Alloimmunization In Sickle Cell Disease Research Articles

Molecular genotyping versus serological diagnosis for RH blood group typing in sickle cell patients

ABSTRACT Objectives High rate of alloimmunization in sickle cell disease (SCD) patients poses a significant challenge in finding compatible blood unit. Accurate determination of the blood group genotype of them can help reduce the alloimmunization risk. Tetra ARMS PCR is a novel method that has been utilized recently to investigate SNPs in diseases in a fast and reliable way. Methods Our study included 104 SCD and sickle thalassemia (Sβ) patients referred to Baghaei-2-Hospital of Ahvaz in 2019 using a nonrandom sampling method. Blood samples were collected for serological and molecular tests. Rh genotyping was performed using Tetra ARMS PCR and compared with the serological results. Results Based on the Tetra ARMS PCR method, out of 104 patients, 7 (6.7%) were d/d, 40 (38.5%) were D/d, 57 (54.8%) were D/D, 25 (24%) were C/C, 59 (56.7%) were C/c, 20 (19.3%) were c/c, 4 (3.8%) were E/E, 25 (24%) were E/e, and patients 75 (72.2%) were e/e. There were discrepancies in the serological and molecular results for 11 patients. Conclusion Use of Tetra ARMS PCR in combination with serological methods for determining the Rh blood group system in donors and transfusion-dependent patients represents a remarkable transformation in the field of immunohematology.

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.

ANTI-D ALLOIMMUNIZATION IN SICKLE CELL DISEASE (SCD) PATIENTS WITH WEAK RHD VARIANT ALLELES

ANTI-D ALLOIMMUNIZATION IN SICKLE CELL DISEASE (SCD) PATIENTS WITH WEAK RHD VARIANT ALLELES

P-035: RED BLOOD CELL ALLOIMMUNIZATION IN SICKLE CELL DISEASE PATIENTS IN THE DEMOCRATIC REPUBLIC OF THE CONGO

Purpose: Data regarding red blood cell (RBC) alloimmunization in sickle cell disease (SCD) patients are scarce in the Democratic Republic of the Congo (DRC) which is the third most affected country in the world by sickle cell disease. We aimed to determine the prevalence of red blood cell alloimmunization and alloantibody specificity in SCD patients in Kisangani (DRC). Materials and methods: We conducted a multi-hospital, multi-site based cross-sectional study among 125 SCD patients at Kisangani and 136 at the Centre Hospitalier Régional de la Citadelle (CHR) of Liège (Belgium). Data were collected using a survey form. Diagnostic con?rmation of SCD was made by high-performance liquid chromatography coupled to mass spectrometry. Alloantibodies were screened using the agglutination technique on gel cards and their specificity determined using 11 and/or 16 cell panels. Statistical analyses were carried out using SPSS. Results: The prevalence of RBC alloimmunization was 9.6% for samples collected at Kisangani versus 22.8% (including historical antibodies) at the CHR and increased to 10% at Kisangani versus 29.3% at the CHR when considering only patients transfused in the last five years prior to the study. At Kisangani as well as at CHR, the median age of alloimmunized patients was higher than that of non-alloimmunized patients, 15.5 years (IQR:4.8-19.8) vs 10 (IQR: 6.5-17) and 24 years (IQR:14-31) vs 17(IQR:12-24), respectively. The median number of blood units was higher in both Kisangani and CHR immunized patients compared to non-immunized patients, 8(IQR:5-11) vs 5(IQR:3-13) and 41(IQR:6-93) vs 6.5(3-37) respectively. At Kisangani(N=14), the most frequent antibodies were anti-D (28.6%), anti-C (21.4%), anti-M (14.3%,) and anti-N (14.3%) versus anti-E (13.6%), anti-S (13.6%), and anti-Lea (11.4%) at the CHR(N=44). Conclusion: Red blood cell alloimmunization is a common complication of transfusion therapy in SCD patients in the DRC and is mostly directed against the RH system. In a low income context in the DRC, blood transfusion with a minimum ABO, D, C, E antigen matching could significantly reduce the incidence of this complication. The authors do not declare any conflict of interest

Frequency of Allo-Immunization in Sickle Cell Disease: Case of the Patients at the Laquintinie Hospital in Douala-Cameroon

