagenesis assay.

BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.

Transfusion management and hemoglobin-based oxygen carrier treatment in a patient with anti-Rh17 antibody.

BACKGROUND: A 54-year-old Hispanic OPos female with known history of anti-Rh17 antibodies was diagnosed with Philadelphia-Chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Rh17, also known as Hr0, is a high-frequency antigen composed of several epitopes on the RhCE protein. Anti-Rh17 antibodies can be made by individuals with missing or varied C/c, E/e antigens. Anti-Rh17 antibodies are clinically significant given multiple case reports of hemolytic disease of the fetus and newborn (HDFN). Finding compatible units for patients with anti-Rh17 can be particularly difficult given that only 1 in 100,000 people are Rh17 negative. STUDY DESIGN AND METHODS: Search for compatible units was conducted by the American Rare Donor Program (ARDP) with no leads. After chemotherapy induction and despite erythropoiesis stimulating agent administration, the patient's hemoglobin continued to trend down to a nadir of 2.8 g/dL. Here we report transfusion of incompatible pRBC to this patient with critically symptomatic anemia. HBOC-201 (Hemopure) was obtained and administered under an emergency compassionate/expanded access designation from the Food and Drug Administration (FDA) under an emergency Investigational New Drug (IND) application. RESULTS AND DISCUSSION: Overall difficulties in this case included the challenge of finding compatible units, dilemma of transfusing incompatible units in a patient with severe anemia and obtaining alternatives to blood products. This case report demonstrates the successful use of HBOC-21 in treating life-threatening anemia.