Risk Of Hemolytic Transfusion Reactions Research Articles

Establishing a Rabbit Blood Bank: An Assessment of Storage Lesion

Introduction: In trauma settings, hemorrhage and its related complications are one of the major causes of mortality. Blood, blood products, and fluid replacement are the mainstay of treatment in such cases. However with storage, the viability and shelf life of stored products start to decline, reducing their quality when administered. This, in turn, necessitates a validated and flexible model that can be tailored to transfusion needs without affecting product quality. In this study, we established a leporine blood banking model that meets established human requirements to support translational resuscitation studies. Crossmatching was performed to assess risk of hemolytic transfusion reaction. To assess the storage lesion, we analyzed hematology and hemostatic function out to 28 days of cold storage. Methods: Juvenile (4-8 months old) male New Zealand White rabbits from Envigo (strain Hsd:Hra NZW (SPF)) (n=11) were anesthetized with their vitals monitored to ensure adequate plane anesthesia. Whole blood and plasma-designated units were collected via direct cardiac exsanguination. Units were collected (n=11) in 100 milliliter (mL) animal blood bags containing 24% CPDA and stored at 4°C upright. Initially, samples (first n=3) were analyzed on days 0 (collection day), 3, 6, 9, 12, 16, and 21. Sampling was then adjusted to days 0, 4, 7, 10, 14, and 21 for the following n=2, and day 28 was also added for the remaining n=6. Complete blood counts (CBC), rotational thromboelastometry (ROTEM), and collagen agonist impedance aggregometry (IA) were performed to assess changes over storage duration. Before ROTEM analysis, units were recalcified with 1 M CaCl2 1:100 Vol:Vol to account for the increased citrated concentration. Statistical analysis was performed using one-way ANOVA with Dunnett's test for multiple comparisons. Results: Red blood cell count showed no significant change from baseline mean 3.905 M/uL to 4.157 M/uL over 28 days (mean difference: 0.2523 M/uL, p=0.8379). Hematocrit and hemoglobin levels, initially at mean 26.34% and mean 8.755 g/dL respectively, also remained stable (hematocrit: mean difference: 1.01%, p=0.7257; hemoglobin: mean difference: 0.267 g/dL, p=0.9172) with means of 26.34% and 8.488 g/dl on day 28. Platelet count, starting at mean of 228.2 K/μL, finalized at 161.0 M/ μL did not significantly change (mean difference: 67.2 M/μL, p=0.3402). Hemolysis, however, increased significantly from 0.2191% to 1.008% (mean difference: 0.7889, p=0.0333), though all measures remained below 2%. ROTEM INTEM clotting time (CT) and clot formation time (CFT) showed no significant changes from baselines of 188.7 sec to 201.3 sec and 68.0 sec to 179.4 sec respectively (CT: mean difference: 12.57 sec, p=0.3171; CFT: mean difference: 111.4 sec, p=0.2118). INTEM maximum clot firmness (MCF) decreased significantly from 56.86 sec to 25.60 sec (mean difference: 31.26 sec, p=0.001). ROTEM EXTEM CT and CFT exhibited no significant changes from baselines of 76.73 sec and 88.32 sec to 125.5 sec and 586.2 sec respectively (CT: mean difference: 48.77 sec, p=0.2367; CFT: mean difference: 497.9 sec, p=0.3677). EXTEM MCF, like INTEM MCF, decreased significantly from 61.05 sec to 23.10 sec (mean difference: 37.95 sec, p <0.001). IA AUC showed no significant change from baseline 15.73 to 8.99 (mean difference: 6.74, p=0.6488). Conclusion: Our study provides a successful methodology for establishing stable leporine whole blood unit manufacturing and storage. This model can be customized to different transfusion requirements and could be utilized to manufacture components. We plan on expanding our sample size with continuous monitoring for all parameters. Our group will also investigate other available blood collection methods (such as vascular access ports for more frequent unit collections) to refine our model, enhance safe and sustainable production and optimize collection strategies.

Red Blood Cell (RBC) Antigen Confirmation Workflow – A Positive Impact at an Academic Medical Center

