Red Blood Cell Alloimmunization Research Articles

Red Blood Cell Alloimmunization in Pregnancy: A Review of the Pathophysiology, Prevalence, and Risk Factors

Red Blood Cell Alloimmunization in Pregnancy: A Review of the Pathophysiology, Prevalence, and Risk Factors

Maternal red blood cell alloimmunization prevalence in the United States

Hemolytic disease of fetus and newborn (HDFN) is a life-threatening disease mediated by maternal alloimmunization to red blood cell (RBC) antigens. Studies of maternal alloimmunization prevalence in the United States (U.S.) lack national data. This study describes prevalence and trends in alloimmunization in pregnancy in the U.S. RBC antibodies (abs) were identified in a large, nationwide, commercial laboratory database from 2010-2021. The cohort comprised pregnancies for which the year of lab collection and patient's state of residence were available. Data were normalized based on U.S. Centers for Disease Control and Prevention estimates of live births and weighted by year and U.S. Census Division. Cochrane-Armitage tests assessed temporal trends of alloimmunization. Of 9,876,196 pregnancies, 1.5% (147,262) screened positive for RBC abs, corresponding to an estimated prevalence of 1,518/100,000 pregnancies. Of identified RBC abs, anti-D comprised 64.1% (586/100,000 pregnancies). Prevalence of other high-risk RBC abs for HDFN included anti-K (68/100,000) and anti-c (29/100,000). Incidence of all three high-risk abs increased from 2010-21 (all p<0.001). Among almost 10 million pregnancies in the US, comprising an estimated 14.4% of all pregnancies, 1.5% screened positive for RBC abs. Almost three-quarters (74.3%; 683/100,000) of RBC abs identified were high-risk for HDFN. Though prevalence of anti-D is difficult to interpret without the ability to distinguish alloimmunization from passive immunity, it remains problematic in HDFN, ranking second only to anti-K in critical titers. Given the sequelae of HDFN, new initiatives are required to reduce the incidence of alloimmunization in patients of reproductive potential.

The influence of HLA allele and haplotype on RhE alloimmunization among pregnant females in the Chinese Han population.

Anti-E alloantibody is the most common and important red blood cell(RBC) alloantibody during pregnancy. The study aimed to determine the correlation between RhE alloimmunization and human leukocyte antigen (HLA)allele polymorphism, as well as haplotype diversity, among pregnant individuals in the Chinese Han population. All individuals included in our study were RhE-negative pregnant women of Chinese Han ethnicity, confirmed through serological testing. Pregnancy could be the only potential stimulating factor in RBC alloimmunization. Given the serological testing, the participants were divided into anti-E (responders) and non-anti-E-producing group (non-responders). The class I and II classical HLA genotyping were determined using next-generation sequencing, and the HLA genotype and haplotype frequencies were compared between the responders and non-responders. In total, 76 responders and 94 non-responders were enrolled in this study. Comparison results showed that all HLA class I alleles had no difference between the two groups. For HLA class II phenotypes, responders had higher frequencies of HLA-DRB1*09:01, HLA-DQA1*03:02 and HLA-DQB1*03:03 phenotypes than non-responders, and the differences were statistically significant (pc < 0.05). In addition, the haplotype frequency of HLA-DRB1*09:01-DQA1*03:02-DQB1*03:03 in the RhE responders was significantly higher than in the non-responders (31.58% vs. 12.77%; odds ratio, 3.154; 95% confidence interval, 1.823-5.456; pc value, 1.25 × 10-3). Our findings indicated that HLA-DRB1*09:01, HLA-DQA1*03:02 and HLA-DQB1*03:03 might be susceptible alleles for RhE alloimmunization among Chinese Han pregnant females. These three susceptible alleles constituted the unique three-locus haplotype in the RhE responders and collaborated to RhE alloimmunization.

Receipt of RhD-positive whole blood for life-threatening bleeding in female children: A survey in alloimmunized mothers regarding minimum acceptable survival benefit relative to risk of maternal alloimmunization to anti-D.

