Relevance of blood groups in transfusion of sickle cell disease patients

Blood groups are clinically significant in sickle cell disease (SCD) as transfusion remains a key treatment in this pathology. The occurrence of a delayed haemolytic transfusion reaction (DHTR) is not rare and is a life-threatening event. The main cause of DHTR is the production of alloantibodies against red blood cell antigens. The high rate of alloimmunization in SCD patients is mainly due to the differences of red blood groups between patients of African descent, and the frequently Caucasian donors. From an immuno-haematological point of view, DHTR in SCD patients has specific features: classical antibodies known to be haemolytic can be encountered, but otherwise non significant antibodies, autoantibodies and antibodies related to partial and rare blood groups are also frequently found in individuals of African descent. In some cases, there are no detectable antibodies. As alloimmunization remains the main cause of DHTR, it is extremely important to promote blood donation by individuals of African ancestry to make appropriate blood available.     

Association of HLA-DRB1 and DQB1 alleles with red blood cell alloimmunization in Chinese

Few systematic investigations have assessed the correlations between red blood cell (RBC) antibodies and human leukocyte antigen (HLA)-DRB1 alleles in the Chinese population. In this case-control study, we investigated whether specific HLA-DRB1 alleles were associated with RBC alloimmunization by calculating the odds ratios for the frequencies of HLA alleles associated with alloimmunization to different RBC antigens. Three hundred and eight patients harboring RBC alloantibodies were analyzed as the case group, and the frequencies of the HLA-DRB1and HLA-DQB1 alleles in control individuals were analyzed by collecting data from the China Marrow Donor Program (including more than 1.6 million healthy people). HLA alleles were genotyped by single specific primer-polymerase chain reaction. The development of anti-C was associated with DRB1*07, DQB1*06, and DQB1*08; anti-C,e was associated with DRB1*07 and DQB1*06; and anti-E and anti-M were associated with DQB1. Other associations were identified between anti-E and DRB1*09 and between anti-Le^a and DRB1*01. Thus, our findings confirmed that HLA-DRB1 and DQB1 restriction played an important role in the generation of RBC alloantibodies in Chinese individuals.     

Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

Prevalence and specificity of some rare red cell pan-reactive alloantibodies against high frequency red cell antigens among hospitalized Saudi patients

Some patients'' sera react with all available donors'' red cells and a compatible donor is difficult or impossible to be found. These may either be due to a complex mixture of antibodies or the presence of alloantibodies against high-frequency antigens (HFAs). The aim of this study is to identify the prevalence and characteristics of antibodies to HFAs in Saudi Arabian patients. A total of 23 out of 172 000 patients who received blood transfusions had rare alloantibodies to HFAs at an incidence of 0.013%. Twenty-three patients suspected with pan-reactive alloantibodies against HFAs had their red cells tested using antisera to HFAs, while their plasma was tested against a selected panel of red blood cells with rare phenotypes. Anti-Ge2 antibody was found in the highest number of patients (56.5%), whereas anti-U, anti JK3, anti H, anti-RH 29, anti-hr^s, anti-Kn^a, anti-Ch, anti-Rg, anti-Yt^a, and anti-Cr^a antibodies were found in the remaining patients (43.5%). This study suggests that although antibodies such as anti-Ge2, anti- Kn^a, anti-Ch, anti-Rg, anti-Yt^a , and anti-Cr^a are not clinically significant, they cause a delay in the provision of compatible blood. Whereas, anti-U, anti JK3, anti H, anti-RH29 and anti-hr^s are clinically significant antibodies. An understanding of antibody characteristics to HFAs and the widespread use of the extended red cell phenotype and antibody identification panel will both be helpful for the diagnosis of these HFAs.     

Sero-prevalence of RBC alloantibodies among Han and minority patients in Xinjiang

The aim of the study was to examine the sero-prevalence and frequency of red blood cell (RBC) alloantibodies (RAAbs) and to investigate the risk factors for producing RAAbs among the Han and Uyghurs in Xinjiang. The RBC antibody screening test and identification were conducted for 45,163 Han and 70,633 Uyghurs admitted to the First Affiliated Hospital of Xinjiang Medical University from October 2010 to December 2014. RBC alloantibodies against the Rh antigens were the most frequent, including anti-E(21.7% in the Han, 21.6% in Uyghurs) and anti-D(18.5% in the Han, 18.2% in Uyghurs). Notably, the sero-prevalence of anti-K and anti-Fy^a was also high in Xinjiang. Transfusion and pregnancy were risk factors among both the Han and Uyghurs; furthermore, Uyghurs had a higher sero-prevalence of RBC antibodies compared to that of Han because of a higher incidence of these risk factors. We concluded that RBC alloantibodies against the Rh factor showed the highest frequency, and antibodies against Asian-related high-frequency antigens, including Fy^a and low-frequency antigens, such as K were notably high in Xinjiang.     

