Red Blood Cell Antigens Research Articles

A newly devised flow cytometric antibody binding assay helps evaluation of dithiothreitol treatment for the inactivation of CD38 on red blood cells.

Anti-CD38 monoclonal antibodies, including daratumumab and isatuximab, often interfere with pretransfusion testing. Dithiothreitol (DTT) treatment of red blood cells (RBCs) negates this interference. However, the optimum DTT concentration and treatment time have not been well defined. Here, we quantified CD38 on RBCs before and after DTT treatment using a flow cytometric antibody binding assay (FABA) to specify the optimum conditions for CD38 inactivation. For FABA, untreated or DTT-treated RBCs were incubated with fluorescein isothiocyanate-labelled anti-CD38 antibody, in the presence or absence of 100-fold or more excess of unlabelled anti-CD38 antibody, and then analysed by flow cytometry (FCM). Dissociation of CD38-positive and control histograms was determined from the D-value using the Kolmogorov-Smirnov test. The results from FABA were compared with those from conventional FCM, indirect antiglobulin test (IAT) and Western blotting. The results from FABA were more consistent than those from conventional FCM. The D-value was found to be reliable in the analysis of difference between CD38 before and after DTT treatment. Our data showed that 0·0075mol/l DTT for 30min is sufficient to inactivate CD38 on RBCs. These results were stable and consistent with the findings from IAT. Flow cytometric antibody binding assay is an objective way of evaluating the efficacy of DTT treatment for CD38 on RBCs. This approach allows the detection of a small number of cell surface antigens and will be useful for assessing the various chemical treatments to denature RBC antigens.

Diversity of variant alleles encoding Kidd, Duffy, and Kell antigens in individuals with sickle cell disease using whole genome sequencing data from the NHLBI TOPMed Program.

Genetic variants in the SLC14A1, ACKR1, and KEL genes, which encode Kidd, Duffy, and Kell red blood cell antigens, respectively, may result in weakened expression of antigens or a null phenotype. These variants are of particular interest to individuals with sickle cell disease (SCD), who frequently undergo chronic transfusion therapy with antigen-matched units. The goal was to describe the diversity and the frequency of variants in SLC14A1, ACKR1, and KEL genes among individuals with SCD using whole genome sequencing (WGS) data. Two large SCD cohorts were studied: the Recipient Epidemiology and Donor Evaluation Study III (REDS-III) (n = 2634) and the Outcome Modifying Gene in SCD (OMG) (n = 640). Most of the studied individuals were of mixed origin. WGS was performed as part of the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine (TOPMed) program. In SLC14A1, variants included four encoding a weak Jka phenotype and five null alleles (JKnull ). JKA*01N.09 was the most common JKnull . One possible JKnull mutation was novel: c.812G>T. In ACKR1, identified variants included two that predicted Fyx (FY*X) and one corresponding to the c.-67T>C GATA mutation. The c.-67T>C mutation was associated with FY*A (FY*01N.01) in four participants. FY*X was identified in 49 individuals. In KEL, identified variants included three null alleles (KEL*02N.17, KEL*02N.26, and KEL*02N.04) and one allele predicting Kmod phenotype, all in heterozygosity. We described the diversity and distribution of SLC14A1, ACKR1, and KEL variants in two large SCD cohorts, comprising mostly individuals of mixed ancestry. This information may be useful for planning the transfusion support of patients with SCD.

Autoantigen presentation by splenic dendritic cells is required for RBC-specific autoimmunity.

Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4+ T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBC-derived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAbfl/fl and Cre-expressing transgenic animals to generate mice that lack I-Ab expression on particular cell subsets. OTII CD4+ T cell proliferation was assessed in vivo in HOD+ I-Abfl/fl xCre+ mice and in vitro upon coculture with sorted APCs. Analysis of HOD+ I-Abfl/fl xCre+ mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4+ T cells. Subsequent in vitro coculture experiments revealed that both CD8+ and CD8- DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.

Erythrocyte Alloimmunization in Children with Sickle Cell Disease

Alloimmunization is an immune response against red blood cell (RBC) antigens due to sensitization during RBC transfusion. RBC alloimmunization is more common in sickle cell disease (SCD) with prevalence between 18 and 47%.

Blood group typing from whole-genome sequencing data.