Low-titer group O whole blood (LTOWB) for treatment of hemorrhagic shock sometimes necessitates transfusion of RhD-positive units due to short supply of RhD-negative LTOWB. Practitioners must choose between using RhD-positive LTOWB when RhD-negative is unavailable against the risk to a female of childbearing potential of becoming RhD-alloimmunized, risking hemolytic disease of the fetus and newborn (HDFN) in future children, or using component therapy with RhD-negative red cells. This survey asked females with a history of red blood cell (RBC) alloimmunization about their risk tolerance of RhD alloimmunization compared to the potential for improved survival following transfusion of RhD-positive blood for an injured RhD negative female child. A survey was administered to RBC alloimmunized mothers. Respondents were eligible if they were living in the United States with at least one red cell antibody known to cause HDFN and if they had at least one RBC alloimmunized pregnancy. Responses from 107 RBC alloimmmunized females were analyzed. There were 32/107 (30%) with a history of severe HDFN; 12/107 (11%) had a history of fetal or neonatal loss due to HDFN. The median (interquartile range) absolute improvement in survival at which the respondents would accept RhD-positive transfusions for a female child was 4% (1%-14%). This was not different between females with and without a history of severe or fatal HDFN (p = .08 and 0.38, respectively). Alloimmunized mothers would accept the risk of D-alloimmunization in a RhD-negative female child for improved survival in cases of life-threatening bleeding.

Challenging the dogma: Red blood cell-directed autoimmunity as risk factor for red blood cell alloimmunisation after blood transfusion.

Red blood cell autoimmunity and alloimmunity are potentially linked. Quantification of this association can tailor extensively matched red blood cell transfusions in patients with autoimmunity. Using an incident new-user cohort comprising 47 285 previously non-transfused, non-alloimmunised patients, we compared transfusion-induced red blood cell alloimmunisation incidences in direct antiglobulin test (DAT)-positive and control patients. Additionally, we performed case-control analyses to handle potential confounding by clinical immunomodulators. Among (IgG and/or C3d) DAT-positive patients (N = 380), cumulative red blood cell alloimmunisation incidences after 10 units transfused reached 4.5% (95% confidence interval [CI] 2.5-8.2) versus 4.2% (CI 3.9-4.5, p = 0.88) in controls. In case-control analyses, alloimmunisation relative risks among DAT-positive patients increased to 1.7 (CI 1.1-2.8). Additional adjustments for pre-DAT transfusion exposure or the extent of Rh/K mismatching did not impact results. In conclusion, while patients with DAT positivity show an intrinsically increased alloimmune red blood cell response, their absolute risk is comparable to control patients due to counteracting co-existing immunosuppressive conditions. Consequently, isolated DAT positivity in patients lacking overt haemolysis or complicated alloantibody testing does not seem to warrant extended matching strategies.

Transfusion in hemoglobinopathies and red blood cell alloimmunization: data from Sicily, Sardinia and Malta.

Hemoglobinopathies are a group of diseases that include those due to globin gene mutations, such as thalassemia major (TM) and thalassemia intermedia (TI) or due to alteration of hemoglobin structure such as sickle cell disease (SCD), as well as a combination of these conditions such as thalasso-drepanocytosis (TD). They constitute the most frequent hereditary anemias requiring blood transfusion. In April 2022, a questionnaire was sent to the Transfusion Services (TS) of Sicily, Sardinia and the Maltese National Blood Transfusion (MNBT) service. The questionnaire was divided into a generic part including the number of patients followed and the type of hemoglobinopathy, and a section relating to transfusion therapy, including the number of units transfused, whether red blood cells (RBC) were washed and, finally, a section relating to the presence or absence of alloantibodies and their identification. Data was retrieved for 2,574 patients: 68.6% TM, 15.4% TI, 10.3% TD, 4.1% SCD, and 1.6% other hemoglobinopathies (OHA). The number of RBC units transfused was 76,974, equivalent to 24.5% of all the RBCU transfused from the total number of patients followed. The number of washed RBCU was 21.1% of all the units used; 337 patients (37%) were diagnosed with alloantibodies, the majority of which were patients with SCD (20.6%). Of the 485 alloantibodies found, 90.3% were identified. The antibodies found most frequently were related to the Kell system (41.7%) followed by antibodies to the Rhesus system (37.9%); 29.7% of patients had more than one antibody. From our study, certain indications can be formulated: complete the National Registry for patients with hemoglobinopathies; create a Registry of alloimmunized patients to ensure transfusion therapy is as safe as possible, considering antibody evanescence; and 3) increase the recruitment of blood donors of diverse ethnicities.

