non-ABO Blood Group Research Articles

Is Indirect Coombs Test by Pooled Cells Sufficient for Antibody screening? An Eye-Opening Case Series

The ideal way to screen for the presence of alloantibodies is by antibody screening panels which represent all clinically significant antigens in appropriate dosage. Most centers use pooled O-cells for antibody screening as it has antigens of non-ABO blood group systems that are prevalent in a representative population. Pooled O-cells sometimes fail to detect antibodies to less prevalent red blood cell antigens with reduced expression or show a dosage phenomenon. Despite pooling 4 to 5, O-donor segments, sometimes, it is difficult to detect clinically significant antibodies. False-negative indirect Coombs test by pooled O-cells may delay getting a compatible unit for elective cases where type and screen policy is used. Donor units with weak antigenic expression or units showing dosage can come compatible despite being antigen positive and lead to a hemolytic reaction. We report two cases where antibody screening by pooled O-cells was negative; still, cross-match was incompatible. Antibody screening with a three-cell panel was positive. Antibody identification with 11-cell panels confirmed the alloantibody to be anti-E. The present cases emphasize the importance of three-cell panels over pooled O-cells.

Red blood cell alloimmunization and sickle cell disease: a narrative review on antibody induction.

The high prevalence of red blood cell (RBC) alloantibodies in people with sickle cell disease (SCD) cannot be debated. Why people with SCD are so likely to form RBC alloantibodies, however, remains poorly understood. Over the past decade, a better understanding of non-ABO blood group antigen variants has emerged; RH genetic diversity and the role this diversity plays in RBC alloimmunization is discussed elsewhere. Outside of antigen variants, the immune systems of people with SCD are known to be different than those of people without SCD. Some of these differences are due to effects of free heme, whereas others are impacted by hyposplenism. Descriptive studies of differences in white blood cell (WBC) subsets, platelet counts and function, and complement activation between people with SCD and race-matched controls exist. Studies comparing the immune systems of alloimmunized people with SCD to non-alloimmunized people with SCD to race-matched controls without SCD have uncovered differences in T-cell subsets, monocytes, Fcγ receptor polymorphisms, and responses to free heme. Studies in murine models have documented the role that recipient inflammation plays in RBC alloantibody formation, with human studies reporting a similar association. Murine studies have also reported the importance of type 1 interferon (IFNα/β), known to play a pivotal role in autoimmunity, in RBC alloantibody formation. The goal of this manuscript is to review existing data on factors influencing RBC alloantibody induction in people with SCD with a focus on inflammation and other immune system considerations, from the bench to the bedside.

CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model.

Approximately 3–10% of human red blood cell (RBC) transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that “non-responders” may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA) antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA) specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb)) demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken together, our data suggest that recipients may indeed become tolerized to antigens expressed on RBCs, with the recipient’s immune status upon initial RBC exposure dictating future responses. Although questions surrounding mechanism(s) and sustainability of tolerance remain, these data lay the groundwork for future work investigating RBC immunity versus tolerance in reductionist models and in humans.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG.

Red blood cell alloimmunization: new findings at the bench and new recommendations for the bedside.

To summarize recent discoveries from clinical studies and animal models that contribute to understanding the alloimmune response to non-ABO blood group antigens. Several studies have confirmed high rates of alloimmunization among patients requiring chronic red blood cell (RBC) transfusion. Moreover, 'triggers' for alloantibody development in the transfusion setting have been identified, with a number of investigations linking recipient inflammation to a higher likelihood of alloimmunization. Additional associations between human leukocyte antigen expression and CD4 T-cell markers in 'responder' or 'nonresponder' humans have been revealed. Recent animal studies have described novel mechanistic properties by which the alloimmune response is governed, including the critical role played by dendritic cells in transfusion-associated alloimmunization. New light has also been shed on the properties of alloantibodies developed as a result of pregnancy, as well as mechanisms through which such alloimmunization may be prevented. Many of the clinical/biological factors that contribute to the RBC alloimmune response have been further elucidated. This knowledge will be applied to identify individuals most likely to mount an immune response to RBC antigens, such that appropriate resources and strategies for preventing alloimmunization (or mitigating its harmful effects) can be implemented.