Many questions can be explored thanks to whole-genome data. The aim of this study was to overcome their main limits, software availability and database accuracy, and estimate the feasibility of red blood cell (RBC) antigen typing from whole-genome sequencing (WGS) data. We analyzed whole-genome data from 79 individuals for HLA-DRB1 and 9 RBC antigens. Whole-genome sequencing data was analyzed with software allowing phasing of variable positions to define alleles or haplotypes and validated for HLA typing from next-generation sequencing data. A dedicated database was set up with 1648 variable positions analyzed in KEL (KEL), ACKR1 (FY), SLC14A1 (JK), ACHE (YT), ART4 (DO), AQP1 (CO), CD44 (IN), SLC4A1 (DI) and ICAM4 (LW). Whole-genome sequencing typing was compared to that previously obtained by amplicon-based monoallelic sequencing and by SNaPshot analysis. Whole-genome sequencing data were also explored for other alleles. Our results showed 93% of concordance for blood group polymorphisms and 91% for HLA-DRB1. Incorrect typing and unresolved results confirm that WGS should be considered reliable with read depths strictly above 15x. Our results supported that RBC antigen typing from WGS is feasible but requires improvements in read depth for SNV polymorphisms typing accuracy. We also showed the potential for WGS in screening donors with rare blood antigens, such as weak JK alleles. The development of WGS analysis in immunogenetics laboratories would offer personalized care in the management of RBC disorders.

USING DROPLET DIGITAL PCR TO SCREEN FOR RARE BLOOD DONORS: PROOF OF PRINCIPLE

Abstract Background Digital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). Methods Nucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. Results DI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. Conclusion The proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Antigen-Specific Immunoadsorption With the Glycosorb® ABO Immunoadsorption System as a Novel Treatment Modality in Pure Red Cell Aplasia Following Major and Bidirectional ABO-Incompatible Allogeneic Hematopoietic Stem Cell Transplantation.

Pure red cell aplasia (PRCA) after ABO-incompatible allogeneic hematopoietic stem cell transplantation (HSCT) is caused by persisting host-derived isohemagglutinins directed against donor red blood cell (RBC) antigens. ABO antigen-specific immunoadsorption (ABO-IA) with Glycosorb®, commonly used for desensitization therapy in ABO-incompatible living donor renal transplantation, specifically eliminates circulating isohemagglutinins and might represent a novel treatment option for post-HSCT PRCA. In this prospective observational (n = 3) and retrospective (n = 3) analysis of six adult HSCT-recipients with PRCA, ABO-IA was initiated at 159 (range: 104–186) days following HSCT. The median treatment frequency was 4.5 (range: 3.9–5.5) sessions/week. ABO-IA-treatment led to a continuous decrease in isohemagglutinin titers. Reticulocytes increased to ≥30 G/L after 17.5 (range: 4–37) immunoadsorption sessions over 28.5 (range: 6–49) days and continued to rise after that. By the end of the 3-month follow-up period after discontinuation of ABO-IA, all patients showed a sustained remission of PRCA and were independent of erythropoietin-stimulating agents and transfusions. No case of infection or graft-versus-host disease was observed. After a median follow-up of 22.03 (range: 6.08–149.00) months after ABO-IA-treatment, all patients were alive and showed a stable RBC engraftment of the donor blood group. Our data provide the first evidence for ABO-IA as an effective treatment for post-HSCT PRCA.

Type 1 Interferon Gene Signature Promotes RBC Alloimmunization in a Lupus Mouse Model.

Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that promote clinically significant hemolytic events. Multiple studies have reported an increased frequency of RBC alloimmunization in patients with autoimmunity. However, cellular and molecular mechanisms that underlie autoimmunity-induced alloimmunization have not been reported. Patients with systemic lupus erythematosus (SLE) have a high frequency of alloimmunization and express a type 1 interferon (IFNα/β) gene signature. Thus, we utilized the pristane-induced lupus mouse model to test the hypothesis that inflammation in lupus promotes RBC alloimmunization, and to examine the potential role of IFNα/β. Intraperitoneal injection of pristane, a hydrocarbon oil, led to autoantibody production, glomerulonephritis, and pulmonary hemorrhage in wild type (WT) mice. Pristane treatment significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISGs) in peripheral blood and peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL glycoprotein, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane induced comparable levels of inflammatory cells and cytokines in mice lacking the IFNα/β receptor (IFNAR1–/–) or the IFNα/β-inducing transcriptions factors (IRF3/7–/–), compared to WT mice. However, pristane-treated IFNAR1–/– and IRF3/7–/– mice failed to produce ISGs and produced significantly lower levels of transfusion-induced anti-KEL IgG, compared to WT mice. Thus, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. To our knowledge, this is the first examination of molecular mechanisms contributing to RBC alloimmunization in a model of autoimmunity. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of the IFNα/β gene signature to the elevated frequency of alloimmunization in patients with SLE.