The alloantibody response in the allogeneically pregnant rat. II. Primary pregnancy-induced anti-RT1Aa alloantibodies are not as cross-reactive as secondary pregnancy-induced or conventionally raised alloantibodies

We compared the cross-reactive pattern of alloantibodies from conventional anti-DA alloimmunizations with anti-RT1Aa alloantibodies brought about by secondary and primary pregnancy-induced alloimmunizations caused by pregnancies by DA males. As expected, conventional alloimmunization produces alloantibodies that are cross-reactive; secondary pregnancy-induced anti-RT1Aa alloantibodies induced by pregnancies by DA males in females previously immunized against DA were as cross-reactive as conventionally raised antibodies. But primary pregnancy-induced anti-RI1Aa alloantibodies brought about by DA pregnancies in unimmunized females were not as cross-reactive as alloantibodies from conventional or secondary pregnancy-induced alloimmunizations. The cross-reactive pattern of the alloantibodies from primary pregnancy-induced alloimmunizations was distinctive for each of the three responding strains, but the cross-reactive pattern observed for a particular strain was not uniformly seen in all the responding females of that strain. One such pattern between two congenic strains defines an Ir gene between two RT1 phenotypes that are known to be high responders to RT1Aa.     

The prevalence of alloantibody in the population of Xinjiang area of China

Alloantibodies are the major cause of hemolytic transfusion reaction and newborn hemolytic disease. It is highly recommended to screen alloantibodies before transfusion and pregnancy. This report applied the microcolum gel method to screen for available alloantibodies, enrolling 20,098 patients from January 2016 to December 2017 in the Xinjiang General Hospital. Seventy-two patients were found alloantibody-positive, at an overall positive rate of 0.35%. The distribution of alloantibody varied according to age, gender or treatment. The patients aged 70 plus and the patients admitted in the Department of Hematology and Department of Gynecology & Obstetrics had a higher incidence rate of alloantibodies. By using 10 screening cell panel systems, 9 types of alloantibodies including anti-D, anti-E, anti-e, anti-C, anti-c, anti-M, anti-s, anti-Fy^b and anti-Ce were identified. This study suggests that the transfusion of red blood cells(RBCs) and pregnancy are the main causes of alloantibodies.     

Anti-leuko-platelet allo-immunization in blood replacements. Do transfusions of platelets from single donors present an advantage over platelets from multiple donors?

42 patients with acute leukaemia, treated with cytotoxic drugs, have been evaluated retrospectively: --group I: 11 patients received packed red blood cells and platelets from single donors; --group II: 6 patients received packed red blood cells and platelets from multiple donors; --group III: 25 patients received packed red blood cells and platelets from single or multiple donors and granulocytes transfusions. There was no difference in age, sex, time of follow up, number of transfusions, in the three groups. The rate of alloimmunization defined as lymphocytotoxicity against more than 20% of a panel of 24 lymphocytes, was 33% (36% group I--33% group II--32% group III). This study shows that platelets from single donors are of no use in preventing or delaying alloimmunization. On the other hand, their major interest is to provide alloimmunized patients with compatible platelets.     

Irregular antibody screening by Groupamatic at the Paris (C.N.T.S.) and Toulouse (C.R.T.S.) blood transfusion centers

We report here the experience of 3 years of irregular antibody automated screening with Groupamatic, that is to say of 661,511 samples tested in Paris and 269, 162 samples tested in Toulouse. The positive reactions in Paris were 16,296 (2.46%) out of which 2,021 irregular antibodies were identified (0.30%). The positive reactions in Toulouse were 8,266 (3.07%) out of which 2,138 irregular antibodies were identified (0,79%). The difference between the number of screened positive reactions and the identified one is due to false positive reactions (half of the cases) and to autoantibodies whose number is roughly the same than the number of identified alloantibodies. During the years 1970, 1971 and 1972, the irregular antibodies were systematically screened in Toulouse on all blood donors with a manual technique on opaline plate using enzyme treated red cell tests (papain). 7,147 positive reactions were detected, out of 240,080 tests (2.98%). 1,954 (0,81%) were alloantibodies and 1,529 (0.64%) autoantibodies; 3.455 were false positive reactions and 209 were non identified antibodies. These figures are superimposed with those obtained with Groupamatic during the following years, thus pointing out the interest of this equipment.     