Analysis of risk factors for red blood cell alloimmunization and its incidence in hemoglobinopathy patients at a tertiary care center in Odisha, Eastern India

Abstract BACKGROUND: Sickle cell disease (SCD) and thalassemia are common hemoglobinopathies in Eastern India. Regular blood transfusion in these patients leads to alloimmunization. Finding compatible red blood cell units remain a challenge in these patients delaying the transfusion support. AIMS: This study aims to estimate the frequency of alloimmunization and predicts the associated risk factors. METHODOLOGY: A retrospective study was conducted for 2 years. Data were retrieved from the immunohematology work-up record. The Chi-square statistics determined the risk factors associated with alloimmunization. Multivariate regression analysis was used to identify independent risk factors of alloimmunization, adjusting the confounders. A P &lt; 0.05 was considered as statistically significant. RESULTS: Forty-seven patients out of 132 developed antibodies. Of these, 41 presented with only alloantibodies, three patients had only autoantibodies, and the rest 3 had both autoantibodies and alloantibodies. Most commonly observed alloantibodies were against Rh, Kidd, Duffy, and S antigens. SCD appeared a significant risk factor for the development of alloimmunization in the Chi-square test (P = 0.003). However, multivariate analysis revealed only age of more than 2 years at the first transfusion, and up to ten transfusions are independent risk factors of alloimmunization (P &lt; 0.05). CONCLUSION: We found a high incidence of alloimmunization in multitransfused hemoglobinopathies patients in this region. This study also shows the first transfusion at an age of more than 2 years and up to 10 transfusions are independent predictors of alloimmunization.

Decreasing alloimmunization-specific mortality in sickle cell disease in the United States: Cost-effectiveness of a shared transfusion resource.

Red blood cell alloimmunization and consequent delayed hemolytic transfusion reaction (DHTR) incidence and mortality in patients with sickle cell disease (SCD) are high. A shared transfusion resource has decreased both in other countries, while in the United States cost concerns persist. We conducted a Markov cohort simulation of a birth cohort of alloimmunized patients with SCD to estimate lifetime DHTR incidence, DHTR-specific mortality, quality-adjusted life expectancy (QALE), and costs with the implementation of a shared transfusion resource to identify antibody history versus without (i.e., status quo). We conducted our analysis using a lifetime analytic time horizon and from a United States health system perspective. Implementation of shared transfusion resource projects to decrease cumulative DHTR-specific mortality by 26% for alloimmunized patients with SCD in the United States, relative to the status quo. For an average patient population of 32 000, this intervention would generate a discounted increment of 4000 QALYs at an incremental discounted cost of $0.3 billion, resulting in an incremental cost-effectiveness ratio of $75 600/QALY [95% credible interval $70 200-81 400/QALY]. The results are most sensitive to the baseline lifetime medical expenditure of patients with SCD. Alloantibody data exchange is cost-effective in 100% of 10 000 Monte Carlo simulations. The resource would theoretically need a minimum patient population of 1819 patients or cost no more than $5.29 million annually to be cost-effective. By reducing DHTR-specific mortality, a shared transfusion resource in the United States projects to be a life-saving and cost-effective intervention for patients with SCD in the United States.

Distinct RBC alloantibody responses in type 1 interferon-dependent and -independent lupus mouse models.

During transfusion of red blood cells (RBCs), recipients are exposed to both ABO and non-ABO 'minor' antigens. RBC donor units and recipient RBCs are not routinely matched for non-ABO antigens. Thus, recipients are exposed to many RBC alloantigens that can lead to RBC alloantibody production and subsequent clinically significant hemolysis. RBC alloantibodies also significantly limit the provision of compatible RBC units for recipients. Prior studies indicate that the frequency of RBC alloimmunization is increased during inflammatory responses and in patients with autoimmune diseases. Still, mechanisms contributing to alloimmune responses in patients with autoimmunity are not well understood. More than half of adult patients with systemic lupus erythematosus (SLE) produce type 1 interferons (IFNα/β) and express IFNα/β stimulated genes (ISGs). Previously, we reported that IFNα/β promote RBC alloimmune responses in the pristane mouse model, which develops a lupus-like phenotype that is dependent on IFNα/β signaling. However, it is unclear whether IFNα/β or the lupus-like phenotype induces alloimmunization in lupus models. Therefore, we tested the hypothesis that IFNα/β promotes RBC alloimmune responses in lupus by examining alloimmune responses in IFNα/β-independent (MRL-lpr) and IFNα/β-dependent (pristane) lupus models. Whereas pristane treatment significantly induced interferon-stimulated genes (ISGs), MRL-lpr mice produced significantly lower levels that were comparable to levels in untreated WT mice. Transfusion of murine RBCs that express the KEL antigen led to anti-KEL IgG production by pristane-treated WT mice. However, MRL-lpr mice produced minimal levels of anti-KEL IgG. Treatment of MRL-lpr mice with recombinant IFNα significantly enhanced alloimmunization. Collectively, results indicate that a lupus-like phenotype in pre-clinical models is not sufficient to induce RBC alloantibody production, and IFNα/β gene signatures may be responsible for RBC alloimmune responses in lupus mouse models. If these findings are extended to alternate pre-clinical models and clinical studies, patients with SLE who express an IFNα/β gene signature may have an increased risk of developing RBC alloantibodies and may benefit from more personalized transfusion protocols.