Abstract Introduction/Objective Introduction: Blood group antigens, present on the surface of red blood cells (RBCs), play a crucial role in transfusion medicine. These antigens are recognized serologically and form the basis of RBC phenotyping. Understanding the significance of antigen-negative RBCs is essential for mitigating the risk of hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN). This study aims to assess the impact of implementing a new RBC antigen confirmation workflow on transfusion service efficiency and patient outcomes. Methods/Case Report Method: The study involved collaboration between our Transfusion Services Department (TS), the Blood Center supplier, and the Apheresis department. A comprehensive review of historical practices and FDA recommendations regarding RBC antigen labeling was conducted. Changes were implemented to streamline antigen confirmation testing and labeling processes. The impact on TS workload, turnaround time, and patient care metrics was evaluated. Results (if a Case Study enter NA) Results: Implementation of the new RBC antigen confirmation workflow led to a significant reduction in antigen confirmation tests performed by TS, with a 607% decrease observed from 2020 to 2023. Concurrently, the workload for reference lab workups increased by 337% over the same period. This shift in workload distribution allowed TS staff to focus more on patient reference workup cases and case review. The improved workflow efficiency resulted in enhanced turnaround times for providing patients with antigen-negative RBCs, ultimately improving the patient experience. Conclusion Conclusion: The adoption of the new RBC antigen confirmation workflow has resulted in compliance with FDA labeling requirements, efficient utilization of TS staff resources, and decreased turnaround times for blood delivery. These improvements have positively impacted patient care by ensuring timely access to compatible blood products. Future steps include developing reports to track turnaround time for patients with special blood product needs and continuing collaboration with the blood supplier and the Apheresis department to optimize inventory management and transfusion processes.

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

What is the optimal pretransfusion testing interval for multi-transfused patients? The University Hospital of Brest experience.

This is a retrospective study of post-transfusional allo-immunizations from 2015 to 2020. For each patient, the time interval between the last negative research and the allo-immunization was investigated. 189 patients developed allo-antibodies. In 16 patients (8,5%), the interval for allo-immunization was 24hours, 48hours and 72hours in 4, 8 and 4 patients respectively. 12 patients were transfused after the discovery of the allo-antibodies. That means if we have chosen a delay of validity of 72hours, then 9 patients would have been transfused with a negative result. Checking for allo-antibodies before RBC transfusion with an interval of 24hours (and not 72hours) is pertinent in order to assure an optimal transfusion safety and to limit the risk of hemolytic transfusion reactions. A pretransfusion testing interval of 24hours for multi-transfused patients should be considered.

The Immunohematologic and Clinical Evaluation of Increased Risk for Red Blood Cell Alloimmunization among Multi-Transfused Pediatric Patients in a Tertiary Medical Institution in Central Luzon, Philippines

Abstract Introduction/Objective Multiple blood transfusion is a therapeutic treatment for patients with hematologic disorders. However, patients who undergo repeated blood transfusions are at higher risk of developing red blood cell alloimmunization. This may further delay the availability of compatible blood and pose a higher risk for hemolytic transfusion reactions. Methods/Case Report A total of 55 patients, with an age range of 2-19 years, were included in the study. All patients had hematologic disorders and underwent chronic blood transfusion. In this retrospective study, Student’s t-test and Fisher’s exact tests were used to compare the means and percentages, respectively, under 5% level of significance. R ver 4.0.3 was utilized in the analyses. Results (if a Case Study enter NA) Out of the 55 pediatric patients, 10 (18.2%; CI95%: 9.1 to 30.9%) were found to be alloimmunized. Two patients had multiple antibodies whereas the others had one specificity, having a total of 12 detected alloantibodies. The most prevalent alloantibody was anti-E (60%), followed by anti-Jka (20%), anti-Mia (20%), anti-S (10%), and anti-K (10%). All obtained alloantibodies showed no correlation (p>0.05) with the age group of the patients. Subsequently, it was not evident that average blood transfusion (p=0.949) and packed red blood cells transfused per year (p=0.782) significantly differ between alloimmunized and non-alloimmunized patients. Similarly, there was no sufficient evidence that the age group (p=0.723), sex (p=1.000), blood group (p=1.000) and hematologic disorder (p=0.949) were associated with their alloimmunization status. Conclusion The alloimmunization incidence rate of 18.2% in this study was higher compared to related literatures, and most of the detected alloantibodies were of IgG type which are commonly clinically significant. The study showed no significant correlation between the specific risk factors and the development of alloimmunization. For patient care, it is imperative to include precautions such as extended RBC phenotyping and provision of antigen matched RBCs to prevent unnecessary transfusion reactions caused by alloimmunization.

