Red Blood Cell Alloantibodies Research Articles

Red Blood Cell Alloantibody Titration - Does the Titration Method Matter?

Red blood cell (RBC) alloantibody titration is a quasi-quantitative method to assess antibody concentration and is considered a useful means of estimating maternal alloimmunization during pregnancy. Traditionally, titration is performed using conventional tube test (CTT). The gel microcolumn agglutination-based method (GMA) has been proven reliable for many immunohematology tests. Our study compared CTT with GMA of two different, commercially available GMA systems for RBC alloantibody titration. Serum samples with significant RBC-alloantibodies were evaluated in our study. Each sample was titrated concurrently with CTT, with ID-DiaMed-GmbH, Cressier, Switzerland (GMA1), and with DG Gel Coombs Diagnostic Grifols, Passeig Fluvial, Spain (GMA2). One hundred thirty-seven titration tests including 50 anti-D, 25 anti-Kell, 10 anti-E, 9 anti-Jka, 8 anti-c, 5 anti-Cw, 5 anti-Fya, 7 anti-M, 6 anti-Kpa, 3 anti-Lua, 1 anti-e, 3 anti-G, and 2 anti-Cha were performed and evaluated. Samples tested by CTT versus GMA1 and GMA2 generated mostly equal or higher titers by GMAs. The results of both comparisons were in good agreement (W = 0.91, p < 0.0001, and W = 0.92, p < 0.0001, respectively). For all antibody specificities, the mean absolute difference in titers ranged from 1 - 3 for both GMA1 and GMA2 versus CTT. Samples tested by GMA1 vs. GMA2 were in almost perfect agreement (W = 0.95, p < 0.0001). Although both GMAs were found slightly more sensitive than CTT for alloantibody titration, the differences were not significant and the agreement between all methods was very good, possibly indicating GMA as a suitable alternative to CTT in RBC antibody titration.

Elimination of HLA antibodies by platelet adsorption

The presence of HLA antibodies in patient serum or plasma may make antibody identification difficult. These HLA antibodies may mask the presence of clinically significant red blood cell (RBC) alloantibodies. Because platelets strongly express HLA antigens, it is possible to remove HLA antibodies by adsorbing the patient's serum or plasma using a platelet pool. Large numbers of random platelets are pooled to ensure a wide variety of HLA types are present. Elimination of the reactivity after adsorption suggests the presence of HLA antibodies in the patient's serum or plasma. The adsorbed patient sample may then be used to evaluate RBC alloantibodies without HLA antibody interference.

Extended phenotyping of blood group antigens: Towards improved transfusion practices

Even though the International Society of Blood Transfusion has defined 38 blood group systems, only ABO and RhD are matched while selecting a compatible unit for blood recipients of Indian origin. Genetic disparity between a donor and a patient with reference to minor blood group antigens creates the risk of alloimmunization. The presence of red blood cell alloantibodies further creates the potential for serologic incompatibility, makes the selection of appropriate units for future transfusion more difficult, delays blood transfusion, and presents the risk of hemolytic disease of the newborn. Hence, there is a need to carry out extended blood group typing for antigens of clinical importance among donors and patients so that prophylactic antigen-matched blood can be given to a patient. Provision of antigen-matched blood will help in management of alloimmunized transfused-dependent patients carrying alloantibodies and/or autoantibodies. Typing of blood group antigens among large number of donors will also help in the development of antigen-negative inventories, develop indigenous red cell panels, and identify rare donors. Selection of blood group antigens for typing is important as it may not be always feasible to phenotype all the antigens. Based on the prevalence and immunogenicity of an antigen in a population, specific antigens have to be selected for typing so that the risk of alloimmunization reduces by strategic level of antigen matching. The aim of this review is to highlight the need for extended blood group phenotyping in different case scenarios and the methods available to do so. This review was performed by searching for keywords such as blood transfusion, red cell alloimmunization, partial antigen matching, rare blood donors, multitransfused patients, transfusion in thalassemics, molecular blood group genotyping, serology, and reagent red cell panels in PubMed and Google databases.

Intrapartum Management of Sickle Cell Anemia With Rare Antibody and Minimal Blood Availability

Sickle cell disease (SCD) can pose serious maternal and fetal risk in pregnancy. Transfusion, both during and outside of pregnancy, can improve patient morbidity and mortality but carries risk of alloimmunization, complicating future management. This case describes a 29-year-old gravida 1, para 0 woman with sickle cell anemia and rare red blood cell alloantibody (anti-Rh46) who presented with severe vaso-occlusive crisis at 29 weeks with hemoglobin of 7.6 g/dL. Only one unit of compatible blood existed in the country. Planning for transfusion with least-incompatible blood was made. She ultimately underwent cesarean section at 31 weeks and 2 days for abnormal fetal testing. This case highlights that blood products should be utilized judiciously because their adverse effects, like alloimmunization, can increase patient morbidity and mortality.