Management of Delayed Hemolytic Transfusion Reaction in a Patient with Sickle Cell Disease: A Case Report

Background: Sickle cell disease (SCD) is a group of inherited blood disorders characterized by abnormalities in hemoglobin (Hb), leading to distortion in shape of red blood cells (RBCs) and their breakdown, potentially blocking blood flow and resulting in pain and anemia. This can significantly impact the patient’s quality of life. Patients often require multiple blood transfusions to prevent complications associated with SCD by reducing the concentration of damaged hemoglobin. However, one of the significant complications of blood transfusions is Delayed Hemolytic Transfusion Reaction (DHTR), which occurs due to RBC alloimmunization (i.e., formation of antibodies against donor RBC antigens). DHTR can manifest as a sudden decrease in hemoglobin levels, which may progress to rapid multiorgan failure and death. Thus, prompt diagnosis is crucial for timely management. This case report highlights the diagnosis and management of a 56-year-old African American female with SCD who presented with bilateral severe knee pain and low Hb levels. Case Presentation: The patient’s symptoms, past medical history, and investigation findings led to a diagnosis of DHTR. Management was conducted within the framework of Bloodless Medicine, incorporating strategies to minimize blood loss and optimize erythropoiesis. Treatment included the use of erythropoietin (EPO) to manage hemoglobin levels and the administration of opioids and supplemental oxygen for symptom relief. Conclusion: Our case report highlights the challenges and various management strategies employed in treating DHTR in SCD patients. It emphasizes the importance of early recognition of the complication and compares the risks and benefits of additional blood transfusions in such patients. By presenting this case, we aim to contribute to the growing literature on DHTR in SCD patients and highlight the need for further research and optimized management strategies in this population.

Antibody Screening, Identification and Red Cell Alloimmunisation Analysis in Multi-Transfused Patients at a Tertiary Care Hospital, Amritsar, India

Introduction: Alloimmunisation to red blood cell antigens, resulting from genetic disparities between donors and recipients, is one of the risks associated with blood transfusions. Antibody screening cells are used to detect unexpected antibodies. The risk of alloimmunisation is higher in patients who have undergone multiple blood transfusions. Aim: To estimate the frequency of various Red Blood Cell (RBC) alloantibodies and to determine the types of antibodies present in repeatedly transfused patients. Materials and Methods: This cross-sectional study was conducted on 200 patients with a history of multiple blood transfusions from October 1, 2019, to April 30, 2021, at the blood centre of Sri Guru Ram Das Institute of Medical Sciences and Research (SGRDIMSR), Amritsar, Punjab, India. Antibody detection and identification were performed, and the results were recorded. The data was statistically analysed using the Statistical Package for Social Sciences (SPSS) version 26.0 to draw relevant conclusions. The observations were tabulated in the form of numbers and percentages. Categorical data was analysed using the Chi-square test. The level of significance was determined as p≤0.05. Results: The study included 200 patients who were on multiple transfusions. The most common blood group among the patients was B positive (39%), followed by O positive (26%). The majority of patients (73.50%) had solid malignancies, followed by 28 (14%) thalassemia patients and 25 (12.50%) patients with chronic kidney disease. Solid malignancies included patients with breast cancer, cervical cancer, ovarian cancer, prostate cancer, and liver cancer. Alloantibodies were found in 15 patients (7.50%), of which 11 had solid malignancies and 4 had thalassemia. The most frequent antibody detected was the anti-K antibody (40%). Alloantibody formation was observed in both males and females. However, no statistical significance was found between gender and alloimmunisation (p=0.940). Conclusion: The effect of alloimmunisation can be avoided by routine RBC antibody screening before blood transfusion, especially in patients with a history of multiple blood transfusions. These measures decrease the incidence of red blood cell alloimmunisation and delayed hemolytic transfusion reactions in multi-transfused patients.