The effectiveness of tetragonola honey combinations Aff.biroi and royal jelly as immunomodulators : immunomodulators modelling in facing the plague of COVID-19

COVID-19 epidemic caused serious concern around the world. Innate and adaptive immunity prevents occurring acute infection. Immunomodulatory activity tests and increased leucocyte cells in rat have been carried out using honey (H) and royal jelly (RJ). This study aims to prove the combination of honey and royal jelly in increasing the leucocyte profile in male Sprague Dawley rats. This study used 28 rats divided into 7 groups, namely the negative control and positive, single dose group H and single dose RJ and the combination dose 1, dose 2, and dose 3, then on day the 8th and 11th rats were injected intraperitoneally with 2% sheep red blood cell antigen as much as 0.5 ml. Leucocyte profile were carried out on the 8th and 15th days. The results showed significant difference on leukocyte count (p = 0.000), but there was a decrease in the normal range on day 15th. The highest leucocyte count was showed on the honey and the combination 3 group. Leukocyte differential showed no significant difference (p> 0.05). The honey group and the dose combination 3 showed an increase in the number of segment neutrophils. The difference in lymphocyte reduction was seen between the honey and royal jelly group, with the dose combination 3, respectively. There are significant differences in granulocyte cells enhancement between the honey group and the dose combination 3. We concluded that single dose honey can be an immunomodulatory. Dose combination 3 can be an alternative for further research for immunomodulatory supplement product.

Transfusion-Related Alloimmunization to Red Blood Cell Antigens in Japanese Pediatric Recipients.

Red blood cell (RBC) transfusion to neonates is thought to rarely provoke an immune response. Neonatal testing guidelines suggest that antibody screening is not necessary when the mother has no antibodies. Alternatively, maternal blood samples can be used for antibody screening and cross-matching. However, the guidelines are based on small-scale studies of white-dominant populations. Furthermore, transfusion-related alloimmunization is less well established among children and adolescents as a whole among Japanese and East Asians. To elucidate the incidence of transfusion-related alloimmunization among neonates, children, and adolescents, and whether current guidelines are applicable to Japanese populations, a nationwide retrospective multicenter cohort survey was conducted in 50 tertiary-care hospitals in Japan. Between 2001 and 2015 inclusive, recipients of at least 1 allogeneic RBC transfusion were categorized into groups A-F according to their age at the time of transfusion: (A) neonates <1 month; (B) infants 1 to <12 months; (C) children 1 to <5 years; (D) prepubescents 5 to <10 years; (E) young pubescents 10 to <15 years; and (F) adolescents/young adults 15 to <20 years. Excluding maternally derived antibodies and naturally occurring, cold-reactive, and/or nonspecific antibodies, 69 (0.61%) of 11350 RBC recipients <20 years old formed at least 1 clinically significant alloantibody. The alloimmunization rate differed significantly (P < .0001) by age: none (0%) of 3407 in group A; 11 (0.46%) of 2410 in group B; 18 (0.76%) of 2361 in group C; 9 (0.80%) of 1119 in group D; 12 (1.15%) of 1043 in group E; and 19 (1.88%) of 1010 in group F. Clearly different incidences of alloimmunization emerged in group A compared to B, C, D, E, or F, as confirmed by logistic regression analysis adjusted by numbers of donor exposure. Alloimmunization did not occur from RBC transfusions within the first month of life and rarely occurred (0.46%-0.80%) after transfusion within the first decade of life. Alloimmunization occurred in 1.15%-1.88% of young pubescents and adolescents/young adults. These findings support the use of guidelines developed in Europe and the United States for East Asian pediatric recipients.