Diagnostic value of laboratory monitoring to predict severe hemolytic disease of the fetus and newborn in non-D and non-K-alloimmunized pregnancies.

Pregnant women are routinely screened for red blood cell (RBC) antibodies early in pregnancy. If RBC-alloantibodies are detected, repeated laboratory testing is advised to timely identify pregnancies at high risk for severe hemolytic disease of the fetus and newborn (HDFN). We assessed for RBC alloantibodies, other than anti-D or anti-K, cut-offs for the titer and the antibody dependent cellular cytotoxicity (ADCC) test to select high-risk cases. To advise on test repeat intervals, and to avoid unnecessary testing, we evaluated the chance for exceeding the cut-offs for Rh antibodies other than anti-D, Jk, Fy, and S/s antibodies. Diagnostic value of antibody titer and ADCC test was determined with data from a prospective index-cohort study, conducted in 2002-2004. Laboratory test outcomes were from a recent observational cohort (2015-2016). A titer cut-off of ≥16 showed a sensitivity of 100% (95% CI:73-100%) and a positive predictive value (PPV) of 17% (95% CI:14%-20%). The percentage of pregnancies reaching a titer above the cut-off of ≥16 varied from 0% for anti-Jka /Jkb (n = 38) to 36% for anti-c (n = 97). The ADCC test showed no cut-off with a 100% sensitivity. However, in cases with a titer ≥16 and an ADCC test ≥30% a PPV of 38% was obtained to detect severe HDFN. A titer cut-off of ≥16 is adequate to detect all cases at risk for severe HDFN; the ADCC test may add a more accurate risk estimation. Repeated testing is recommended in pregnancies with anti-c. In pregnancies with other Rh antibodies a repeated test in the third trimester is recommended.

How do I incorporate red cell genotyping to improve chronic transfusion therapy?

Children with transfusion dependent anemia, such as sickle cell disease (SCD) and thalassemia, are at an increased risk for developing red blood cell (RBC) alloantibodies due to their lifelong need for transfusion therapy. With the advent of genotyping, extended RBC antigen typing can be incorporated into chronic transfusion therapy programs (CTTPs) to improve patient care and provide antigen matched blood for this population of patients. The hospital, blood center (BC), and hematology clinic caring for children requiring long-term transfusion support developed a CTTP. Genotyping was performed at entry to determine patient RBC antigen type. Limited versus extended antigen matching of transfusions was provided based on known RBC antibodies. Fifty patients with the following disorders were enrolled: 20 with SCD, 23 with thalassemia, and 7 with other disorders. At enrollment, nine (18%) had RBC alloantibodies, including six (30%) of patients with SCD and three (13%) with thalassemia. Two children developed antibodies after enrollment; one warm autoantibody following limited "CEK" matched RBCs and one patient with a hemizygous variant RHD allele developed anti-D. Six (30%) patients with SCD had variant RHCE alleles; two had homozygous variant alleles and four had a variant present along with a wild type allele. We demonstrate how a CTTP can be developed in a community hospital through collaboration with the blood supplier, hospital, and clinical care team. A model of incorporating RBC genotyping informs risk for alloimmunization and allows consideration of transfusion strategy for providing prophylactic antigen matched blood.

Results of Unrelated Donor Hematopoietic Stem Cell Transplantation for Sickle Cell Disease in Europe on Behalf of Paediatric Diseases (PDWP) and Inborn Errors Working Parties (IEWP) of the EBMT