<i>Background:</i> Allo-immunization is an immune response to foreign antigens after exposure to genetically different cells or tissues. Allo-immunized patients with sickle cell disease (SCD) appear to experience worse survival compared to non allo-immunized patients. Therefore, to contribute to the management of patients living with SCD and improve the blood transfusion process, we aimed to determine the frequency of allo-immunization in SCD’s patients. To realize this study, we carry out a cross sectional study in one Hospital of Douala-Cameroon. <i>Method: </i>Plasma was prepared from collected blood sample, electrophoresis was done and depending on the migration on the gel; the type of electrophoresis was determine. Blood group ABO/Rh was done. After electrophoresis, depending on the result obtained those that was not homozygote (SS) and heterozygote sickle cell anemia (AS) was discarded. The check for irregular agglutinins was done using the indirect Coombs test. Clinical and biological characteristics of the different participants were studied and analyzed using a Statview statistical software. <i>Result:</i> We obtained 104 sickle cell patient, out of these we had 55% of positive RAI, with an average transfusion of 7.35. Also 83 patients were homozygote sickle cell anemia for a mean age of 15.75 years old (ranging from 1 to 52 years). The result obtained shows that the number of blood transfusion for homozygote sickle cell patient increases with age and that homozygote sickle cell patient received more blood transfusion than heterozygote patient. It can also be said that allo-immunization in these patients originate from the multiple blood transfusion received that were not fully compatible and that it affects more children (from 1 – 15), they accounted for about 53.85% of the total population. Our result obtained implies that in the future, sickle cell patient will have difficulty in finding compatible blood for treatment and is a real problem since transfusion is the major standard of care for patient suffering from sickle cell anemia in Cameroon. The absence of treatment may lead to the death of the patient. To help reduced allo-immunization, extended compatibility test in all the red blood cell system and routinely screening for the presence of irregulars agglutinins may reduce the frequency of allo-immunization.

Red Blood Cell Alloimmunization in Sickle Cell Disease: Advantage of Ethnic Heritage between Donors and Patients in Jeddah, Saudi Arabia

Abstract Introduction/Objective Red blood cell (RBC) transfusion is frequently required for patients with sickle cell disease (SCD). Development of alloantibodies in these patients complicates the blood bank process needed to identify these antibodies and to find compatible RBC units. The rate of alloimmunization has been reported as high as 47% in one study and 34.2% in another study from Eastern region of Saudi Arabia. The purpose of this study was to determine incidence and rate of RBC alloimmunization in the Saudi population in the Western region in SCD. Methods/Case Report A retrospective analysis of the immunohematological and transfusion history of a total of 161 SCD patients was reviewed, of which there 95 males and 66 females. All patients had erythrocytapheresis, ranging from one to 24 full red cell exchange sessions. A total of 490 red cell exchanges were performed and 4,914 units of blood were used. Extended compatibility to RhCcEe and K antigen was performed. Patient who developed alloimmunization to any of RhCcEe and K antigen were matched for Kidd, Duffy and MSN antigens for subsequent RBC requirements. Results (if a Case Study enter NA) The RBC alloimmunization incidence was 18% with a rate of 0.6 antibodies per 100 RBC transfusions. Alloimmunization in females was significantly higher than in the patients. Eighteen (11.2%) female patients demonstrated antibodies as compared to eleven (6.8%) male patients. Twelve patients (7.4%) had a history of at least one alloantibody and 17 (10.6%) had more than one. Antibodies found were directed against E (7.4%), K (5.6%), and D, C, c, S, M, Lea, Jk a, Chido/Rodgers, Fy a. Seven (4.3%) patients also had warm autoantibodies. Conclusion RBC alloimmunization incidence and rate in our study was lower to those reported in less heterogeneous population of donors and patients. Nonetheless, RBC alloimmunization still occurs in patients with SCD, often due to Rh variants or lack of consistency in the application of prophylactic antigen matching between institutions. Therefore, we believe that this rate can still be further reduced if all centers in the region establish transfusion programs to include at least RhCcEe and K phenotypic compatibility and communication mechanisms between major treating centers and transfusion centers in smaller cities to minimize the risks of exposing the patient to different RhCcEe and K phenotype and of developing RBC alloimmunization.

Increased Prevalence of Alloimmunization in Sickle Cell Disease? Should We Restore Blood Donation in French Guiana?

Patients with sickle cell disease often undergo frequent blood transfusions. This increases their exposure to red blood cell alloantigens of donor units, thus making it more likely that they produce alloantibodies. This cross-sectional study aimed to describe the prevalence of allo-immunization in patients with sickle cell disease who were monitored at Cayenne Hospital in 2016. Of the 451 patients recruited during the study period, 238 (52.8%) were female. There were 262 (58.1%) homozygous sickle cell and 151 (33.5%) compound heterozygous sickle cell patients. The median age of the participants was 23.09 years (range, 0.5–68). We noted different red blood cell extended phenotypes: -in the Duffy system, the Fya- Fyb–profile was found in 299 patients (66%);—for the Kidd system, the most represented profile was Jka+ Jkb-, with 213 patients (47%). The Jka antigen was present in 355 patients;—in the MNS system, the S-s+ profile was found in 297 patients (66%);—the Lea antigen of the Lewis system was absent in 319 patients. The most frequent Rh phenotype in our patients was D+ C- E- c+ e+ K-, representing 51% of the patients. A total of 6,834 transfused packed red blood cell units were recorded. Sixty-eight patients (23%; 95% confidence interval, 20–25%) had detectable RBC alloantibodies. In multivariate logistic regression, only the mean number of single transfusions was statistically higher for the alloimmunized patients (p < 0.04). Thirteen (19%) of the patients with alloimmunization developed a delayed hemolytic transfusion reaction, thus representing 4.4% of the total number of transfused patients. Whether differences between donors from France vs. recipients from French Guiana could explain this high prevalence of alloimmunization to be examined. In conclusion, careful transfusion strategies for patients with RBC alloantibodies should allow further reduction of the rate of alloimmunization.