Frequency and specificity of Red blood cell alloantibodies in multitransfused Egyptian patients with hematological and nonhematological malignancies

BackgroundRepeated blood transfusions can result in the production of alloantibodies against one or more red blood cell (RBC) antigens, which can complicate future transfusions. AimThis study aims to determine the frequency and specificities of RBC alloantibodies in multitransfused adult cancer patients admitted at the National Cancer Institute, Cairo University. MethodsThis cohort study enrolled 2000 multitransfused cancer patients diagnosed with different types of malignancies; they were screened for RBC alloantibodies using Serascan Diana 3 and Identisera Diana 11-cell identification panels (Diagnostic Grifols, Spain). ResultsOf the 2000 patients tested, 25 had autoantibodies and were excluded from the study. Of the remaining 1975 patients, 181 patients had a total of 267 different alloantibodies (9.16%), with some having more than 1 antibody detected. Our study showed that more female patients (63%) than male patients (37%) had acquired RBC alloantibodies, and a higher prevalence of alloantibodies in patients with nonhematological malignancies (14%) compared with those with hematological malignancies (6.5%). The highest percentage of alloantibodies belongs to the Rh blood group system, followed by the Kell system, then Duffy, MNS, Kidd, and Lewis. Patients who received combined chemotherapy and immunotherapy exhibited a lesser antibody response compared to other patients. ConclusionThe prevalence of alloimmunization in our study is comparable to previous reports on oncology patients. Repeated blood transfusions, which can lead to alloimmunization, often complicate future transfusions. Therefore, we recommend extending phenotype matching for patients who are presumed to depend on blood transfusions in the long term.

Development and validation of a universal blood donor genotyping platform: a multinational prospective study

AbstractEach year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non–self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

The potential role of antibodies against minor blood group antigens in renal transplantation.

Blood group antigens are red blood cell (RBC) surface markers comprising specific carbohydrate moieties attached to the glycolipids and glycoproteins within the membrane. In addition to the major ABO blood group antigens, at least 35 minor blood group antigens have been defined to date. These antigens have immunogenic potential and may cause a transfusion reaction. There is evidence for renal expression of antigens from the Kidd, MNS, Duffy and Lewis groups and therefore the potential for antibodies directed against these antigens to cross-react in a transplanted kidney. In individuals lacking a specific RBC antigen, antibodies may develop after de novo exposure to that antigen, in addition to the potential presence of pre-existing innate antibodies. Relatively little attention has been paid to non-ABO system antibodies, with most reports in the literature focusing on transfusion reactions rather than on any putative role in allograft rejection. Here, we review each of these antigens in the context of renal transplantation and what limited evidence there is on how such immunological risk may be assessed and managed.

Using droplet digital PCR to screen for rare blood donors: Proof of principle

BackgroundDigital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). MethodsNucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. ResultsDI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. ConclusionThe proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Anti-C5 antibody treatment for delayed hemolytic transfusion reactions in sickle cell disease.

Delayed hemolytic transfusion reaction (DHTR) is an unpredictable severe complication of transfusion in patients with sickle cell disease (SCD). It presents clinically as a vaso-occlusive crisis (VOC), often associated with the failure of one or more organs, after the transfusion of packed red blood cells (pRBC).1,2 Hyperhemolysis is encountered in the most severe forms. Both transfused and autologous red blood cells (RBC) are lysed. The mechanisms underlying DHTR remain unclear. Alloantibodies against RBC antigens were initially thought to underlie the pathophysiology, but no such antibodies are detected in about a third of the cases.3 RBC degradation products, such as hemoglobin and heme, are released into the bloodstream during intravascular hemolysis. These elements and heme-loaded membrane microvesicles have recently been implicated in inflammation and organ injury in DHTR.4 Complement is activated via the classical pathway, by alloantibodies, and/or via the alternative pathway, by free heme.5 Hemedependent complement deposits on the endothelium contribute to organ damage.6 Due to these vascular lesions, hyperhemolysis often progresses to multiple organ failure and, in some cases, death.