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is, to date, the only curative treatment for sickle cell disease (SCD). Because a human leukocyte antigen (HLA) matched sibling donor is not always available, alternative stem cell sources such as unrelated or haploidentical related donors have been explored. The likelihood of finding a 10/10 (HLA-A, B, C, DRB1 and DQB1) matched donor varies among ethnic groups, with the lowest probability among individuals of African descent. To date, few series of SCD patients transplanted with an unrelated donor (UD) have been reported, but the high rates of rejection and chronic graft versus host disease (cGvHD) have limited its widespread application. Patients and methods: We report the results of a retrospective, registry based, survey on 70 UD HSCT performed in patients (pts) with SCD from UD in 22 European Society for Blood and Marrow Transplantation (EBMT) centers between 2005 and 2017. Data were collected from the EBMT database and missing information was updated by the centers. Median follow up was 38 (range 2-154) months. Most pts were HbSS (n=54; 78%), had positive serology for CMV (80%), and a Karnofsky score &gt;80% (98%). Eighteen pts had a major ABO incompatibility. Recurrent vaso-occlusive crisis (n=58), cerebral vasculopathy (n=23) and acute chest syndrome (n=24) were the main indications for HSCT. Red blood cell (RBC) transfusions pre-HSCT were reported in 97% of pts of whom 53% received more than 20 transfusions; 14% of the transfused pts had RBC alloantibodies. Hydroxyurea pre-HSCT was used in 65% of pts. Median age at HSCT was 9.6 years (range 2-43) with 87% of pts being ≤ 16 years. Stem cell source was bone marrow (BM) in 55 pts (79%) and peripheral blood (PBSC) in 15 (21%). The median number of infused TNC /kg was 3.6 x 108 for BM and 7.1 x 108 for PBSC; the median number of infused CD34/kg was 4.4 x 106 for BM and 8.3 x 106 for PBSC. HLA matching at high resolution typing was 10/10 (HLA-A, B, C, DRB1 and DQB1) in 31, 9/10 in 17 and 8/10 in 4 of the patient-donor pairs; intermediate resolution typing was available for 10 (10/10 or 9/10) and the HLA information was missing for the remaining 8 patient-donor pairs. The most frequent conditioning regimens were fludarabine-thiotepa-treosulfan (64%) and busulfan- cyclophosphamide (12%). GvHD prophylaxis was cyclosporine plus methotrexate in 59%. Anti-thymocyte globulin was used in 90% and alemtuzumab in 9% of pts. Results: The cumulative incidence (CI) of neutrophil engraftment at 60 days was 93% (95% CI 76-100), with median time to engraftment of 18 days; platelet engraftment at 180 days was 90% (95% CI 83-98) with a median time of 20 days. Ten pts had graft failure (5 primary and 5 secondary) of whom 6 had a second transplant and were all alive at last FU (median 9.5 months after second HSCT). The CI of grade II-IV aGVHD at 100 days was 23% (95% CI 15-36), and 8 pts (11%) had grade III-IV. Acute GVHD was more frequent in patients who received PBSC (PBSC 42.9%, BM 18.2%, p=0.062). Three-year CI of cGVHD was 23% (95% CI 15-36), 7 pts (10%) had limited and 9 (13%) extensive cGvHD. Three-year overall survival (OS) was 90±4%; three-year event free survival (EFS) (considering death and graft failure as events) was 76±6%; HLA matching between donor and recipient was the most important factor for OS and EFS. Considering only pts-donor pairs with high resolution HLA typing available (n=52), 3-year OS was 96±4% in 10/10 group compared to 77±11% in 9/10 plus 8/10 group (p 0.065), 3-year EFS was 85±7% vs 62±12% (p 0.040), respectively. No significant differences between the groups were observed in CI of neutrophil engraftment, aGVHD and cGVHD. Conclusion: UD HSCT is a valid option for SCD patients who lack an HLA-identical sibling donor. Nevertheless, efforts are still needed to improve outcomes after UD HSCT. Our results indicate that using a 10/10 HLA matched UD improves both OS and EFS compared to donors with 1 or more mismatches; so, when such a matched unrelated donor is not found, using an haplo relative or an unrelated cord blood as donor source should be evaluated. A prospective trial is in preparation to evaluate the use of haploidentical donors for HSCT in SCD (EudraCT number: 2018-002652-33). Disclosures No relevant conflicts of interest to declare.

Validation of Daraex to Resolve Daratumumab-Induced Interferences in Pre-Transfusion Screen Tests