Red cell transfusion and alloimmunization in sickle cell disease.

Red cell transfusion remains a critical component of care for acute and chronic complications of sickle cell disease. Randomized clinical trials demonstrated the benefits of transfusion therapy for prevention of primary and secondary strokes and postoperative acute chest syndrome. Transfusion for splenic sequestration, acute chest syndrome, and acute stroke are guided by expert consensus recommendations. Despite overall improvements in blood inventory safety, adverse effects of transfusion are prevalent among patients with sickle cell disease and include alloimmunization, acute and delayed hemolytic transfusion reactions, and iron overload. Judicious use of red cell transfusions, optimization of red cell antigen matching, and the use of erythrocytapheresis and iron chelation can minimize adverse effects. Early recognition and management of hemolytic transfusion reactions can avert poor clinical outcomes. In this review, we discuss transfusion methods, indications, and complications in sickle cell disease with an emphasis on alloimmunization.

Alkaline Phosphatase Is Associated with Red Cell Alloimmunization in the Pulmonary Hypertension and Hypoxic Response (PUSH) Sickle Cell Disease Cohort

Background: Blood transfusion is a common therapy for sickle cell disease (SCD). Although, highly effective, a major limitation is development of alloantibodies to minor blood group antigens on donor red cells. Alloimmunization has a prevalence of 2-5% for transfusions in the general population, but it is significantly higher in SCD. Risk factors for alloimmunization have been poorly characterized, although number of lifetime transfusions is an important risk factor. Alloimmunization has been clinically observed in children with a prevalence of about 7%. With development of each antibody, blood donor matching becomes increasingly difficult and expensive with an increased risk for transfusion reactions and diminished availability of compatible red cell units for treatment of SCD. The ability to identify risk factors for developing alloantibodies would be beneficial for clinicians. To identify markers for alloimmunization in SCD, we have analyzed children and adults who developed this complication. Methods: We analyzed The Pulmonary Hypertension and Hypoxic Response in Sickle Cell Disease (PUSH) study, which enrolled n=468 pediatric and n=59 adult SCD subjects. In both children and adults, alloimmunization cases were defined as a history of at least 1 alloantibody. Controls in both cohorts were defined as subjects with no history of alloantibodies and receipt of more than 10 lifetime red cell transfusions. All others within the study who did not meet these criteria were assigned to a third comparison group. To identify differences between cases, controls and all others, we performed univariate analyses (using ANOVA or Kruskal Wallace where appropriate) for clinical parameters and laboratories. Case control comparisons were also performed for selected variables and plasma levels for 11 cytokines. Results were further analyzed using regression modeling. Results: The overall prevalence of alloimmunization was 7.3% among children (34/468 subjects; median age 12, range 3-20 years) compared to 28.8% in adults (17/59 subjects; median age 37, range 18-73 years). When only considering those with &amp;gt;10 lifetime transfusions, the prevalence was considerably higher at 29.3% and 54.8% in children and adults, respectively. At the same time, 8 pediatric (23.5%) and 5 adult (29.4%) alloimmunization cases had received fewer than 10 transfusions. In a 3-way pediatric cohort comparison (cases, controls and all others), risk factors associated with alloimmunization included SS genotype, older age and markers of more severe disease (higher ferritin, WBCs, platelets and total bilirubin). Comparison of cases to controls showed alkaline phosphatase (P=0.05) was significantly lower in cases, whereas AST (P=0.02) was significantly higher even with adjustment for age. Levels of plasma cytokines MCP-1 (P=0.01) and IFNgamma (P=0.08) were lower in cases from a subset of the pediatric cohort. In adults, only 4/59 (6.8%) subjects had never received a lifetime transfusion (all non-SS). In the adult 3-way comparisons, only SS genotype and higher ferritin were associated with alloimmunization. The adult case control analysis showed higher absolute monocyte count (P=0.02), absolute eosinophil count (P=0.04) and absolute basophil count (P=0.008) in association with alloimmunization cases. In addition, alkaline phosphatase was again significantly lower among cases (P=0.02) as seen in the pediatric cohort. There were no significant differences in cytokine levels among adults. Conclusions: When considering only transfused SCD patients, the prevalence of alloimmunization is higher than 30%. As seen in prior studies, higher lifetime red cell transfusions are an important risk factor especially among adults where most patients have received transfusions. Children who develop alloantibodies appear to have laboratory markers of more severe disease, but this is not observed in adults. A novel association observed across both pediatric and adult subjects is a significantly lower serum alkaline phosphatase in those with alloantibodies. The results of this study suggest a need for improved tracking of red cell transfusion therapy in the US for SCD patients due to a high prevalence of alloimmunization. Further study is also needed to elucidate the significance of the alkaline phosphatase association. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Ironwood: Research Funding; Imara: Research Funding.