The Effect of ABO Immune Complexes on Endothelial Cell Integrity As Measured By Electric Cell-Substrate Impedance Sensing (ECIS) and Confocal Microscopy

Background: ABO non-identical transfusions have been associated with increased risk of hemolytic transfusion reactions. We previously reported that the ABO immune complexes (ABO-IC) formed in vitro as a result of ABO non-identical exposure affect platelet function, red blood cell integrity, and clot kinetics (Vox Sang. 2016;110:219-26). We developed an in vitro model as a means to investigate the interaction between ABO-IC and endothelial cells (EC).Methods: The ABO-IC were prepared by combining equal volumes of concentrated O plasma (high titers of anti-A and anti-B) with A plasma (high concentration of A antigens). Anti-A titers were measured before and after mixing to confirm the presence of ABO-IC. ABO-known human pulmonary vessel EC were then seeded onto gold microarray plates to prepare for electric cell-substrate impedance sensing (ECIS). The trans-endothelial electrical resistance (TER) was recorded for 48 hours after the addition of media, PBS, thrombin, O, A, B, and/or AB plasmas added 1:1 with media, as well as various concentrations of ABO-IC. Statistics were performed using the chi-square test to compare TER values at various timepoints to baseline. Human pulmonary EC were cultured on glass-bottomed petri dishes for confocal microscopy. The cells were stained with fluorescent green calcein AM dye and assessed for viability at 100x magnification at baseline and again 15 minutes following the addition of ABO-IC.Results: Substantial decreases in TER were observed over the course of 2 hours in O and A EC when treated with non-identical plasma, and sustained over 10 hours (p<0.05 for all). Significant decreases in TER were also observed in cells treated with various concentrations of ABO-IC, over 2 hours and were found to be dose-dependent and independent of ABO type (p<0.05 for all). Unlike the ABO-IC, exposure to thrombin revealed a marked decrease in TER after 2 hours, although the final TER measurement revealed complete recovery (Table 1a-c). Incubation with ABO-identical plasma showed slight decreases in TER over time that were not statistically significant when compared to incubation with cell media (Figure 1a-b). Physical damage to the EC membrane were observed via confocal microscopy after 15 minutes of exposure to ABO-IC (Figures 2a-b).Discussion: The exposure of ABO-IC to human pulmonary vessel EC revealed significantly decreased monolayer integrity via ECIS and confocal microscopy. The decreases seen in the TER of EC incubated with non-identical plasma and ABO-IC indicate the potential for capillary leak as a possible reaction to ABO non-identical transfusion. We have shown that the substantial decrease in TER when exposed to plasma is ABO type-dependent, and that the damage caused to the cells happens within a few hours and is irreversible over 48 hours. The confocal images reveal a significant change in the morphology of the EC membrane within 15 minutes of exposure to ABO-IC. These preliminary data suggest that anti-A and ABO-IC may significantly affect EC viability and function. This in vitro model provides potential novel mechanisms for the detrimental effects of ABO non-identical transfusion on EC. Further investigation of biomarkers for cell viability and integrity in response to exposure to ABO-IC is underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Variation of anti-A and anti-B titers in group O potential blood donors: A pilot study.

Resuscitation with fresh whole blood is vital to preserving life on the battlefield. Transfusing low titer O whole blood (LTOWB), defined as anti-A and anti-B titer levels of <1:256, is safe because LTOWB alleviates the risk for hemolytic transfusion reactions. Because of possible variations in titer levels over time, a study was needed using US Navy and Marine Corps personnel to assess how these titers change across two assessments. Retrospective data from group O marines and sailors (M = 25 years of age; range, 19-35 years) stationed in the San Diego region were acquired from the Armed Services Blood Program and the Composite Health Care System. Of 972 group O donors between January 2016 and November 2019, 55 donors with 2 samples were identified (N = 55). Analysis included contrasting rates of high (≥1:256) and low (<1:256) anti-A and anti-B titers on the initial and second blood tests, along with the time between testings. The average time between testing was 332 days (range, 35-1,121 days), which far exceeded the recommended 90-day interval (p < 0.00001). Only 45% met the 90-day recommendation. Titer status changed frequently, from low to high (anti-A, 18%; anti-B, 13%; LTOWB to not LTOWB, 21%) or from high to low (anti-A, 62%; anti-B, 78%; not LTOWB to LTOWB, 62%). Anti-A and anti-B titers change frequently enough to warrant testing immediately before deployment and even during deployment. The observed time elapsed between testing is unacceptably long. The present pilot study provides a foundation for a larger formal study to more fully characterize titer changes over repeated testing. Diagnostic test, level IV.

Clinical and epidemiological profile of alloimmunized and autoimmunized multi-transfused patients against red blood cell antigens in a blood center of Minas Gerais