IMMUNE ANTIBODIES IN MOKSHA AND ERZYA ETHNIC GROUPS OF THE REPUBLIC OF MORDOVIA

ABO system's immune antibodies are common, but not as persistent as natural anti-A and anti-B antibodies. They are the result of hyper immunization of an iso- or heteroimmune nature and belong to the IgG class. The study of serum for the presence of immune antibodies of the ABO, Rh system is necessary for the diagnosis of hemolytic disease cases of newborns with ABO, Rh-conflict pregnancy. Clinically significant immune antibodies of erythrocyte systems capable to cause in vivo destruction of red blood cells are of great importance in transfusiology and immunohematology. The most common cause of their occurrence is alloimmunization with red blood cell antigens during childbirth. The aim of this study was to determine the characteristics of the immune anti-erythrocyte antibodies in residents of the Republic of Mordovia belonging to the ethnic groups of Moksha and Erzya. Methods. The cross-sectional study involved 294 people from the Moksha and 387 people and Erzya ethnic groups, randomly selected in 7 districts of the Republic of Mordovia. Detection of IgG anti-A, anti-B ABO systems was performed by inactivation of disulfide bonds in IgM molecules using a 5 % unitiol solution and then by direct agglutination method at indoor temperature with standard red blood cells of group O(I), A(II), and B(III). The study of incomplete anti-erythrocyte antibodies of other systems was performed by indirect Coombs' test using gel technology. Results. The study of immune antibodies of the ABO system revealed from 10.61 to 16.00 % of anti-A antibodies and from 2.27 to 6.38 % of anti-B antibodies in both men and women Moksha and Erzya. We studied immune anti-erythrocyte antibodies: anti-D 0.34 % in Moksha and anti-D 0.26% and anti-Kell 0.26 % antibodies in Erzya. The scale of immunogenicity of red blood cell antigens in the studied groups: A&gt;, B&gt;, D&gt; and K&gt;. The alloimmunization index of transfusion-hazardous antigens was calculated as 0.34 for Moksha and 0.52 for Erzya. Conclusions. Physiologically, the immune system in Moksha and Erzya actively reacts with antibody's production and it is sensitive to immunization by the group of antigens.

Genomic analyses of KEL alleles in alloimmunized thalassemia patients from Iran

ObjectivesSerological methods are unreliable for red blood cells (RBCs) antigen typing in multi-transfused thalassemia patients due to the presence of donor RBCs in the recipient’s circulation and interfering antibodies. Kell blood group system is important in transfusion medicine and Kell antibodies have shown as the most prevalent antibodies in thalassemia patients. We intended to determine the genotype of Kell antigens among Iranian alloimmunized thalassemia patients using molecular methods and compare the results with serological phenotyping. MethodsTwo hundred thalassemia patients participated in this study. Blood group phenotype was performed by the serological method, while the genotype was determined for KEL*01, KEL*02, KEL*03, and KEL*04 alleles using PCR-Sequence Specific Primer (PCR-SSP) method. The genotypes of patients with incompatibility between phenotype and genotype were re-evaluated by Restriction Fragment Length Polymorphism-PCR (RFLP-PCR) and confirmed by DNA sequencing in all cases. ResultsTen patients were found with discrepancies between genotype and phenotype; however, there was a complete agreement between the results of SSP-PCR, RFLP-PCR, and DNA sequencing. Six discrepancies were found in the KEL*01/KEL*02 allele when serologically phenotyped as K-k+. One patient with K-k- and three patients with Kpa-Kpb + phenotype were identified as KEL*01/KEL*02 and KEL*03/KEL*04, respectively. ConclusionIt can be concluded that molecular genotyping is more reliable compared with the serological method, especially in the patients who have received multiple transfusions. Therefore, using a combination of these techniques can lead to a better matched transfusion in these patients.

Alloimmunization and autoimmunization in adult transfusion-dependent thalassemia patients: a report from a comprehensive center in Israel.

Patients with beta thalassemia major (TM) are transfusion-dependent (TD) since early childhood and for life. Development of alloantibodies and autoantibodies against red blood cell (RBC) antigens is increasingly recognized as a significant transfusion hazard, especially among heavily transfused patients. The aim of this study is to assess RBC alloimmunization and autoimmunization rates in TD TM patients treated in our Comprehensive Center of Adult Thalassemia, Hemoglobinopathies and Rare Anemias. TD TM patients, regularly transfused every 2-3 weeks, were included in the study. Clinical and RBC transfusion records, including RBC antibodies, since diagnosis in early childhood, were retrieved from patients' files and from the blood bank database. Forty TD TM patients, > 18 years of age, were included in the study. Alloimmunization was demonstrated in 17 (42.5%) patients. Thirty-four alloantibodies were detected, with the most frequent being RH related (12 of 34, 35.3%) followed by those of the Kell system (8 of 34, 23.5%). Age at first transfusion was positively related to the probability of developing alloantibodies (p = 0.02). Splenectomy was found to be correlated with developing alloantibodies (p = 0.016). Logistic regression analysis of the lifelong probability of developing alloantibodies on the age at first transfusion and splenectomy demonstrates a strong positive relationship (p = 0.002). A substantially high rate of alloimmunization was found among adult TD TM patients. Early initiation of RBC transfusions, avoidance of splenectomy and extended Rh and K antigen matching, can reduce the incidence of alloimmunization in TD TM patients.