A potential association of an interferon gene signature with RBC alloimmunization in sickle cell disease

Abstract RBC transfusion can lead to the production of alloantibodies against RBC antigens. RBC alloimmunization can result in hemolytic events and severely limit the availability of compatible RBCs. Patients with sickle cell disease (SCD) have the highest rate of alloimmunization, compared to any other disease population. However, mechanisms underlying this increased frequency are poorly understood. Inflammation in the recipient has been shown to promote alloimmunization in mice and humans. In mouse transfusion models, type 1 interferons (IFNα/β) and interferon-stimulated genes (ISGs) have been shown to promote alloimmunization. Given the chronic inflammatory state in patients with SCD, we hypothesized that SCD patients may also have an IFNα/β gene signature that may contribute to the increased frequency of alloimmunization. To test this hypothesis, we measured the expression of multiple ISGs, including myxovirus resistance protein 1 (MxA) and Siglec-1 by whole blood immunoassay and flow cytometry, respectively. MxA and Siglec-1 levels were significantly elevated in SCD patients, compared to transfused controls. We then determined the degree to which ISG expression correlated with alloimmunization frequency by linear regression. There was a trend toward elevated MxA expression in patients with 1 or more alloantibodies. Patients with 2 or more alloantibodies had significantly elevated MxA compared to non-alloimmunized transfused patients. These findings suggest the presence of an IFNα/β gene signature in patients with SCD and warrant further investigation of the contribution of IFNα/β pathways toward the profoundly elevated frequency of RBC alloimmunization in patients with SCD.

Increased Expression of Type 1 Interferon Stimulated Genes in Sickle Cell Disease and a Potential Association with RBC Alloimmunization

Background RBC transfusion can lead to the production of alloantibodies against RBC antigens and is a clinically significant issue in transfusion medicine. Patients with sickle cell disease have an increased risk for alloimmunization; 30-50% of SS patients have alloantibodies compared to 3-10% of other hospitalized patients. These alloantibodies can cause dangerous hemolytic transfusion reactions and limit the availability of compatible antigen-negative RBC products. This is of particular importance in SS patients, who commonly make alloantibodies against multiple RBC antigens and need regular transfusions to treat their disease. However, mechanisms underlying the increased frequency of alloimmunization in sickle cell patients are poorly understood. In previous studies, inflammation in the recipient has been shown to promote alloimmunization in both mice and humans. In mouse transfusion models, type 1 interferons (IFNα/β) and Interferon Stimulated Genes (ISGs) have been shown to promote alloimmunization. Other studies have shown that patients with inflammatory autoimmune diseases express an IFNα/β signature, which may contribute to the increased frequency of alloimmunization in these populations. Given the chronic inflammatory state in patients with sickle cell disease, we hypothesize that: SS patients may also have an IFN gene signature that may contribute to the increased frequency of alloimmunization. Methods To test this hypothesis, we initially measured the expression of the ISG, myxovirus resistance protein 1 (MxA), in the blood of previously-transfused patients (n=50) with SS disease (SS, n=13) and without SS disease (ββ, n=37) by whole blood immunoassay (ELISA). We then measured expression of another ISG, Siglec-1 (SS n=5, ββ=24), expressed on monocytes by flow cytometric analysis. To determine the degree to which ISG expression correlated with alloimmunization frequency, expression of MxA in non-alloimmunized patients was compared to the expression in patients with 1 or more alloantibodies. Statistical analysis of 2 groups was completed with a Mann-Whitney U test. Significance between 3 groups was determined using a Kruskal-Wallis test with a Dunn's post-test. Results SS patients had significantly elevated levels of MxA (mean ± standard error of the mean, SS MxA = 8.98 ng/mL ± 2.46) compared to control patients without SS (MxA = 1.25 ± 0.54, p&amp;lt;0.0001). (Figure 1 A). SS patients also had significantly elevated levels of Siglec-1 on blood monocytes, measured by flow cytometric mean fluorescence intensity (MFI, SS MFI = 132.72 ± 42.9, ββ MFI = 64.9 ± 6.17, p&amp;lt; 0.05). (Figure 1 B,C). For all 50 patients, including SS and ββ control patients, there was a trend toward elevated MxA expression in alloimmunized patients. Patients with 2 or more alloantibodies had significantly elevated MxA (MxA 8.16 ± 2.61), compared to non-alloimmunized transfused patients (MxA = 2.05 ± 1.65, p &amp;lt; 0.01) or patients with only 1 alloantibody (MxA = 1.18 ±0.48, p&amp;lt;0.01). There was no significant difference in MxA levels between patients with 0 and 1 alloantibody. Of the 13 patients with SS disease, only 2 patients lacked alloantibodies. (SS with 1 alloantibody, n=3, SS with 2 or more alloantibodies, n=8). Therefore, a correlation between MxA levels and alloimmunization in SS patients could not be assessed. Discussion Factors that contribute to RBC alloimmunization in sickle cell disease are poorly understood. In this study, we found that sickle cell patients had an increase in the expression of ISGs compared to other transfused patients. We also found that MxA levels are increased in patients that have 2 or more alloantibodies compared to patients without alloantibodies. These findings suggest the presence of an IFNα/β gene signature in patients with sickle cell disease. Further studies are needed to determine the relationship between interferon-stimulated responses in sickle cell patients and the increased frequency of alloantibody production. Disclosures No relevant conflicts of interest to declare.