BackgroundThe large diversity of red blood cell antigens favors, especially in multi-transfused patients, the occurrence of autoimmunization and alloimmunization with the risk of hemolytic transfusion reactions. Thus, this study aimed to determine the rates of alloimmunization and autoimmunization in these individuals, as well as the types of alloantibodies and their systems, clinical and epidemiological aspects and the frequency of autoimmunity in alloimmunized and non-alloimmunized patients. MethodsIn a retrospective study, 153 multi-transfused patients from 2006 to 2014 were evaluated. Sixty-eight had onco-hematological diseases, 64 had hemoglobinopathies and 21 had chronic renal failure. Descriptive analyses were carried out with the proportions being compared using the chi-square test, with the significance level set at 5%. ResultsThe Rh system was the most frequently involved (53.11%) and anti-E and anti-K (Kell system) were the most prevalent alloantibodies (21.87% each). Autoantibodies were found in ten patients (6.54%) with the percentages of autoimmunization in alloimmunized and non-alloimmunized individuals being 29.16% and 2.32%, respectively (p=0.0001). There was a significant difference between autoimmunization and the number of transfusions (16.21% in 6–10 vs. 5.26% <6 vs. 2.56% >10; p=0.0203) and diseases (19.04% in chronic renal failure vs. 6.25% in hemoglobinopathies vs. 2.94% in onco-hematological diseases; p=0.0329). ConclusionThe results show a strong correlation between alloimmunization and autoimmunization. Moreover, they reinforce the need for further studies on the clinical and epidemiological profile of multi-transfused patients in relation to alloimmunity and autoimmunity, especially the latter, for a better understanding of its etiopathogenesis and physiopathogenesis.

C3 Modulates RBC Antigen to Negatively Regulate Antibody Response

Background:Red blood cell (RBC) transfusion can result in the development of alloantibodies that can make it difficult to find compatible RBCs for future transfusions and increase the risk of hemolytic transfusion reactions. Despite the consequences of RBC alloimmunization, the factors that regulate this process remain relatively unknown. Recent studies suggest that complement deposition on an antigen surface can significantly enhance the immune response to foreign antigen. As many anti-RBC alloantibodies fix complement and RBCs otherwise lack known adjuvants, early antibody-mediated complement deposition may serve as a key regulator that enhances antibody production. To test this, we employed the KEL RBC model system, which employs RBCs that transgenically express the human KEL antigen specifically on RBCs (KEL RBCs). Using this system, we examined the immunological consequence of KEL RBC exposure following transfusion into C57BL/6 wild-type (WT) or complement component 3 (C3) knockout (KO) recipients.Methods: KEL RBCs were transfused into WT or C3 KO recipients, followed by serum collection on days 3, 5, 7, 14, and 21 post-transfusion. Antibody development in WT or C3 KO recipients was examined by flow crossmatch, where serum was incubated with KEL RBCs followed by antibody detection with fluorescently-tagged secondary anti-IgM and anti-IgG antibodies using flow cytometry. To determine the impact of complement deposition on the level of detectable antigen on the RBC surface, RBCs were labeled with the lipophilic dye, DiI, prior to transfusion and then sampled 1, 2, 3, 5, 7 and 9 days post-transfusion. The level of detectable KEL antigen, complement deposition, KEL RBC survival and antibody bound to the RBC surface was measured by flow cytometry. To examine the effect of complement deposition on the level of KEL protein in the RBC membrane post-transfusion, RBCs stroma was isolated at various time points post transfusion, followed by western blot analysis for the KEL protein.Results: While KEL RBCs induced robust anti-KEL antibody formation and C3 deposition in WT recipients, similar exposure to KEL RBCs in C3 KO recipients actually resulted in an unexpected increase in IgM and IgG anti-KEL antibodies when compared to WT recipients. To determine the consequence of C3 deposition, we examined the potential impact of antibody engagement and complement fixation on KEL antigen levels. Consistent with a potential role for complement in directly impacting KEL antigen availability to the immune system, KEL RBCs transferred into WT recipients experienced a decrease in the level of detectable KEL antigen over time that paralleled the development of anti-KEL antibodies and C3 deposition. In contrast, C3 KO recipients failed to experience the same degree of KEL antigen reduction despite the development of significant anti-KEL antibodies over this same time period. Western blot analysis of RBCs post-transfusion revealed that loss of detectable KEL antigen on the RBC surface paralleled a complete lack of detectable KEL antigen in RBC membranes, indicating that C3 may actually facilitate the removal of KEL from the RBC surface.Conclusion: These results suggest an unexpected role for C3 in negatively regulating antibody responses following RBC transfusion. The impact of C3 on the developing alloantibody response strongly suggests that C3-mediated loss of antigen over time likely reduces antigen availability to the immune system, thereby facilitating the inhibition of antibody production over time. These results not only provide novel insight into potential impact of antigen modulation on the development of an immune response to a RBC alloantigen, but also suggest a completely unexpected role for complement in negatively regulating alloantibody production. In doing so, these results suggest that unique differences in complement activity and overall activation following RBC alloantigen exposure between individuals may represent a previously unrecognized factor that influences alloantibody formation following RBC transfusion. DisclosuresNo relevant conflicts of interest to declare.