Frequency of red blood cell allo-immunization in patients undergoing blood transfusion at the Uganda Cancer Institute

Background: There is limited data on red blood cell (RBC) alloimmunization in patients with cancer in sub-Saharan Africa (SSA). We examined the frequency of RBC alloimmunization in transfused patients with cancers in Uganda.Methods: A randomized control trial was conducted on participants at the Uganda Cancer Institute. Eligible participants were age ≥15 years and required blood transfusion. Participants were randomized to receive either leucoreduced or non-leucoreduced blood transfusion. Participants’ plasma samples were screened for RBC alloantibodies at enrolment and 3-4 weeks after blood transfusion using a 2-cell panel of reagent group O RBCs using the tube method. Antibody identification was performed using a 10-cell panels of reagent RBCs. Participants were considered alloimmunized if antibodies to RBC antigens were identified.Results: A total of 277 participants were randomized (leucoreduced blood, n=137; non-leucoreduced blood, n=140). Overall, the most represented diagnoses were gynaecological cancers (n=88, 31.8%), acute leukaemia (n=35, 12.6%), and gastrointestinal cancers (n=25, 9.0%). Concomitant HIV infection was present in 26 (9.4%) participants. Most participants received &lt;5 units of blood during the study. No study participant developed allo-antibodies.Conclusion: There was no RBC alloimmunization in participants with cancers. Routine RBC allo-antibody screening in all patients with cancer in SSA requires further research.

Red Blood Cell Alloimmunization Prevention Practices in Pregnancy at Tertiary Care Level Hospitals in Islamabad, Pakistan: a Cross-sectional Study

Red Blood Cell Alloimmunization Prevention Practices in Pregnancy at Tertiary Care Level Hospitals in Islamabad, Pakistan: a Cross-sectional Study

Risk factors for red blood cell alloimmunization in multi-transfused oncology patients at Moi Teaching and Referral Hospital, Kenya

Background : Multiple blood transfusions may result in the production of alloantibodies against one or more red blood cell antigens which might make it more challenging to execute subsequent transfusions. Despite age and gender being risk factors for transfusion and being associated with alloimmunization frequency, they are not routinely taken into account before transfusion. This study assessed red blood cell alloimmunization and its association with risk factors among multi-transfused oncology patients at Moi Teaching and Referral Hospital Cancer Centre, Kenya.&#x0D; Methodology : The study employed a cross-sectional study design and focused on multi-transfused oncology patients at the Moi Teaching and Referral Hospital Cancer Centre. A sample size of 162 was used in the study based on Fisher's exact test formulae and a consecutive sampling technique was applied. The gel-based antibody screening and identification were performed with "ID-Diacell I-IIIII®" panel cells. The frequency, mean, median, and dispersion of descriptive statistics were shown and the association between alloimmunization with the number of transfusions, age and sex were determined by Spearman's correlation analysis. Statistical significance was established at P&lt; 0.05 and statistical tests were run at a 95% level of significance.&#x0D; Results : This study established no association between alloimmunization and the number of transfusions (P= 0.753). There was also no association between alloimmunization and age (P= 0.159). However, there was a significant positive association between alloimmunization with gender (P= 0.01). The study had a 6.2% prevalent rate of red blood cell alloimmunization, females had a greater prevalent rate than male patients. Anti-E and anti-K were the most prevalent alloantibodies.&#x0D; Conclusion and Recommendation : There is a need to improve current blood grouping and cross-match practices in most Kenyan hospitals by performing antibody screening and antibody identification tests. This study suggests routinely assessing alloimmunization in patients getting several transfusions while taking gender into account.

Magnitude of Red Blood Cell Alloimmunization Among Pregnant Women Attending Antenatal Care at Wolaita Sodo University Comprehensive Specialized Hospital, Southwest Ethiopia.