Genetic Association Studies of Red Blood Cell Transfusion Related Alloimmunization: A Systematic Review and Meta-Analysis

Introduction: Blood transfusions are an important treatment modality for patients with either acute or chronic onset anemia such as trauma, sickle cell disease, and hematological malignancies. Transfusion poses a risk for alloimmunization, which may lead to potentially lethal transfusion reactions. A promising strategy to prevent alloimmunization is extensive matching on blood groups, yet this is a costly procedure and should be reserved for patients at highest risk for alloimmunization. Identification of genetic variants that increase the risk for alloimmunization might help to identify high-risk patients and could be used as a screening tool for patients receiving multiple transfusions. Objectives: To summarize all available evidence on genetic risk factors for alloimmunization after blood transfusion. Design: Systematic review with meta-analysis of observational studies. Studies were only included in the meta-analysis if polymorphisms were tested at least 3 times, and if ethnic background of the population and the control populations were comparable between studies. Data sources: The online databases Embase, MEDLINE and the Cochrane Library were search for relevant articles with search terms: 1) transfusion, 2) alloimmunization 3) genetics. The search was last updated March 2018. Eligibility criteria: 1) Primary study that assessed the association of genetic polymorphisms with transfusion related alloimmunization, 2) a human population, 3) studies with at least 50 patients, 4) full text availability. Data extraction: Two reviewers independently screened articles for eligibility, extracted data using a standardized data extraction form. Extracted data included study setting, study population, participant demographics, baseline characteristics, study methodology, comparisons and outcome, and risk of bias. Primary outcome measure: Alloimmunization after one or more blood transfusions. Risk of bias assessment: The quality of the included studies was assessed by the Q-genie tool for genetic association studies. Results: A total of 2045 cases and 24084 controls were derived from 18 genetic case-control studies that were included in this systematic review. Most commonly studied disease group was sickle cell disease (SCD) (8 studies). Three studies included patients with different diseases and seven studies did not report the underlying disease. Eleven studies identified the association of HLA polymorphisms with alloimmunization and 8 studies focused on non-HLA variants. Overall quality of the included studies was moderate (11 studies), 2 studies were of high quality, and 5 studies were ranked as poor. HLA-DRB1*04 (Odds Ratio 7.16, 95%CI 3.87-13.22, P&lt;0.00001) and HLA-DRB1*15 (OR 3.01, 95%CI 1.84-5.53, P&lt;0.0001) were by meta-analysis significantly associated with anti-Fy(a) formation, although there was considerable heterogeneity (I2=78% and 55% respectively). Moreover, HLA-DRB1*10 (OR 2.64, 95%CI 1.41-4.95, P=0.002), HLA*DRB1*11 (OR 2.11, 95%CI 1.34-3.32, P=0.001), and HLA-DRB1*13 (OR 1.71, 95%CI 1.26-2.33, P=0.0006) were overall associated with anti-Kell formation. Heterogeneity was less prominent with an I2 of 0%, 54% and 19% respectively (Figure 1). No other variants were eligible for meta-analysis. Non-HLA variants were tested less extensively, as most variants were reported by only 1 study. Polymorphisms of genes in the immunomodulatory pathways were assessed most frequently. Of these variants, FC-gamma-receptor 2C.nc-ORF was associated with a decreased risk of alloimmunization in SCD (OR 0.26, 95%CI 0.11-0.64, p=0.003). All other associations that were described as significant by the original articles were summarized in Figure 2. Discussion: There is limited evidence supporting the role of genetic risk factors for alloimmunization. The results of our meta-analysis suggest that several HLA polymorphisms potentially influence antigen presentation of the Duffy(a) and Kell antigen. Once confirmed by experimental studies, these polymorphisms could be used as a screening tool for the prevention of alloimmunization among frequently transfused patients. Overall, the effect of genetic variants on alloimmunization has mostly been assessed by small studies, hampering reliable interpretation of the results. Future studies should include large and well-defined cohorts when performing genetic analysis on this complicated subject. Disclosures No relevant conflicts of interest to declare.