The importance of antibody screens after transfusions

The importance of antibody screens after transfusions

High Percentage of Evanescent Red Cell Antibodies in Patients with Sickle Cell Disease Highlights the Need for a National Antibody Database

Introduction: From 20 to 50% of patients with sickle cell disease are alloimmunized to red cell antigens from transfusions and/or pregnancies. Pre-transfusion testing (e.g. antibody screening) is essential to avoid hemolytic transfusion reactions from clinically significant antibodies, such as those to Rh, Kell, Duffy and Kidd antigens, among others. Unfortunately, alloantibodies may evanesce over time, becoming undetectable or even leading to a negative antibody screen. Furthermore, antibodies to low frequency antigens in the donor pool, mostly from Caucasians, may not be detected because they are not typically expressed on standard reagent red cells used for testing. Both of these facts contribute to the risk of hemolytic transfusion reactions despite negative pre-transfusion screening. To mitigate this risk, individual transfusion services maintain antibody histories of all patients indefinitely, and must refer to them prior to issuing every unit for transfusion.Patients and Methods: We analyzed data collected from the electronic medical records of every adult (19 and older) patient with sickle cell disease who had one or more positive antibody screen in our institution during a 2-year period from 2013-2015 to determine: 1. antibody specificities; 2. percentage of patients with at least one non-reacting alloantibody; 3. specificities of evanescent antibodies; and 4. number (percentage) of patients with antibodies to low frequency antigens.Results: We identified 71 patients, of which 5 were excluded because they only had a cold-reacting anti-M (n=2), anti-Lewis, a warm autoantibody (WAA), or a High Titer Low Avidity (HTLA) antibody (n=1 each). Thus, 66 patients were included in the analysis (62% females). The age range of the study-group was 19-59 years old (mean ± SD, 33 ± 11 years), similar between males and females (p = 0.43). Males tended to have an antibody screen ordered more often during the study-period, with a trend toward statistical significance (9.4 versus 5.0 times; p = 0.06). The total number of clinically significant alloantibodies was 218 with mean and median number per patient of 3.3 and 3.0, respectively. In addition, 16 patients also had a WAA. Anti-E was the most common alloantibody, followed by anti-C and anti-K; the table shows the antibodies identified at least once in 10% or more of the patients, as well as how often they were not reacting. Of 9 patients with anti-D, 7 were Rh positive, consistent with the propensity of African-Americans to express a D variant. Of note, 30 patients (45%) had antibodies to one or more low frequency antigen such as Cw, V, Vs, Goa, Jsa, Kpa, Lutheran b, Wra, and Ytb; 6 patients (9%) only had alloantibody(ies) to these antigens. Only 12 patients had a positive antibody screen every time they were tested. However, they had significantly fewer tests (average and median of 2, range of 1-6) compared with 54 patients with at least one antibody screen completely negative (average and median of 5 tests, range of 1-30) (p < 0.0001). Table 1Rh systemKellDuffyKiddMNSsOtherAntibody specificityDCEVGoaKJsaFyaFybJkbSAUSNumber of patients93040147201518912177Percentage of total antibodies4%14%18%6%3%9%7%8%4%5%8%3%Times not reacting516218312121078123Percent evanescent56%53%53%57%43%60%80%56%78%67%71%43%AUS-antibody of unknown specificity; Fya-Duffy a; Fyb-Duffy b; Jkb-Kidd b;Conclusions: Our results demonstrate that 81% of patients with sickle cell disease with a history of red cell alloantibodies had at least one test in which the antibody was not detectable. Delayed hemolytic transfusion reactions are the major risk of evanescent alloantibodies not known to the transfusion service preparing the units. Such reactions may even be fatal, especially if not promptly recognized, since they may be confused with a pain crisis. In addition, we noticed that almost half of the patients had antibodies to low frequency antigens that could have been missed during pre-transfusion testing. Considering that patients with sickle cell disease often suffer from lack of continuity of care, unawareness of their antibody history may lead to life-threatening transfusion reactions. We suggest that a national, or even regional, database of red cell alloimmunization in this patient population is warranted for increased transfusion safety. DisclosuresNo relevant conflicts of interest to declare.

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P(k)-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1 (k) phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1 (k) or P2 (k) erythrocyte units are preferentially selected for transfusion to P(k) patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels.