Daratumumab is a CD38-directed antibody increasingly used for the treatment of adult patients with multiple mieloma. The membrane of red blood cells express CD38 and thus samples from patients treated with daratumumab show agglutination in red blood cell antibody screen tests performed prior to transfusion. This interference hinders the detection of red blood cell alloantibodies. Published literature has described a method to eliminate CD38 in red blood cells with DTT (Chapuy, 2016). This technique is cumbersome, requires positive and negative controls as DTT destroys Kell antigens and can produce in vitro hemolysis. The increasing number of multiple myeloma patients treated with daratumumab poses the need for a simple and straightforward technique with applicability in standard transfusion centers. DaraEx (Inno-Train) is a new anti-CD38 neutralizing agent that overcomes daratumumab-induced interferences detected in pre-transfusion tests without the major drawbacks associated with the DTT technique. Our aim was to validate and implement DaraEx as the method of choice to solve daratumumab interferences detected in pre-transfusion screen tests in a tertiary care center. A two-step approach using in vitro and in vivo samples was designed to validate the new method. First, we compared DaraEx efficacy in vitro to the reference DTT method in two samples spiked with daratumumab to achieve a concentration of 10mg/mL (Sample A: serum from a patient without known red blood cell alloantibodies; Sample B: serum from a patient with alloantibody anti-c). Red blood cells in the screen test (3 red blood cell screen; ID-DiaCell I-II-III) as well as positive (E+ red blood cells) and negative controls (K+ red blood cells) were treated with DTT 0.2M solution for 30 minutes at 37ºC and then washed four times with saline. In parallel, red blood cells in the screen test were incubated during 30 minutes at room temperature in a shaker (600rpm) with DaraEx. Red blood cells treated with each of these methods were used for indirect antiglobulin test with our gel card system (BioRad; IH-1000). Preference of method in terms of time needed and result interpretation was evaluated by three hematologists specialized in blood banking and four different technicians. Secondly, we tested pre-transfusion samples from patients treated with daratumumab with the DaraEx technique to check in vivo efficacy. There was a 100% concordance between both techniques (DDT reference method and DaraEx new method) in both in vitro samples. All hematologists and technicians found the DaraEx technique less cumbersome in terms of processing and time to result (2 hours with DTT versus 1 hour with DaraEx) and the interpretation straightforward. Twelve samples with daratumumab-induced interference in pre-transfusion screen tests belonging to 5 patients were tested between January and July 2019. All the interferences detected resolved with DaraEx regardless of time from last daratumumab administration (range: 7-145 days; mean: 57 days). Figure 1 shows screen test with and without treatment with DaraEx in a patient sample. In our experience, DaraEx technique is a simple, fast and efficacious method, regardless of time from last daratumumab administration, to resolve interferences secondary to daratumumab administration without the major disadvantages associated with DTT. Figure 1 Disclosures García Gutiérrez: Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

The Presence and Persistence of Pregnancy-Associated Red Blood Cell Alloantibodies in Blood Donors

Background: Females have higher RBC alloantibody prevalence than males, presumably due to exposure to non-self RBC antigens through pregnancy as well as transfusion. Given the lack of routinely available and longitudinal data on lifelong pregnancy and transfusion histories, few studies have been able to distinguish pregnancy versus transfusion as contributing risk factors for RBC alloantibody induction or for the persistence of RBC alloantibody detectability. We hypothesized that pregnancy would be an important source of persistently detected RBC alloantibodies and investigated this in a longitudinal blood donor database. Study Design and Methods: Donor/donation data were obtained from 4 US blood centers from 2012-2016. RBC antibody screen results and antibody identification (using tube, gel, or solid phase), donor demographics, pregnancy history, and transfusion history were evaluated. Blood donors were included in this analysis if they had an RBC alloantibody detected at any given donation as long as they had at least one subsequent blood donation and antibody screen. Anti-D antibodies detected, but present for &lt; 6 months in females &lt;50 years old were presumed due to passive RhIg and were excluded from this analysis. RBC alloantibody persistence was defined as an RBC alloantibody detected at each subsequent donation following its initial identification and RBC alloantibody evanescence was defined as one or more subsequent donations without re-detection of the alloantibody. Rates of donor alloantibody persistence were calculated as blood donors with persistent alloantibodies divided by blood donors with any alloantibody, and patterns of persistence were characterized by donor sex and presumed alloantibody source exposure. Results: 503 blood donors had a detectable RBC alloantibody and a follow-up antibody screen; 374 (74%) were female and 130 (26%) were male. Of the 374 alloimmunized females, 210 (56%) reported prior pregnancy and no transfusion, 124 (33%) reported prior pregnancy and transfusion, 26 (7%) reported no pregnancy or transfusion, and 12 (3%) reported prior transfusion and no pregnancy. 215/334 (64%) of previously pregnant females had RBC alloantibodies that remained detectable throughout the duration of the study. The mean duration of detectability for persistent antibodies in previously pregnant females was 640 days, compared to a mean time to disappearance of 183 days for evanescent antibodies. In contrast, males and all never-pregnant females were less likely to have persistently detectable antibodies, with 52% (87/167) being in this category (chi square p = 0.008). The mean duration of detectability for persistent antibodies in males and never-pregnant females was 576 days, compared to a mean time to disappearance of 290 days for evanescent antibodies. Previously pregnant females with a reported history of prior transfusion (n=124) had the highest rate (77%) of RBC alloantibody persistence. The alloantibody specificities of previously pregnant females included E (96), D (81), K (78), Fy (21), C (47), MNS (38), and Jk (18). Of these, Fy and non-passive D were most likely to be persistent (each 86%), followed by C (79%), K (74%), E (59%), Jk (55%), and MNS (34%). Conclusion: Data from this multi-center blood donor database, with documented life-long pregnancy and transfusion histories, highlight the role that pregnancy plays in RBC alloimmunization. Importantly, pregnancy was an exposure source in almost all studied RBC alloimmunized female healthy blood donors and was the sole reported exposure in the majority of donors. Although these data cannot definitively identify the source of non-self blood group antigen exposure in blood donors with both pregnancy and transfusion histories, they show that antibodies in previously pregnant females are long lasting. It is possible that length of exposure to non-self RBC antigens, the relatively young age of females during childbearing, long standing fetal white blood cell microchimerism in the maternal circulation, or other variables may impact plasma cell longevity and/or antibody production, and thus the persistence of pregnancy (versus transfusion) associated RBC alloantibodies. Given the numbers and persistence of such RBC alloantibodies, we recommend that pregnancy history be taken into consideration in all RBC alloimmunization studies involving females at or beyond childbearing age. Disclosures Spencer: HemaStrat, LLC: Membership on an entity's Board of Directors or advisory committees.