Maternal red cell alloimmunization occurs when a woman's immune system becomes sensitive to unfamiliar red blood cell antigens. This leads to the production of alloantibodies, which can have serious implications for the fetus and newborn. However, there is a lack of comprehensive information about the extent of red cell alloimmunization in underdeveloped countries like Ethiopia. Therefore, this study aimed to determine the magnitude of red cell alloimmunization among pregnant women attending antenatal care at Wolaita Sodo University Comprehensive Specialized Hospital from September 01 to November 30, 2022. In this institutional-based cross-sectional study, 422 pregnant women were participated and recruited using a systematic random sampling technique. Data on sociodemographic characteristics, obstetric history, and other clinical information were collected using structured questionnaires through face-to-face interview. Blood grouping and indirect antihuman globulin tests were performed. The relationship between red cell alloimmunization and the independent variables was determined using the chi-square test. P-value <0.05 was considered statistical significance. In this study, the blood group distributions among the participants were as follows: O, 177 (41.9%); A, 124 (29.4%); B, 76 (18%); and AB, 45 (3.86%). Among the pregnant women included in the study, a total of 51 (12.08%) were identified as RhD-negative. Out of these RhD-negative women, 5 (9.8%) were found to have developed alloimmunization with red blood cell antigens. Miscarriage and post-partum hemorrhage were found to be important factors associated with the occurrence of red cell alloimmunization in these women. This study showed that one out of ten pregnant women was alloimmunized. Therefore, antenatal blood grouping and indirect antihuman globulin screening should be performed routinely to manage and minimize the undesirable outcomes of alloimmunization during pregnancy.

Identifying Inflammatory Pathways That Promote RBC Alloimmunization in Patients with Sickle Cell Disease

Introduction: During red blood cell (RBC) transfusion, exposure to unmatched non-ABO antigens can lead to production of alloantibodies that promote potentially fatal hemolytic events. Patients with Sickle Cell Disease (SCD) have the highest incidence of RBC alloimmunization. Prior studies indicate that an altered immuno-regulatory and/or pro-inflammatory state may determine the risk of RBC alloimmunization. For example, one study reported that patients with SCD have inflammatory cytokine gene polymorphisms that could be associated with diverse disease presentations and alloimmunization. We previously reported that inflammatory type 1 interferons (IFNα/β) produced by innate immune cells are required for RBC alloimmunization in mice, while others have suggested that NFkB-induced cytokines play a role in murine transfusion models. However, the inflammatory factors that regulate RBC alloimmunization in patients with SCD are poorly understood. Alloimmunized patients with SCD also have elevated levels of anti-RBC autoantibodies, which facilitate erythrophagocytosis. Given the role of erythrophagocytosis by spleen macrophages in processing RBC antigens, we hypothesized that erythrophagocytosis of SCD RBCs may promote inflammatory pathways that regulate alloimmune responses. Methods: RBCs from control subjects (AA), and reticulocytes and mature RBCs from patients with SCD, were opsonized or not with anti-RBC antibodies (anti-RhD) and co-cultured for 3 hrs with human macrophages (hMac) differentiated from monocytes of healthy subjects. Phagocytosis of labeled RBCs was analyzed by flow cytometry. Bulk mRNA sequencing of co-cultured macrophages was performed to identify differential gene expression. Ingenuity Pathway Analysis (IPA) software was used to identify relevant pathways and upstream regulators of differentially expressed genes. To validate sequencing results, cytokine levels from co-culture supernatants were measured by multiplex cytokine assays. Results: Flow cytometric analysis revealed that opsonization was required for erythrophagocytosis in an FcR dependent manner. RNA-seq Differential Expression analysis of macrophages co-cultured with AA opsonized RBCs vs. non-opsonized RBCs identified 166 significantly upregulated and 24 downregulated genes (p&amp;lt;0.0001). IPA analysis revealed that the top 12 canonical pathways were related to inflammation, including TNF, IL-17, NF-kappa-B and NOD-like receptor signaling pathways. TNF signaling was the top predicted upstream regulator pathway with a fold enrichment score of 10.3 (16 genes) (p&amp;lt;0.0001). Co-cultures of mature RBCs from patients with SCD showed comparable results. When comparing macrophages from co-cultures of opsonized reticulocytes vs. non-opsonized reticulocytes from patients with SCD, 174 genes were upregulated and 31 were downregulated (p&amp;lt;0.0001). Pathway analysis also revealed unique signatures for opsonized reticulocytes from patients with SCD. The top 12 canonical pathways included TNF, NF-kappa-B and NOD-like receptor signaling pathways (Figure 1). The top predicted upstream regulators were TNF and the NF-kappa B signaling pathway with a fold enrichment score of 12.3 (22 genes) (p&amp;lt;0.0001) and 10.9 (18 genes), respectively (Figure 1). Finally, to validate the sequencing results, cytokines levels from supernatants of hMac co-cultures with the RBCs from the patients with SCD and control subjects were measured. Erythrophagocytosis of opsonized reticulocytes and AA RBCs, compared to non-opsonized co-cultures, led to production of NFκB-mediated cytokines, including TNF. Our results show that erythrophagocytosis of SCD RBCs promote NFκB-mediated TNF production. These results warrant further investigation of the role of NFκB-mediated inflammation in RBC alloimmunization in patients with SCD.