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.

A locus on chromosome 5 shows African ancestry–limited association with alloimmunization in sickle cell disease

AbstractRed blood cell (RBC) transfusion remains a critical therapeutic intervention in sickle cell disease (SCD); however, the apparent propensity of some patients to regularly develop RBC alloantibodies after transfusion presents a significant challenge to finding compatible blood for so-called alloimmunization responders. Predisposing genetic loci have long been thought to contribute to the responder phenomenon, but to date, no definitive loci have been identified. We undertook a genome-wide association study of alloimmunization responder status in 267 SCD multiple transfusion recipients, using genetic estimates of ancestral admixture to bolster our findings. Analyses revealed single nucleotide polymorphisms (SNPs) on chromosomes 2 and 5 approaching genome-wide significance (minimum P = 2.0 × 10−8 and 8.4 × 10−8, respectively), with local ancestry analysis demonstrating similar levels of admixture in responders and nonresponders at implicated loci. Association at chromosome 5 was nominally replicated in an independent cohort of 130 SCD transfusion recipients, with meta-analysis surpassing genome-wide significance (rs75853687, Pmeta = 6.6 × 10−9), and this extended to individuals forming multiple (>3) alloantibodies (Pmeta = 9.4 × 10−5). The associated variant is rare outside of African populations, and orthogonal genome-wide haplotype analyses, contingent on local ancestry, revealed genome-wide significant sharing of a ∼60-kb haplotype of African ancestry at the chromosome 5 locus (Bayes Factor = 4.95). This locus overlaps a putative cis-acting enhancer predicted to regulate transcription of ADRA1B and the lncRNA LINC01847, both members of larger ontologies associated with immune regulation. Our findings provide potential insights to the pathophysiology underlying the development of alloantibodies and implicate non-RBC ancestry-limited loci in the susceptibility to alloimmunization.

Impact of Red Blood Cell Antigen Matching on Alloimmunization and Transfusion Complications in Patients with Sickle Cell Disease: A Systematic Review.

Red blood cells (RBC) transfusion is critical in managing acute and chronic complications in sickle cell disease (SCD); however, it is complicated by RBC alloimmunization, iron overload, transfusion reactions and infection. Several reports documented an increased incidence of alloantibodies in transfused individuals with SCD, especially for Rh and Kell antigens. As a result, the National Institutes of Health Expert Panel and British Society for Haematology guidelines recommend primary matching for C/c, E/e and K antigens in addition to ABO/RhD for RBC transfusions. However, the evidence supporting these recommendations was cited as limited and understanding of alloimmunization in SCD is evolving. To examine the limitations of the evidence, we undertook a systematic review of evidence behind recommendations for limited and extended serologic and genotypic RBC antigen matching to reduce alloimmunization, autoimmunization and transfusion reactions. Searches of PubMed, Embase, Cochrane, and Web of Science databases using MeSH index and free text terms between 1976 through October 2015 and papers and captured through July 2016 through review references in papers, word of mouth, and ongoing Google Scholar and Medline Alerts identified 303 unique articles. Nineteen articles met inclusion criteria and were classified by the Oxford Centre Evidence Based levels of evidence. Strengthening the Reporting of Observational Studies in Epidemiology checklists were completed for 18 of the 19 studies. There were no prospective randomized controlled trials. Sixteen of the articles were cohort studies, two were cross-sectional studies, and one decision tree model examining costs. Low-quality evidence from observational cohort studies supports that alloimmunization prevalence can be decreased by extending serological RBC antigen matching. Transfusion reactions are generally poorly and inconsistently reported. There was no evidence reporting the effect prophylactic genotypic matching has on alloimmunization, autoimmunization or transfusion reactions. There were no studies comparing prophylactic genotypic matching to serologic matching. High-quality evidence was lacking to support clinical decision making regarding best transfusion practices. Multicenter prospective randomized clinical trials are needed to determine best strategies for reducing the rate of alloimmunization using serologic and genotypic matching.

Red blood cell alloimmunization in sickle cell disease: assessment of transfusion protocols during two time periods.