Mechanisms of alloimmunization in sickle cell disease.

Red blood cell (RBC) transfusion is an important treatment for some complications of sickle cell disease (SCD). On the contrary, transfusion may lead to alloimmunization to RBC antigens, with such alloantibodies putting patients at risk for acute or delayed hemolysis, and increasing the difficulty of finding compatible RBCs. Patients with SCD are more susceptible to developing RBC alloantibodies than other multiply transfused patient populations, for reasons that are not completely understood. In this review, we summarize the available data about risk factors and underlying mechanisms associated with RBC alloimmunization in SCD. Although RBC antigen matching between blood donors and transfusion recipients can decrease alloimmunization, complete matching at all loci is not feasible. Patients with SCD show evidence of increased inflammation at baseline and in times of illness. Resultant changes to the innate and adaptive immune systems presumably influence the development of RBC alloantibodies as well as RBC autoantibodies. The inflammation and immune dysregulation associated with SCD may be therapeutic targets for preventing the formation of antibodies and/or for mitigating the dangers of existing RBC alloantibodies. As long as RBC transfusion therapy remains an important treatment for SCD, the quest to improve its safety profile will continue.

PP-61 - Red blood cell alloantibodies in thalassemia major patients: an analysis from Istanbul

PP-61 - Red blood cell alloantibodies in thalassemia major patients: an analysis from Istanbul

Type 1 IFN signaling critically regulates influenza-induced alloimmunization to transfused KEL RBCs in a murine model.

Only a fraction of red blood cell (RBC) transfusion recipients form alloantibodies, and variables determining responsiveness or nonresponsiveness are poorly understood. We and others have previously shown in animal models that pretreatment with toll-like receptor agonists that mimic different types of infections impacts the magnitude or frequency of RBC alloantibody responses. We hypothesized that influenza infection, coexistent with transfusion, would impact responses to transfused RBCs in a manner dependent on Type 1(α/β) interferon (IFN) signaling and tested this in a murine model. Wild-type mice or mice lacking the ability to respond to Type 1 IFN were infected with influenza prior to the transfusion of transgenic murine RBCs (K1) expressing the human KEL glycoprotein or the triple fusion HOD protein. Alloantibody responses were measured longitudinally after transfusion by flow cytometric crossmatch, and posttransfusion RBC recovery and survival was evaluated. Influenza-infected mice transfused with K1 RBCs developed robust anti-KEL alloantibodies, whereas animals transfused in the absence of infection remained nonresponders; influenza-associated RBC alloimmunization was also observed after transfusion of HOD RBCs. Recipient Type 1 IFN production was critical to the mechanism of action of influenza-induced RBC alloimmunization, with alloimmunization being significantly decreased in mice unable to sense Type 1 IFN (through antibody blockade or genetic approaches). These and other data suggest that Type 1 IFN responses to toll-like receptor agonists or infections regulate RBC alloantibody responses. Studies investigating whether such a correlation exists in humans may be informative.

Red blood cell alloimmunization is associated with lower expression of FcγR1 on monocyte subsets in patients with sickle cell disease.