Alloantibody Formation In Sickle Cell Disease

BackgroundTransfusion of red blood cells (RBC) is a common intervention to treat and prevent complications in sickle cell disease (SCD). However, frequent transfusions may lead to erythrocyte alloimmunization, thereby complicating donor matching procedures and posing patients at risk for hemolytic transfusion reactions. Little information is available about the risk of alloimmunization of sickle cell patients living in European countries. In the Netherlands extensive matching procedures to prevent alloimmunization were introduced a decade ago, but the effect on alloimmunization has not been evaluated yet. AimsThe primary aim of this study is to evaluate the cumulative incidence of first alloantibody formation in a Dutch cohort of transfused SCD patients, and to compare this with a general Dutch RBC-transfused population. In addition, the effect of extended RBC matching protocols on the incidence of alloantibody formation in SCD and potential clinical determinants of alloimmunization will be assessed. MethodsWe conducted a retrospective cohort study and collected data on SCD patients (genotypes HbSS, HbSC, HbSβ0 and HbSβ+ thalassemia), diagnosed in three Dutch Sickle Cell Treatment Centers that received non-extended matched (ABO, RhD) RBC transfusions between 1984-2004 and extended matched (at least ABO, Rhesus phenotype, Kell) RBC transfusions between 2004-2011.In addition, we compared this population with a general population of 3 042 patients that received non-extended matched (ABO, RhD) RBC transfusions between 2005-2009 in the Leiden University Medical Center (Zalpuri et al. 2012). Cohorts were not matched for ethnicity. Alloimmunization risk was calculated as Kaplan-Meier incidence with cumulative number of transfusions as time variable. The association with the clinical determinants gender, SCD-phenotype and ethnicity was analyzed with Cox-regression analysis. ResultsA total of 291 SCD patients received 7 957 RBC units. Alloantibody formation occurred in 52 (17.9%) patients. The cumulative incidence of alloimmunization was 9% after 5 RBC units, 15% after 10, 24% after 20 and 34% after 40 RBC units. Multivariate analysis, correcting for the cumulative number of transfusions, demonstrated a significantly increased risk of alloantibody formation in our SCD cohort when compared to a general population of transfused patients (HR 7.5 (95% CI: 5.06-11.14), where the cumulative incidence of alloimmunization was 1.1% after 5, 2.4% after 10, 3.4% after 20 and 6.5% after 40 RBC units. No association could be demonstrated between alloantibody formation and clinical determinants such as gender, SCD-phenotype or ethnicity. However, a significant reduction in alloimmunization was observed in SCD patients that received their first transfusion from the year 2004 onwards, after preventive matching for Rhesus phenotype and Kell was introduced for SCD patients (HR 0.48 (95% CI: 0.24-0.97)). ConclusionThe overall rate of first RBC alloantibody formation in our cohort was 17.9% and the risk of alloimmunization increased substantially with an increasing number of RBC transfusions. A unique comparison with a general cohort of Dutch transfused patients demonstrates a significantly higher risk of alloantibody formation in SCD, acknowledging earlier findings. This may partially be explained by differences in RBC antigens between patients of African descent and the predominantly Caucasian donors. Besides the number of RBC units, no other clinical risk factors for allo-immunization in SCD could be identified. The effectiveness of extended RBC matching protocols in the prevention of alloimmunization for chronically transfused patients in the participating centers was confirmed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

KEL RBC Transfusion Induces IgG Anti-KEL Antibodies Independent Of CD4 T Cells

BackgroundWhile red blood cells (RBCs) transfusion can provide life saving therapy, patients who require chronic transfusion therapy may develop RBC alloantibodies that limit the availability of compatible RBCs for future transfusion and increase the risk of hemolytic transfusion reactions. However, not all patients generate alloantibodies following RBC exposure. Among factors that potentially influence RBC alloantibody formation, several studies suggest that a recipient's MHC class II repertoire may predict an individual's likelihood of responding to a particular RBC alloantigen. However, whether MHC class II antigens are required for efficient alloantibody formation following RBC transfusion remains unknown. As a result, we examined the potential role of MHC class II in the development of RBC alloantibodies following transfusion in a murine model of KEL alloimmunization. MethodsRBCs transgenically expressing the human KEL antigen specifically under a β-globin promoter (KEL RBCs) were transfused into C57BL/6, C57BL/6 MHC class II knock out (KO) or KEL transgenic control recipients. Following transfusion, blood was harvested on days 3, 5, 7, 14, 21 and 28 following transfusion and serum was analyzed for IgM or IgG anti-KEL antibodies by indirect immunofluorescence using flow cytometry with KEL and control C57BL/6 RBCs as targets. To deplete CD4 T cells, mice were injected with anti-CD4 (clone GK1.5) 4 and 2 days prior to transfusion. As a control, additional C57BL/6 recipients were similarly injected with an isotype control. C57BL/6 recipients were also injected in parallel with GK1.5 or isotype control followed by splenocyte examination for CD4 T cell depletion using anti-CD3 and an anti-CD4 clone that recognizes a different CD4 epitope than GK1.5 (clone RM4-5). All experiments were completed at least three times with 3–5 recipients per group per experiment. ResultsTransfusion of KEL RBCs resulted in significant IgM anti-KEL antibody formation that peaked approximately 5 days following transfusion in both C57BL/6 and C57BL/6 MHC class II KO recipients. Similarly, IgG anti-KEL antibodies could also be detected in C57BL/6 or C57BL/6 MHC class II KO as early as 7 days following transfusion and continued to rise to similar peak levels within 14 to 21 days following KEL RBC transfusion. Injection of GK1.5, but not isotype control antibody, depleted CD4 T cells to less than 1% of their original level. Transfusion of KEL RBCs into C57BL/6, CD4 depleted C57BL/6 or isotype control treated C57BL/6 resulted in similar levels of IgM anti-KEL antibody that peaked approximately 5 days following transfusion. Likewise, transfusion of KEL RBCs induced similar levels of IgG anti-KEL antibodies within 7 days following transfusion that also peaked between 14 and 21 days in C57BL/6, CD4 depleted C57BL/6 or isotype control treated C57BL/6 recipients. (All the above differences achieved a p value of <0.05) ConclusionsDespite the potential role of CD4 T cells in facilitating RBC alloantibody formation, these results suggest that significant IgG RBC alloantibody can occur independent of MHC class II or CD 4 T cells. Although it remains possible that CD4 T cells become activated following RBC alloantigen exposure, the lack of CD4 T cell requirement in this model suggests that some patients may be capable of mounting a clinically significant immune response following RBC transfusion in the absence of CD4 T cell help. As a result, MHC antigen presentation of unique RBC alloantigens may not be necessary for RBC alloimmunization to occur. Disclosures:Zimring:Immucor Inc.: Research Funding; Terumo: Research Funding; Haemonetics: Consultancy; Cerus: Honoraria.