Prevention of red blood cell (RBC) alloimmunization in patients with sickle cell disease (SCD) focuses on phenotypic RBC matching. We assessed alloimmunization among transfused patients with SCD after implementing leukoreduction and prophylactic antigen matching (PAM). Retrospective review of transfusion and medical records for SCD patients 18 months to 81 years of age was performed covering two 5-year periods: Period 1, no PAM, occasional leukoreduction, and Period 2, consistent leukoreduction and extended PAM (Rh, Kell, S, Fy, Jk) for patients already alloimmunized. Patients transfused in Period 1 were excluded from Period 2. A total of 293 patients were transfused in Period 1 and 183 in Period 2. Median time between first sample and last type and screen after transfusion was 2.12 years in Period 1 and 1.03 years in Period 2. Initial alloimmunization prevalence was lower in Period 2 (26.2%) versus Period 1 (37.5%) and after subsequent transfusions in Period 2 (23.8%) versus Period 1 (45.7%), although without significant difference after adjusting for number of units transfused, percentage of leukoreduced RBCs, sex, and age. Alloimmunized patients received more nonleukoreduced RBCs in Period 1 than nonalloimmunized. Patients transfused during inflammatory conditions were not significantly more likely to become alloimmunized. The prevalence of initial and subsequent RBC alloimmunization in Period 2 was lower than that in Period 1; however, overall prevalence remained high. We recommend leukoreduced, hemoglobin S-negative Rh and Kell PAM RBCs for transfusion of patients with SCD. Component and recipient factors affecting alloimmunization should be studied further.

Identification of Patient-Specific Factors Associated with Alloimmunization in Sickle Cell Disease

▪Background:Alloimmunization is a frequent complication of red blood cell (RBC) transfusion in sickle cell disease (SCD), arising from disparities between the recipient and donor RBC antigens. This potentially life-threatening complication impacts the utilization of chronic transfusion, a disease-modifying treatment in SCD. Many centers across the country use dedicated donor programs that match SCD recipients to a small group of racially and phenotypically-matched RBC donors in order to minimize exposure to foreign RBC antigens. The pathogenesis of alloimmunization remains an area of active research. Emerging data suggests that a pro-inflammatory state in the recipient plays a role in alloantibody (alloAb) generation. Some SCD patients develop an alloAb after minimal exposure to donor RBC units, whereas others do not. In a subset of these latter patients, multiple exposures to disparate RBC units will not lead to alloAb formation. To further understand this phenomenon, we sought to identify patient-specific factors in SCD that are associated with a higher risk of alloimmunization.Methods:We retrospectively analyzed clinical, laboratory, and transfusion-related data for all pediatric and adult SCD patients treated at the University of Missouri between 2000-2017. Descriptive statistics were expressed for continuous variables. Two-tailed Student's t-tests and Chi-squared analyses were used to determine statistical significance.Results:Of a total of 130 SCD patients identified, 89 patients (58 HbSS, 25 HbSC, and 6 HbSβ0/+ patients) had adequate transfusion, clinical, and laboratory data for inclusion in this study. The overall alloimmunization rate was 28%, with genotype-specific alloimmunization rates of 36% (HbSS), 4% (HbSC), and 50% (HbSβ0/+). Neither age nor gender significantly differed between the alloimmunized and non-alloimmunized groups.Alloimmunized SCD patients received, on average, 4 times more lifetime RBC units as compared to the non-alloimmunized group (264 units vs. 65 units, respectively, p = 0.002). Among all SCD patients, individuals who received more than 5 units per year of life were more likely to become alloimmunized (61% vs. 16%, p = 0.02, Figure 1). The most common alloantibodies identified in our patients were C, E, and Kell (Figure 2). Warm autoantibodies were identified in 36% of all alloimmunized patients versus only 5% of non-alloimmunized patients (p = 0.0001).Lastly, alloimmunization was associated with lower hemoglobin (8.66 ± 1.17 vs. 9.52 ± 1.54, p = 0.01) and higher creatinine (0.71 ± 0.36 vs. 0.56 ± 0.22, p = 0.02) among all SCD patients. In addition, higher LDH (549 ± 180 vs. 386 ± 127, p = 0.01) was observed in pediatric SCD patients who were alloimmunized. Both pediatric and adult SCD patients with the HbSS genotype demonstrated higher creatinine in the alloimmunized group (p = 0.03). An increased incidence of leg ulcers was found to be associated with alloimmunization (12% vs. 0%, p = 0.005, Table 1) amongst all of the clinical variables analyzed. Hydroxyurea therapy was not associated with a decreased prevalence of alloimmunization (Table 2).Conclusions:We identified an association between alloimmunization and higher LDH, lower hemoglobin, and higher creatinine in our SCD patient population. In addition, increased alloimmunization was seen in patients with a higher exposure to RBC units, both cumulatively and on a per life year basis. These data suggest that SCD patients with increased hemolysis or recipients of more than 5 RBC units per life year are associated with a higher risk of developing an alloAb. Larger studies will be required to define a causative relationship between this clinical SCD phenotype and a higher risk of alloimmunization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nonclassical FCGR2C haplotype is associated with protection from red blood cell alloimmunization in sickle cell disease