Despite the clinical significance of red blood cell (RBC) alloantibodies, there are currently no laboratory tests available to predict which patients may be at risk of antibody formation after transfusion exposure. Given their phagocytic and inflammatory functions, we hypothesized that differences in circulating monocytes may play a role in alloimmunization. Forty-two adults with sickle cell disease (SCD) were recruited, with data extracted from the electronic medical record and peripheral blood analyzed by flow cytometry for total monocytes, monocyte subsets (CD14 high/CD16 low+ classical monocytes, CD14 high/CD16 high+ intermediate monocytes, and CD14 intermediate/CD16 high+ non-classical/inflammatory monocytes), and FcγR1 (CD64) expression. Thirteen "non-responder" patients (non-alloimmunized patients with documented RBC transfusion at the study institution) were compared to 20 alloimmunized "responder" patients, who had a total of 44 RBC alloantibodies identified. There were no significant differences in the percentages of total monocytes, monocyte subsets, or measured cytokines between non-responders and responders. However, non-responders had higher CD64 expression on classical monocytes (MFI mean 3424 ± standard deviation 1141) compared to responders (MFI mean 2285 ± 1501), p = 0.029, and on intermediate monocytes (MFI mean 3720 ± 1191) compared to responders (MFI mean 2497 ± 1640), p = 0.033. Monocytes and the inflammatory milieu increasingly are being appreciated to play a role in some complications of SCD. The differences in FcγR1 expression on monocyte subsets noted between responders and non-responders, which cannot be directly explained by the serum cytokines evaluated, warrant further investigation.

The role of improved pre‐transfusion testing in the prevention of delayed serologic transfusion reactions among blood recipients in Uganda: a Randomized Controlled Trial (IPAT Study)

Background and objectivesThe goal of pre‐transfusion testing (PTT) is to provide patients with beneficial and safe transfusions. In Uganda, PTT includes ABO/RhD typing plus room temperature (RT) saline cross‐matches without red‐blood‐cell (RBC) alloantibody screening. The aim of the IPAT study was to assess the role of improved PTT in the prevention of delayed serologic transfusion reactions (DSTRs).Materials and methodsIn this randomized controlled trial, patients at Mbarara Hospital in Uganda, with a history of RBC exposure, were randomized 1:1 to have either RBC alloantibody screening (SCREEN group) or room temperature saline cross‐matches (CONTROL group) during PTT. ‘Home‐made’ reagent RBCs from group O RhD‐positive volunteers were used for antibody screening in the indirect antiglobulin test. Participants were evaluated for RBC alloantibody production 7–14 days after transfusion. Post‐transfusion haemoglobin estimation and direct antiglobulin tests (DATs) were also performed.ResultsWe randomized 220 patients to either the SCREEN or CONTROL group. Both study arms had similar demographic and transfusion characteristics at baseline. There were 19 (17·3%) individuals in the CONTROL group with DSTRs compared to 8 (7·3%) in the SCREEN group at the time of follow‐up (P = 0·02). Overall, post‐transfusion DATs were positive in 7 (3·5%) patients but there was no associated decrease in haemoglobin levels.ConclusionRed‐blood‐cell alloantibody screening is associated with occurrence of significantly fewer DSTRs. The use of ‘home‐made’ reagent cells during PTT in Uganda is feasible. We recommend a change in the local PTT policy to consider the introduction of RBC alloantibody screening.

Red cell alloimmunization among haemato-oncologic patients in a teaching hospital in Malaysia

Background: The aim of the study is to estimate the prevalence and specificity of red blood cell alloantibody among haemato-oncologic patients and to correlate the association of antibody development with other factors such as age, gender, race, number of packed red blood cell (RBCs) transfused and diagnosis. Materials and Methods: This prospective cross-sectional study was conducted in Transfusion Medicine Unit, HUSM. Clinical and serological data of 216 haemato-oncologic patients who who received blood transfusions were collected and analysed. The blood samples were subjected to the standard immunohematological procedure for RBC antibody screening and identification using reagents of Diamed-ID Gel microtyping system Results: RBC alloimmunization rate among haemato-oncologic patients was 3.2%. Red cell antibodies were detected in seven patients. Four patients developed single antibody, while three develop multiple antibodies. However, we noted all seven patients were Malay with lymphoproliferative disease and majorities were adult male (71.4%) patients. Conclusions: The prevalence of RBC alloimmunization in post transfusion haemato-oncology patients was low despite of multiple transfusions and it is comparable with other diseases, which required multiple transfusions. Therefore, it is important to prioritize the extended antigen matching to patients that will be most benefit.