Risks of hemolysis due to anti‐A and anti‐B caused by the transfusion of blood or blood components containing ABO‐incompatible plasma

The increasing use of fresh blood group O whole blood in acute trauma medicine makes it important to reevaluate the issue of hemolytic reactions related to the transfusion of ABO-incompatible plasma. This review summarizes and evaluates published articles and case reports concerning hemolytic reactions in connection with the transfusion of group O whole blood or blood products to nongroup O recipients. In 1945-1986, 15 nonmilitary publications reported hemolytic transfusion reactions with group O blood/blood products. All patients recovered except for two fatalities. Late in World War II and during the Korean and Vietnam wars and onward in Iraq and Afghanistan only "low anti-A, anti-B titer" group O whole blood has been used as universal blood. In spite of a large number of units transfused, there are no reports of hemolytic reactions. Twenty-five publications report hemolytic reactions after transfusion of group O platelets to nongroup O recipients. In all patients but one, the titer of the implicated A- or B-antibody was >100 (saline) or >400 (antiglobulin) and all cases with an infused volume of incompatible plasma <200 mL were related to anti-A or anti-B antiglobulin titers >1000. In emergency lifesaving resuscitation, the risk of hemolytic transfusion reactions from transfusion of group O blood to nongroup O recipients constitutes risk that is outweighed by the benefits. A low titer of anti-A/B will minimize the risk for a hemolytic reaction, particularly if the screening is repeated after an immunization episode, e.g., blood transfusion, vaccination, or pregnancy.

Sensitization to Multiple Rh Antigens by Transfusion of Random Donor Platelet Concentrates in a -D- Phenotype Patient

The -D- phenotype is a rare Rh phenotype that strongly expresses D antigen without C, c, E, or e antigens. In -D- phenotype individuals, anti-Rh17 (Hro) is commonly found if there is a history of pregnancy or transfusion with red blood cells (RBCs) that express C, c, E, or e antigens. We report the first case of a -D- phenotype patient with multiple Rh antibodies including anti-Rh17 who had a history of two occasions of transfusion with eight random donor platelet concentrates two and six years ago. We found that a trivial amount of RBCs in the platelet components was able to trigger sensitization to RBC antigens, especially the highly immunogenic and clinically significant Rh antigens, including C, c, E, e or CcEe polypeptides. To avoid unnecessary sensitization and to minimize the risk of hemolytic transfusion reactions in patients with this rare Rh phenotype, a modified strategy for pretransfusion screenings needs to be discussed in the field of transfusion medicine.

ABO antibody titers are not predictive of hemolytic reactions due to plasma-incompatible platelet transfusions.