P142 Hyperacute rejection in a kidney transplant recipient with no HLA antibodies

A 65 year old, blood group O, African-American male with 0% CPRA received a blood group O deceased donor kidney transplant with a negative crossmatch (XM). During surgery, the transplanted kidney became dusky within minutes of restoring blood flow. There was no suspicion of anti-HLA or anti-ABO antibodies which could mediate hyperacute rejection, so a thrombotic event or anatomical defect was suspected. The OPO consented to allocate the second kidney from the same donor; it was transplanted within hours, and the same events transpired. Three days later, the recipient had bleeding and pain at the graft site, and donor vein and artery grafts had to be removed. We sought to identify the factors which caused the allografts loss. Both kidneys were sent for microscopic and C4d evaluation. Recipient HLA antibodies were tested by FlowPRA and Luminex Single Antigen Beads (LSAB). XM were performed by CDC and flow cytometry. Recipient serum was tested for MICA antibodies by LSAB, endothelial cell (EC) antibodies by XM, AT1R antibodies by ELISA, and non-ABO blood group antibodies. Molecular blood typing of the donor DNA was performed. Histopathology identified karyorrhectic nuclear debris in the glomeruli, small fibrin thrombi, and neutrophilic infiltrates, consistent with hyperacute rejection, although IHC failed to show definite C4d deposition. All FlowPRA, XM, LSAB, MICA, and EC XM tests were negative. The AT1R result was 14 U/mL (at risk). Molecular testing confirmed the donor as Group O. Testing ruled out the rare Bombay phenotype, and the recipient was negative for all non-ABO blood group antibodies, except for weak cold agglutinins only seen at 4 °C. The events during the surgery and the pathology findings are consistent with humoral hyperacute rejection. Confoundingly, the recipient has no HLA antibodies and had negative CDC and flow XM with the donor. However, none of the other testing has been able to identify an antibody with a strength that would explain hyperacute rejection. The only identified antibodies thus far are anti-AT1R and cold agglutinins. We are continuing to investigate more non-HLA antibodies that may have a synergistic impact on graft rejection.

Detection rate of blood group alloimmunization based on real-world testing practices and kinetics of antibody induction and evanescence.

Failure to detect non-ABO blood group alloantibodies places patients at risk for hemolytic reactions. Suboptimal alloantibody detection could result from posttransfusion testing performed too early, too late, or not at all. Testing performed too early may precede antibody induction, while testing performed too late could miss antibodies that have evanesced. Taking these factors into account, our goal was to determine the percentage of alloantibodies detected with real-world testing practices. The alloantibody detection rate in a general hospital setting was determined based on the frequency and timing of antibody testing after red blood cell (RBC) transfusions and rates of antibody induction and evanescence. Intervals to follow up testing after RBC transfusions (n=561 RBC units in 100 random patients) were determined retrospectively. Best-fit lines and equations for antibody induction and evanescence were computed on previously published data. Nearly half (271/561; 48.3%) of RBC infusions had either no follow-up antibody screen or testing too soon (<30 days) after transfusion to detect alloimmunization. Of the remaining RBC units, 10.3% (58/561) had follow-up testing 30 to 112 days posttransfusion, 28.7% (161/561) were followed up at more than 112 days, and 12.7% (71/561) were tested at both 30 to 112 days and more than 112 days. By inputting these timing data into best-fit line equations for antibody induction and evanescence, we calculated an alloantibody detection rate of 31.6%. Posttransfusion antibody testing was inadequately timed for optimal alloantibody detection. Real-world compatibility testing was predicted to detect less than one-third of non-ABO antibodies, thereby exposing patients to risks of mismatched transfusion.

Non-ABO blood group systems phenotyping in non-human primates for blood banking laboratory and xenotransplantation

Some biomedical research procedures, such as organ xenotransplantation, usually require intensive hemotherapy. Knowledge of the whole phenotype of blood donor and graft could be useful in the field of xenotransplantation. Human and simian-type categories of blood groups have been established and they can be tested by standard methods used for human blood grouping. The aim of this work was to study the incidence of non-ABO blood group systems in different species of non-human primates, which are employed in biomedical research. The phenotype of Rh, Lewis, Kidd, Kell, MNSs, Lutheran, P and Duffy antigens was investigated in olive baboon (n = 48), chacma baboon (n = 9), Guinea baboon (n = 14), Rhesus macaque (n = 38) and squirrel monkey (n = 30) by using commercial microtyping cards. Kell, Lutheran, Kidd and Duffy antigens have been detected in all species, Rh in squirrel monkey, MNSs in rhesus macaque and squirrel monkey, and Lewis in baboon and rhesus macaque. There were differences in frequency and haemagglutination scores between species regardless of their gender and age. The main differences were found in squirrel monkey when compared with baboons and macaques. This typing system provides a tool to assess the presence of antigens in animals used for experimental procedures, such as xenotransplantation and xenotransfusion.