PS1574 HLA‐DRB1∗15 INCREASES THE RISK OF RED BLOOD CELLS ALLOANTIBODIES IN PATIENTS WITH BETA THALASSEMIA MAJOR

Background:The cornerstone of the management of Beta Thalassemia Major (B‐TM) is lifelong Red Blood Cells (RBC) transfusion. This is associated with the development of RBCs alloantibodies. Human Leukocyte Antigen (HLA) is considered a risk factor for a number of autoimmune disorders. We hypothesized that HLA impacts the rate of alloantibody formation in patients with B‐TM.Aims:To assess the impact of HLA alleles on the risk of RBC alloimmunization in patients with B‐TM.Methods:This is a retrospective cohort study of patients with B‐TM managed at Sultan Qaboos University Hospital (SQUH) between January 1996 till December 2016. All adult and paediatric patients with B‐TM with available HLA typing results were included. Patients with missing data were excluded. Chi‐squared test was used to assess the impact of HLA alleles on alloimmunization and the effect size was estimated using odd ratio (OR). HLA testing was performed using polymerase chain reaction – specific sequence of oligonucleotides (PCR‐SSO). Gel card technique (BIORAD ID‐System; DiaClon, Bio‐Rad, DiaMed GmbH, Switzerland) was used to screen for the RBC alloantibodies.Results:One hundred and sixty‐five patients fulfilled the inclusion criteria without missing information. The median age of all patients was 14 years. Females constituted 48% of the sample and 45% of patients were O RhD positive. The median RBC transfusion was 17 times per year. Patients were first detected to have RBC alloantibody at a median age of 16 years. The commonest RBC alloantibodies found in these patients were anti‐K (50%) and anti‐E (58%). The commonest HLA allele in loci A, B, C, DQ and DRB1 were HLA‐A∗02 (21%), HLA‐B∗51 (14%), HLA‐C∗7 (18%), HLA‐DQ∗05 (47%) and HLA‐DRB1∗16 (32%) respectively. HLA‐DRB1∗15 and HLA‐DQ∗6 were associated with a higher risk of RBC alloantibody formation (HLA‐DRB1∗15: OR 3.57, P = 0.047; HLA‐DQ∗06: OR 4.37, P = 0.025). None of the remaining common alleles (&gt;5%) in Omani population were associated with the risk of alloantibody formation (p values &gt;0.05).Summary/Conclusion:HLA‐DRB1∗15 and HLA‐DQ∗06 increases the risk of RBC alloantibody formation in patients with B‐TM. We recommend extended RBC phenotyping and extended antigen matched blood transfusion in patients carrying these two alleles. These findings will need to be confirmed in a larger multicenter study.

PB2447 RED CELL ALLOIMMMUNIZATION A RETROSPECTIVE SINGLE CENTRE EXPERIENCE IN AN AREA WITH A LOW INCIDENCE OF HAEMOGLOBINOPATHY:

Background:Alloimmunization to Red Blood Cell (RBC) antigens could become a growing issue owing to increasing transfusion programs and an ageing population. The clinical impact of RBC alloimmunization is likely underestimated. It has been recognized that alloimmunization may compromise the therapeutic effect of RBC transfusion. Alloimmunized patients have an increased risk of serious haemolytic transfusion reactions (HTRs) and may not receive adequate RBC transfusion support due to lack of compatible RBC units. Since recently, alloimmunization is not routinely reported to SHOT and there is comparatively little data of its impact on clinical practice.Aims:Identifying transfusion‐related risk factors of alloimmunization against RBC antigens and correlate alloimmunization to the clinical data.Methods:A retrospective cohort study of all RBC‐alloimmunization in a district hospital (UKAS accredited laboratory) between January 2005 and January 2015. The demographics of the patients with alloantibodies, their medical history, and the transfusion‐related adverse events were examined to explore the concept of RBC alloimmunization itself may contribute to morbidity and mortality. Patients with haemoglobinopathies and palliative leukaemia's were excluded. Laboratory data included patients’ ABO/Rh type, antibody screening results, direct antiglobulin test, and alloantibody and autoantibody specificities. Data concerning transfusion reactions were obtained from the hospitals’ transfusion records. The retrospective study was done to find the incidence and type of RBC alloantibodies and to identify factors such as frequency of transfusion, and their association with the development of clinical complications.Results:The study encompasses all the patient whose immunohematological tests were conducted at the Department of Transfusion Medicine Nine patients were identified over the ten year period. Two cases were excluded due to incomplete data. Of the seven patients that had sufficient data the mean blood component requirements were 4.8 (media 3.4‐ 6.3) whereas platelets (PLT) was 1.2 platelet units (range 2.1 – 3.4) respectively. The number of alloimmunized transfused patients in our centre is &lt; 1% ‐ likely bellow than the 1% to 3% commonly quoted, taking into consideration the frequent occurrence of RBC antibody evanescence ‐. The most frequent antibody specificity was anti‐E, followed by Jkb ‐ Jka −Lua, −D, −C and anti‐Wra. Unsurprisingly, we identify alloimmunization was associated with higher numbers of transfused RBC units (3 vs 6; p = 0.001), as well as with longer time under transfusion (4.3 vs. 8.1 months; p = 0.017) as well as increased mortality, in the scenario of previous malignancy.Summary/Conclusion:The clinical course for each of the reported patients provides insight into the direct and indirect consequences of RBC alloimmunization, where patients experienced delayed HTRs or did not receive needed RBC transfusions. Future work examining the clinical impact of RBC alloimmunization should not only consider HTRs but should also address the potential consequences associated with difficulties in obtaining compatible blood.