Human RBC alloimmunization evaluation after exposure to foreign E antigen of the rhesus system in transfusion

Anti-E alloantibody has been one of the most frequently detected clinically significant alloantibodies in previous studies. Red blood cell (RBC) transfusion is unique in its common intravenous introduction of foreign E antigen and provides a valuable opportunity to study the human immunologic response to intravenous foreign E antigen. Patients exposed to foreign E antigen while receiving RBC transfusions are at risk of forming anti-E alloantibody. Valid estimates of anti-E alloimmunization risk are clinically important, but the forming mechanism of anti-E alloimmunization remains unclear. Here, we screened 516 inpatients at risk of exposure to foreign E antigen while receiving RBC transfusions and monitored the development of anti-E alloantibody for up to two years after left hospital. However, only 2 cases of anti-E alloimmunization were identified in this study. Patients who received RBC transfusion had a very high risk of exposure to foreign E antigen, but the anti-E alloantibody production incidence was very low and few anti-E alloantibodies were produced within 3 to 6 months after RBC transfusion in this study. Further research would contribute to our knowledge of the anti-E alloimmunization mechanism and prevent anti-E development, which would be significantly useful in clinical for transfusions, obstetric management, and the evaluation and management of transfusion reactions in laboratory and institutional resources and cost reduction in healthcare system.     

Blood typing in Saimiri sciureus monkeys: influence of anti-red blood cell alloantibodies on Plasmodium falciparum parasitaemia in vivo

The Saimiri sciureus monkey is a well-established host for experimental studies with human malaria parasites. During the course of iterative inoculations with Plasmodium falciparum parasitised red blood cells (RBC), anti-RBC alloantibodies were detected in the sera of two of eight Saimiri monkeys. These anti-RBC antibodies were further used to investigate RBC phenotypes in 35 colony-reared Saimiri monkeys by flow cytometry. Three RBC phenotypes (named I-III) were observed. Their distribution was I (86%), II (11%) and III (3%). Using the Palo Alto FUP-2 strain, a variant P. falciparum line insensitive to hyperimmune serum and the passive transfer of anti-RBC alloantibodies, a dramatic drop in parasite growth was documented in an incompatible monkey.     

Generation of ‘designer erythroblasts’ lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells

Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34⁺CD41⁺CD235ab⁺ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers—CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.     

Determination of antithrombocyte antibodies in the blood serum of patients with idiopathic thrombocytopenic purpura by an immunoenzyme method

Enzyme-linked immunosorbent assay (ELISA) was developed for determination of serum antiplatelet antibodies. Platelets obtained from healthy donors of blood group 0(1) were washed off plasma and sedimented on the bottom of microtest wells. After washing off unattached platelets and blocking of plastic with albumin platelets were incubated with sera under investigation and binding of serum antibodies was detected using antihuman immunoglobulin antibodies conjugated with peroxidase. Ten patients with idiopathic thrombocytopenic purpura (ITP). 1 patient with systemic lupus erythematosus. 1 patient with red blood cell aplasia and 9 healthy donors (negative control) were studied by ELISA. Serum antibodies which effectively bound to platelets were detected in 5 patients with ITP, in patient with lupus erythematosus and in patient with red blood cell aplasia.     

Incidence of unexpected red blood cell antibodies in the north of China

This study aimed to investigate the frequency of unexpected antibodies and evaluate the cumulative incidence of additional unexpected antibodies in Beijing. From January 1, 2011 to December 31, 2014, blood samples from 2,095 patients from 98 medical institutes in Beijing were sent to the Beijing Red Cross Blood Center for antibody identification. Of the unexpected antibodies, 29.5% were autoantibodies and 70.5% were alloantibodies. Anti-E was the most prevalent form of allo-antibodies (n = 445), accounting for 52.9% of the Rh system, followed by anti-M (76.6% of the MNS system) and then 142 cases of anti-C,e, 128 cases of anti-E,c, and 113 cases of anti-Lea. The cumulative incidences of additional antibodies were 0.55% (after the first transfusion), 1.82% (second time), 2.33% (fourth time), 3.07% (firth time), and 4.24% (seventh time). Antibody against the Rh system was the most prevalent, followed by antibodies against MNS, Lewis, Kidd, P1, and Duffy.     