AbstractRed blood cell (RBC) transfusions are of vital importance in patients with sickle cell disease (SCD). However, a major complication of transfusion therapy is alloimmunization. The low-affinity Fcγ receptors, expressed on immune cells, are important regulators of antibody responses. Genetic variation in FCGR genes has been associated with various auto- and alloimmune diseases. The aim of this study was to evaluate the association between genetic variation of FCGR and RBC alloimmunization in SCD. In this case-control study, DNA samples from 2 cohorts of transfused SCD patients were combined (France and The Netherlands). Cases had a positive history of alloimmunization, having received ≥1 RBC unit. Controls had a negative history of alloimmunization, having received ≥20 RBC units. Single nucleotide polymorphisms and copy number variation of the FCGR2/3 gene cluster were studied in a FCGR-specific multiplex ligation-dependent probe amplification assay. Frequencies were compared using logistic regression. Two hundred seventy-two patients were included (130 controls, 142 cases). The nonclassical open reading frame in the FCGR2C gene (FCGR2C.nc-ORF) was strongly associated with a decreased alloimmunization risk (odds ratio [OR] 0.26, 95% confidence [CI] 0.11-0.64). This association persisted when only including controls with exposure to ≥100 units (OR 0.30, CI 0.11-0.85) and appeared even stronger when excluding cases with Rh or K antibodies only (OR 0.19, CI 0.06-0.59). In conclusion, SCD patients with the FCGR2C.nc-ORF polymorphism have over a 3-fold lower risk for RBC alloimmunization in comparison with patients without this mutation. This protective effect was strongest for exposure to antigens other than the immunogenic Rh or K antigens.

Medical and economic implications of strategies to prevent alloimmunization in sickle cell disease.

The pathogenesis of alloimmunization is not well understood, and initiatives that aim to reduce the incidence of alloimmunization are generally expensive and either ineffective or unproven. In this review, we summarize the current medical literature regarding alloimmunization in the sickle cell disease (SCD) population, with a special focus on the financial implications of different approaches to prevent alloimmunization. A review of EMBASE and MEDLINE data from January 2006 through January 2016 was conducted to identify articles relating to complications of SCD. The search was specifically designed to capture articles that evaluated the costs of various strategies to prevent alloimmunization and its sequelae. Currently, there is no proven, inexpensive way to prevent alloimmunization among individuals with SCD. Serologic matching programs are not uniformly successful in preventing alloimmunization, particularly to Rh antigens, because of the high frequency of variant Rh alleles in the SCD population. A genotypic matching program could offer some cost savings compared to a serologic matching program, but the efficacy of gene matching for the prevention of alloimmunization is largely unproven, and large-scale implementation could be expensive. Future reductions in the costs associated with genotype matching could make a large-scale program economically feasible. Novel techniques to identify patients at highest risk for alloimmunization could improve the cost effectiveness of antigen matching programs. A clinical trial comparing the efficacy of serologic matching to genotype matching would be informative.

Genomic Applications for Transfusion Support

Red blood cell (RBC) and platelet antigen determination by DNA methods (genotyping) has become an important part of the practice of transfusion medicine over the past decade.The results are reliable and highly correlated with serologic phenotyping results, and are superior in some situations. Application of RBC genotyping to provide more precise transfusion therapy for various patient populations will be discussed, with a particular emphasis on those requiring chronic RBC therapy or with complex serologic reactivity. Current DNA arrays can test for a large number of blood group antigens, but routine typing of the ABO and Rh systems is not currently feasible due to the large number of genetic mutations.Patients with sickle cell disease (SCD) are one of the most challenging populations to safely transfuse, in part due to high rates of alloimmunization against Rh antigens despite prophylactic matching for D, C, and E antigens. Genotyping of the RH loci has revealed that RHD and RHCE variants are prevalent in African Blacks. Nearly 90% of patients with SCD have at least one altered RH gene and contributes to Rh alloimmunization in patients with SCD. The role of RH variation and alloimmunization in SCD will be reviewed, and the feasibility and approach to genetic RBC matching of patients and donors will be discussed. The use of targeted DNA arrays, whole exome sequencing, or whole genome sequencing to identify common and variant RH alleles will be addressed, including limitations of each method. DisclosuresNo relevant conflicts of interest to declare.