The persistence and evanescence of blood group alloantibodies in men

Non-ABO blood group (BG) alloantibodies can disappear over time, confounding compatibility testing and predisposing patients to delayed hemolytic transfusion reactions. The goal of this study was to analyze BG antibody disappearance after transfusion-related alloimmunization in men. The transfusion service records of 18,750 military veterans at a Department of Veterans Affairs (VA) medical center were screened to identify male patients with one or more BG alloantibodies and who had at least one type and screen performed after initial antibody detection (n = 304). Antibodies were categorized as to whether they were present at a patient's first antibody screening test ("preexisting") or after initial negative testing ("hospital-acquired"). Overall, the evanescence of hospital-acquired antibodies (108/222; 48.6%) was significantly higher (p < 0.0001) than that of preexisting antibodies (36/185; 19.5%). Half (54/108) of evanescent, hospital-acquired alloantibodies disappeared within 6 months of detection, and all disappeared by 10 years. Evanescence of hospital-acquired antibodies was dependent on duration of follow-up testing, because antibodies tested 5 or more years after initial development demonstrated the highest evanescence rate (35/55; 64%; p < 0.01). Some evanescent antibodies (9/407; 2.2% of total antibodies) disappeared and reappeared one or more times without known antigenic reexposure ("multiple evanescence"). Among commonly observed alloantibodies, disappearance varied with antigenic specificity. Approximately two-thirds of BG alloantibodies disappeared within 5 years of induction, a rate higher than previously reported in mixed-sex populations. Evanescence did not appear to be a random, first-order decay process, as evidenced by the lower evanescence rate of preexisting antibodies.

Immune complex binding by immunocamouflaged [poly(ethylene glycol)‐grafted] erythrocytes

Immune complexes (IC) are constantly formed at low levels in normal individuals. In humans, the red blood cell (RBC) complement receptor 1 (CR1) plays the dominant role in the IC binding and clearance. Over the last several years, we have investigated the potential utility of immunocamouflaged (methoxypoly(ethylene glycol) [mPEG] grafted) RBC to attenuate the risk of alloimmunization. Because the grafted polymer nonspecifically camouflages membrane proteins, its effects on CR1 detection and IC binding were assessed. The dose dependent (0-2.5 mM) effects of activated mPEG (CmPEG, 5 kDa; and BTCmPEG, 5 and 20 kDa) on CR1 detection and the binding of artificially generated IC [C3b coated alkaline phosphatase and antialkaline phosphatase complexes] to control and pegylated RBC was investigated by flow cytometry. In contrast to selected non-ABO blood group antigens, grafted mPEG did not effectively camouflage CR1. Surprisingly, however, even very low grafting concentrations of mPEG (>or=0.3 mM) resulted in a >or=95% loss in IC binding. Further reductions in grafting concentration (0.15 and 0.03 mM mPEG) still yielded decreased IC binding of approximately 60 and 40%, respectively. Importantly, unactivated mPEG had minimal effects on IC binding. These data demonstrate that even small amounts of grafted mPEG interfere with the multivalent CR1-IC interaction necessary for high affinity IC binding, hence large volume transfusions of mPEG-RBC may be contraindicated in patients with pre-existing IC disease. Whether this concern is of clinical significance in healthy humans is less clear due to dilutional effects and the presence of secondary clearance pathways.

A statistical appraisal on the relationship between non-ABO blood group systems and diseases.

The present review was visualised as a search for relationship between non-ABO blood group systems including the AB(H) secretor types and susceptibility to representative group of diseases associated with various systems of the body. The materials were collected from all publications available to us during 1950–1967. For statistical evaluation, the method of Woolf (1955) was followed. It becomes evident from the various reports that many of the authors were aware of the possible biases, and did their best to avoid them. Three degrees of probabilities have been considered. The possible association of certain diseases to particular blood groups or types has been discussed. It is the main purpose of this review to show, where there are possible relationships, and to encourage further investigations in the same direction.