Risk Estimation of HNA-3 Incompatibility and Alloimmunization in Thai Populations

Severe transfusion-related acute lung injury (TRALI) is often due to antibodies in blood components directed against human neutrophil antigen (HNA)-3a. This study aimed to report the genotype frequencies of the HNA-3 system and to estimate the potential risk of HNA-3 incompatibility and alloimmunization in two Thai populations. Eight hundred DNA samples obtained from 500 unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok and 300 samples from the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand were included. HNA-3 genotyping was performed using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The observed frequencies of the HNA-3a/3a, HNA-3a/3b, and HNA-3b/3b genotypes were 0.528, 0.380, and 0.092 in central Thais and 0.600, 0.350, and 0.050 in northern Thais, respectively. The frequencies were used to estimate HNA-3 incompatibility and risk of HNA-3a alloimmunization. The HNA-3 incompatibility in central Thais (33.28%) was higher than northern Thais (28.75%), corresponding to a significantly higher probability of HNA-3a alloimmunization (P<0.05) similar to Japanese and Chinese populations. This study showed the high risk of HNA-3 incompatibility and alloimmunization, especially in central Thai blood donors. A molecular-based identification of the HNA-3 genotype of female donors is suggested to reduce the risk of TRALI following plasma and whole blood allogeneic transfusion.     

Detection of anti-CD32 alloantibody in donor plasma implicated in development of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) occasionally causes serious symptoms that may be fatal to recipients. Polymorphonuclear neutrophils (PMNs) and alloantibodies specific to PMN cell surface antigens are suspected to cause TRALI. The aim of this study is to establish a sensitive and stable procedure of detecting alloantibodies not only in donor blood, but also in recipient's plasma. We have introduced a new method of detecting alloantibodies based on double-determinant enzyme-linked immunosorbent assay (DD-ELISA) and a monoclonal antibody-immobilized granulocyte antigen (MAIGA) test (arbitrarily designated as modified DD-ELISA). We verified the specificity of alloantibodies against PMN cell surface antigens in plasma samples from three normal healthy donors of blood that induced respiratory distress in recipients after a blood transfusion. Anti-CD32 (Fc gamma RIII) alloantibodies were detected in all the plasma samples using two different clones of the monoclonal anti-CD32 antibody. The specificities of these plasma samples could not be identified by the granulocyte immunofluorescence test (GIFT) using typed test cells. Except for the anti-CD32 alloantibodies, one plasma sample was proved to have the anti-HNA-1a alloantibodies. In another plasma sample, the anti-HNA-2a alloantibodies were detected. By modified DD-ELISA, we could clearly specify the presence of alloantibodies in the three plasma samples. Our results also suggest that the anti-CD32 alloantibodies can be generated in vivo and may play some roles in the development of TRALI.     

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.     

Study of equivalents of rhesus antigens in non-human primates]

Human alloantibodies specific of some Rh antigens cross-react with non human primates red blood cells. These crossreactions demonstrated that only African apes express equivalents of Rho (D) and hr' (c). The antigenic resemblance between these two human antigens and their primate homologues is confirmed by the reactivities of human anti-D and anti-c monoclonal antibodies. The use of a human Rh cDNA probe allowed to confirm by Southern blot hybridization that nonhuman primates possess Rh-like genes. The number of Rh-like genes per haploid genome was deduced from the results obtained with exon-specific probes.     

Characterization with dithiothreitol (DTT) of IgM auto-lymphocytotoxic antibodies in dialyzed patients awaiting kidney transplants

A group of 42 sensitized dialysis patients showing high reactivity (81%-100%) against a random panel of peripheral blood lymphocytes (PBL), were analyzed for the presence of autoreactive lymphocytotoxic antibodies. The test was performed at different temperatures (4 degrees C, 22 degrees C, 37 degrees C) with dithiothreitol (DTT) against autologous PBL, EBV-induced autologous B lymphoblasts (A-BCL), K562 cells and T lymphocytes. Thirteen of 42 patients had IgM auto-lymphocytotoxic antibodies. The broadly reactive IgM autoantibodies could be inactivated by treatment with DTT 5 mM and allowed the identification of the presence of autoantibodies alone or in combination with anti-IgG alloantibodies.