Recipient factors influencing red blood cell alloimmunization

Red blood cell (RBC) alloantibodies develop in a subset of individuals following exposure to non‐self RBCs through transfusion, pregnancy or other activities; these antibodies can lead to difficulty locating compatible RBCs, acute or delayed haemolytic transfusion reactions, or haemolytic disease of the newborn. Alloimmunization is underestimated due in part to antibody evanescence, the random nature of post‐transfusion antibody screens, fragmented medical care and the lack of widespread antibody registries. Factors that influence who will develop detectable alloantibodies are not well understood. Transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many RBC units (and many non‐self blood group antigens). Individuals with sickle‐cell disease (SCD) and myelodysplastic syndrome (MDS) are more likely to form RBC alloantibodies than most other patient populations. Individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher‐than‐average risk of forming RBC alloantibodies. Inflammation, in a broad sense, is one common thread amongst these diagnoses associated with high prevalence rates of RBC alloimmunization. Reductionist murine models support some types of inflammation (including viral‐like stimuli) around the time of RBC exposure as being associated with an increased likelihood of alloantibody formation. Strategies other than transfusion avoidance or extended antigen matching beyond ABO/Rh would be beneficial to prevent new RBC alloantibody formation, especially in patients at highest risk.

Incidence of Red Blood Cell Alloantibody Formation after Platelet Concentrate Transfusions

Incidence of Red Blood Cell Alloantibody Formation after Platelet Concentrate Transfusions

Red blood cell alloantibodies are associated with increased alloimmunization against human leukocyte antigens.

Alloantibodies recognizing human leukocyte antigens (HLA) can cause immune-mediated refractoriness to platelet transfusion. An association between HLA alloimmunization and red blood cell (RBC) alloimmunization has been suggested but remains uncertain. We tested for HLA alloantibodies in 660 patients with and without RBC alloantibodies. Calculated panel reactive antibody (cPRA) values were determined for HLA alloimmunized patients. Current and historical diagnoses and blood product exposure were catalogued. Variables associated with high-level HLA alloimmunization (cPRA ≥ 90%) were evaluated. The cohort included 366 women and 294 men with median age of 66 years (interquartile range [IQR], 53-76). The number of patients with and without RBC alloantibodies was 447 and 213, respectively. Among patients with and without RBC alloantibodies, 20.6% and 8.5% had a cPRA ≥ 90%, respectively (p < 0.0001). In univariate analyses of men and nulliparous women and previously pregnant women, the median number of RBC alloantibodies was significantly higher in patients with a cPRA ≥ 90% (p < 0.0001). The number of RBC alloantibodies remained an independent predictor of a cPRA ≥ 90% in multivariate analysis (odds ratio [OR] 1.50, 95% confidence interval [CI] 1.22-1.85). Other independent predictors of a cPRA ≥ 90% were parity (OR 1.26, 95% CI 1.08-1.47), age (OR 0.98, 95% CI 0.97-1.00), history of renal disease (OR 1.88, 95% CI 1.02-3.48), and number of non-leukoreduced products transfused (OR 1.02, 95% CI 1.00-1.04). RBC alloimmunization was significantly associated with HLA alloimmunization with a cPRA ≥ 90%. RBC alloimmunization status combined with specific components of the clinical history may estimate the risk of high-level HLA alloimmunization.