Prevalence of red-blood-cell and non-red-blood-cell-targeted autoantibodies in alloimmunized postpartum women.

Alloantibodies against red-blood-cell (RBC) antigens often coincide with alloantibodies against leucocytes and platelets and sometimes with autoantibodies towards various antigens. Chimerism may be one of the factors responsible for the combination of allo- and autoantibodies. Women with alloantibodies against RBC antigens causing haemolytic disease of the fetus and neonate may need to receive intrauterine transfusions. These transfusions increase not only maternal antibody formation but also fetomaternal bleeding and may enhance fetal chimerism. We determined the prevalence of and risk factors for autoantibodies against some common clinical target antigens, in alloimmunized women after IUT. We tested for autoantibodies against RBC, anti-thyroid peroxidase, anti-extractable nuclear antigens, anti-cyclic citrullinated proteins and anti-tissue transglutaminase. Women with and without autoantibodies were compared for age; number of RBC alloantibodies, pregnancies and IUTs, and other factors that may play a role in immunization. Non-RBC-targeted autoantibodies were present in 40 of 258 tested women (15·5%, with 90% anti-TPO specificity), comparable to the prevalence reported in healthy Dutch women of these ages. Surprisingly, compared with women who had a single RBC alloantibody, a significantly higher proportion of women with multiple RBC alloantibodies had autoantibodies (5·3% and 18·4%, respectively; odds ratio 4·06, 95% CI: 1·20-13·7). Other characteristics of women with and without autoantibodies were not different. Multiple RBC alloantibodies after extensive allogeneic exposure during pregnancy and presumed increased fetomaternal chimerism are not associated with (selected) autoantibodies. Lack of allo-RBC multi-responsiveness seems associated with decreased auto(-TPO) antibody formation.

Utilising red cell antigen genotyping and serological phenotyping in sickle cell disease patients to risk-stratify patients for alloimmunisation risk.

Alloimmunisation and haemolytic transfusion reactions (HTRs) can occur in patients with sickle cell disease (SCD) despite providing phenotype-matched red blood cell (RBC) transfusions. Variant RBC antigen gene alleles/polymorphisms can lead to discrepancies in serological phenotyping. We evaluated differences between RBC antigen genotyping and phenotyping methods and retrospectively assessed if partial antigen expression may lead to increased risk of alloimmunisation and HTRs in SCD patients at a tertiary centre in Canada. RBC antigen phenotyping and genotyping were performed by a reference laboratory on consenting SCD patients. Patient demographic, clinical and transfusion-related data were obtained from a local transfusion registry and chart review after research ethics board approval. A total of 106 SCD patients were enrolled, and 91% (n = 96) showed additional clinically relevant genotyping information when compared to serological phenotyping alone. FY*02N.01 (FY*B GATA-1) (n = 95; 90%) and RH variant alleles (n = 52, 49%; majority accompanied by FY*02N.01) were common, the latter with putative partial antigen expression in 25 patients. Variability in genotype-phenotype antigen prediction occurred mostly in the Rh system, notably with the e antigen (kappa: 0.17). Fifteen (14.2%) patients had a history of alloimmunisation, with five having HTR documented; no differences in clinical outcomes were found in patients with partial antigen expression. Genotype/extended-phenotype matching strategies may have prevented alloimmunisation events. We show a high frequency of variant alleles/polymorphisms in the SCD population, where genotyping may complement serological phenotyping. Genotyping SCD patients before transfusion may prevent alloimmunisation and HTRs, and knowledge of the FY*02N.01 variant allele increases feasibility of finding compatible blood.