Real-World Feasibility of RH Genotype-Matched Red Blood Cells for Chronically Transfused Patients with Sickle Cell Disease

Introduction Most red blood cell (RBC) alloimmunization in sickle cell disease (SCD) is caused by sensitization to Rh (D, C, c, E, and e) and K antigens. Provision of serologic Rh and K-matched RBCs has reduced but not eliminated Rh alloantibody formation due to the high genetic variation within the RH loci in Blacks. Over 85% of patients with SCD, as well as Black donors needed to support CEK matching programs, have variant RHD or RHCE alleles that result in loss or alteration of Rh antigens. These variant Rh proteins are not detected by standard serologic tests, and thus, genetically matched RBCs may be necessary to prevent all Rh alloimmunization events. We previously used bioinformatic modeling to show that RH genetic matching was achievable for a cohort of ~200 patients of whom ~50% were chronically transfused, and represented 98 different RHD/RHCE allele combinations, with only 10 additional donations each weekday (~1050 Black donors/month) than required for serologic Rh and K matching. The real-world feasibility of providing RH genotype-matched RBCs to patients requiring regular transfusion has not been tested. Methods We performed a pilot study to evaluate the feasibility of providing RH genotype-matched RBCs from Black and Hispanic donors to non-Rh- and Rh-alloimmunized chronically transfused patients with SCD (1 to 18 years old). Black donor units at the blood center were first screened by serology, HEA BeadChip, and in-house RH SNP assays, and those selected for transfusion to study subjects had comprehensive RHD and RHCE genotyping. Data captured included RH genotypes of matched units identified, transfusion delays, unit age at time of transfusion, and pre-transfusion hemoglobin (Hb) and HbS levels. On-study values were compared to pre-study values obtained in the 6 to 12 transfusion visits prior to study enrollment. Results The study opened in February 2020. Due to the COVID-19 pandemic, donor center collections dropped to a nadir of ~500 Black donor units/month in 2020 but returned to ~1500 units/month currently, similar to pre-COVID numbers. For 14 participants (13 Black, 1 Hispanic), we transfused 327 RH genotype-matched units; 287 (88%) were exact RH genotype-matched (donor with identical genotype, homozygous for a matching RH haplotype, or RH allele match) and 40 (12%) were RH matched with the provision that RHD*RHD and *DAU0 and RHCE*ce and *ce48C are equivalent since no epitopes appear to differ between these alleles. No transfusion visits (n=183) were postponed due to the inability to identify RH genotype-matched units, including during the pandemic and national blood shortages that had a disproportionate impact on Black donations. Using individual patients as factors, ANOVA showed no statistically significant difference in unit age (days, Figure) or pre-transfusion Hb values between pre- and on-study transfusion visits. Although the goal maintenance pre-transfusion HbS levels varied between 30 and 50%, a statistically significant lower mean HbS on- (n=183) vs pre-study (n=139) (ANOVA F-ratio 15.4, p=0.000) (Figure) potentially suggests improved transfused RBC survival with RH genotype matching. This feasibility trial was not powered to detect a difference in alloantibody formation, but no Rh nor non-Rh antibody have been detected. Conclusion Recruitment of minority blood donors is a growing initiative nationally needed to support CEK matching. Our preliminary results suggest that genetic RBC matching at the RH loci may be feasible with Black and Hispanic donors. Donor center RH genotyping can facilitate transfusion of RH genotype-matched RBCs, improve allocation of minority and rare donor units and, at the same time, potentially improve patient care by avoiding Rh alloimmunization for individuals with SCD.

The impact of an antibody investigation algorithm emphasizing specificity on reducing potential false-positive warm autoantibody detection at a Canadian tertiary care centre.