The overall risk of hemolytic transfusion reactions (HTRs) from plasma (minor)-incompatible platelet (PLT) transfusions and the role of a critical anti-A or anti-B titer in predicting and preventing these reactions has not been clearly established. We evaluated all apheresis PLT (AP) transfusions for 3 months. Using the gel titer method, we determined the anti-A and/or the anti-B immunoglobulin (Ig)G titer for all incompatible APs. Reported febrile transfusion reactions and HTRs were recorded; transfusions were not prospectively evaluated by the study team. A posttransfusion direct antiglobulin test (DAT) and eluate were performed after a reported febrile or hemolytic reaction for patients who received plasma-incompatible APs. A total of 647 of 4288 AP transfusions (15.1%) were plasma incompatible. Group O APs (n = 278) had significantly higher anti-A and anti-B titers than group A or B APs (p < 0.0001). No group A or B APs had a titer of more than 128 (0/342). For group O APs, 73 had titers of 256 or greater (26.3%), and 27 had titers of 512 or greater (9.7%). No HTRs were reported to any plasma-incompatible AP transfusion during the study period. Two plasma-incompatible AP transfusions were associated with fever and chills and positive DATs, of which one had a positive eluate. The incidence of a DAT and eluate-positive febrile transfusion reaction in the plasma-incompatible AP population is 0.15% (95% confidence interval, 0.0%-0.86%). A critical anti-A or -B titer is not sufficient to predict the risk of hemolysis in patients receiving plasma-incompatible APs, although underreporting of reactions to the blood bank may limit the generalizability of this study.

Applying molecular immunohematology discoveries to daily transfusion practice

Applying molecular immunohematology discoveries to daily transfusion practice

Emergency uncrossmatched transfusion effect on blood type alloantibodies

Trauma patients receive emergency transfusions of unmatched Type O Rh-negative (Rh-) blood until matched blood is available. We hypothesized that patients given uncrossmatched blood may develop alloantibodies, placing them at risk for hemolytic transfusion reactions (HTRs). Data regarding alloantibody profiles and HTR occurrence were collected from the records of trauma patients at our university-based trauma center who received emergency uncrossmatched blood from July 2008 to August 2010. A total of 132 patients received 1,570 units of packed red blood cells. Mean injury severity score was 28 ± 1.3. Forty-five (34%) patients died: 27 on hospital day 1; the remaining 18 had no evidence of HTR before death. Four Rh- female patients received Rh+ fresh frozen plasma, but none received Rh+ packed red blood cells. Three Rh- male patients received both Rh+ packed red blood cells and fresh frozen plasma, and one received Rh+ fresh frozen plasma. One patient developed anti-Rh D antibodies. None experienced HTR. One female patient had HTR from reactivation of anamnestic JK antibodies. Thirteen (33%) of 39 patients met criteria for HTR based on urinalysis and 29 (40%) of 72 patients tested met criteria for HTR based on hemoglobin and bilirubin values. Only one patient had confirmed HTR. High rates of injury recidivism in trauma patients increase the likelihood of multiple blood transfusions during their lifetime. Rh- patients who receive Rh+ blood are at risk of developing anti-Rh antibodies, putting them at risk for HTR. The conservation of Rh- blood for use in female patients may be detrimental to Rh- male patients. Laboratory diagnostic criteria for HTR are nonspecific in the trauma population and should be used with caution.

Delayed hemolytic transfusion reaction in children with sickle cell disease

Transfusion is a cornerstone of the management of sickle cell disease but carries a high risk of hemolytic transfusion reaction, probably because of differences in erythrocyte antigens between blood donors of European descent and patients of African descent. Patients may experience hemolytic transfusion reactions that are delayed by from a few days to two weeks and manifest as acute hemolysis (hemoglobinuria, jaundice, and pallor), symptoms suggesting severe vaso-occlusive crisis (pain, fever, and acute chest syndrome), and profound anemia, often with reticulocytopenia. This case-series study aims to describe the main characteristics of this syndrome, to discuss its pathophysiology, and to propose a management strategy. We identified 8 pediatric cases of delayed hemolytic transfusion reactions between 2006 and 2009 in the database of the Necker Hospital, France. All patients had received cross-matched red cell units compatible in the ABO, RH, and KEL systems. We reviewed the medical charts in the computerized blood transfusion databases. All patients were admitted to the intensive care unit. We progressively adopted the following strategy: intravenous immunoglobulins, and darbopoietin alpha when the reticulocyte count was below 150×10(9)/L, without further blood transfusion during the acute episode unless absolutely necessary. The median time between the transfusion and the diagnosis of delayed hemolytic transfusion reaction was six days. All patients had severe bone pain; all but one had a high-grade fever. Five patients had hemoglobin levels less than than 4 g/dL and 3 had reticulocytopenia. In 5 patients, no new antibody was found; one patient had weakly reactive antibodies. Only 2 patients had new allo-antibodies possibly responsible for the delayed hemolytic reaction. The initial symptoms of delayed hemolytic transfusion reaction were complex and mimicked other complications of sickle cell disease. In most of our cases, no new antibody was identified, which underlines the complexity of the pathophysiology of this syndrome.