To reduce potential false-positive warm autoantibody (WAA) by solid-phase red cell adherence assay (SPRCA), our centre implemented a new antibody investigation algorithm (AIA) by classifying cases with panreactive SPRCA but negative saline-indirect antiglobulin test as 'antibody of undetermined significance' (AUS) after excluding clinically significant antibodies. We assessed the effects of the new AIA and subsequent alloantibody formation in patients with AUS. Samples from patients with positive SPRCA screens between 1 September 2017 and 31 August 2021 were selected for the study. Frequencies of antibodies classified by the old and new AIAs were compared using Fisher's exact test. Patient demographics, transfusion history and antibody formation in cases of AUS were collected. A significant reduction in potential WAA frequencies from 127/1167 (11%) to 53/854 (6%) was observed (p < 0.001) when compared between the old and new AIAs among 2021 positive SPRCA antibody screens. While no patients with AUS later transitioned to potential WAA using the new AIA, four patients developed alloantibodies, including anti-E, anti-C, both anti-C and anti-E, and anti-Wra . A significant reduction in the frequencies of potential false-positive WAA detection at our centre was observed after implementing the new AIA, leading to less resource and phenotypically matched red blood cell (RBC) use. Some patients still developed subsequent RBC alloimmunization, so clinically relevant alloantibodies should be carefully excluded before determining AUS, taking forming or evanescent antibodies into consideration.

Red blood cell alloimmunization in myelodysplastic syndromes: Associations with sex, DAT-positivity, and increased transfusion needs.

Many patients with myelodysplastic syndromes (MDS) need repeated red blood cell transfusions which entails a risk of immunization and antibody formation. Associations between alloantibodies, autoantibodies and increased transfusion requirements have been reported, but their relationship remains unclear. In this study, we analyzed factors potentially associated with red blood cell alloimmunization, as well as changes in transfusion intensity and post-transfusion hemoglobin increments. In a retrospective cohort study, we linked Swedish MDS patients diagnosed between 2003 and 2017 to transfusion and immunohematology data. Potentially associated factors were analyzed using Cox proportional hazards regression. The transfusion rate after detected alloimmunization was analyzed using a fixed effects Poisson regression. Post-transfusion hemoglobin increments before and after alloimmunization were compared using a mixed effects regression. Alloantibodies following MDS diagnosis were detected in 50 out of 429 patients (11.7%). Female sex and a positive direct antiglobulin test (DAT) were independently associated with alloimmunization, with hazard ratios of 2.02 (95% confidence interval [CI] 1.08-3.78) and 9.72 (95% CI, 5.31-17.74), respectively. The transfusion rate following alloimmunization was increased with an incidence rate ratio of 1.33 (95% CI, 0.98-1.80) and the post-transfusion hemoglobin increment after alloimmunization was 1.40 g/L (95% CI, 0.52-2.28) lower per red blood cell unit (p = .002) compared to before alloimmunization, in multivariable analyses. Alloimmunization against blood group antigens was associated with sex, DAT-positivity, increased transfusion needs, and lower post-transfusion hemoglobin increments. These findings warrant further investigation to evaluate the clinical significance of up-front typing and prophylactic antigen matching in patients with MDS.

Effect of delayed cord clamping on red blood cell alloimmunization: a retrospective cohort study

Although delayed cord clamping has well-known benefits for preterm and term neonates, it has been inadequately assessed in alloimmunized neonates. This study aimed to evaluate the benefits and risks of delayed cord clamping in alloimmunized neonates. This was a retrospective comparative pre-post cohort study conducted from 2003 to 2018 in a tertiary care center in France. All living singleton neonates whose mothers were followed up for red blood cell alloimmunization during gestation and confirmed at birth (N=224) were included. Neonates were either exposed to immediate (n=125) or delayed cord clamping (n=99). Our main outcome was the time from birth to first exchange transfusions and/or transfusions. Secondary outcomes were hemoglobin level at birth, rate of exchange transfusion, number of postnatal transfusions, maximum bilirubin level, and number of phototherapy hours. Hemoglobin at birth was significantly higher in case of delayed cord clamping (mean difference, 1.7 g/dL; 95% confidence interval, 0.7-2.8). Among infants treated with exchange transfusion or transfusion, the time to initial treatment was higher in case of delayed cord clamping (median difference, 8 days; rate ratio, 1.51; 95% confidence interval, 1.09-2.10). There were no significant differences in the need for exchange transfusion, the number of transfusions, the maximum total bilirubin level, nor the number of phototherapy hours. In the subgroup analysis of neonates needing intrauterine transfusion during pregnancy (ie, severe alloimmunization), neonates had a lower rate of exchange transfusion in case of delayed cord clamping (odds ratio, 0.36; 95% confidence interval, 0.15-0.82). Our results indicate a benefit of delayed cord clamping in alloimmunization, regardless of pathology severity, without increased risk of jaundice.