anti-B IgM Research Articles

Red Blood Cell Membrane-Coated Nanoparticles Enable Incompatible Blood Transfusions.

Blood transfusions save lives and improve health every day. Despite the matching of blood types being stricter than it ever has been, emergency transfusions among incompatible blood types are still inevitable in the clinic when there is a lack of acceptable blood types for recipients. Here to overcome this, a counter measure nanoplatform consisting of a polymeric core coated by a red blood cell (RBC) membrane is developed. With A-type or B-type RBC membrane camouflaging, the nanoplatform is capable of specifically capturing anti-A or anti-B IgM antibodies within B-type or A-type whole blood, thereby decreasing the corresponding IgM antibody levels and then allowing the incompatible blood transfusions. In addition to IgM, the anti-RBC IgG antibody in a passive immunization murine model can likewise be neutralized by this nanoplatform, leading to prolonged circulation time of incompatible donor RBCs. Noteworthily, nanoplatform made by expired RBCs (>42 days stored hypothermically) and then subjected to lyophilization does not impair their effect on antibody neutralization. Most importantly, antibody-captured RBC-NP do not exacerbate the risk of inflammation, complement activation, and coagulopathy in an acute hemorrhagic shock murine model. Overall, this biomimetic nanoplatform can safely neutralize the antibody to enable incompatible blood transfusion.

An in vitro comparison of intra-operative isohemagglutinin and human leukocyte antigen removal techniques in pediatric heart transplantation

Background: Highly sensitized pediatric patients awaiting heart transplantation experience longer wait times and thus higher waitlist mortality. Similarly, children less than 2 years of age have increased waitlist times and mortality when compared to their older peers. To improve the likelihood of successful transplantation in these patients, various strategies have been utilized, including peri-operative plasmapheresis. However, limited data exists comparing plasmapheresis techniques for antibody reduction. This study’s aim was to compare the in vitro magnitude of isohemagglutinin titers (IT) and human leukocyte antigen (HLA) antibody removal and the time required between membrane-based plasmapheresis (MP) and centrifuge-based plasmapheresis (CP) incorporated into the extracorporeal (EC) circuit. Methods: Two MP (Prismaflex) and two CP (Spectra Optia, Terumo BCT) circuits were incorporated into four separate EC circuits primed with high titer, highly sensitized type O donor whole blood. Assays were performed to determine baseline IT and anti-HLA antibodies and then at 30-minute increments until completion of the run (two plasma volume exchanges) at two hours. Results: There was a decrease in anti-A and anti-B IgM and IgG titers with both MP and CP. Mean anti-A and anti-B titer reduction was by 4.625 titers (93.7% change) and 4.375 titers (93.8% change) using MP and CP, respectively. At 2 h of apheresis, CP reduced 62.5% of all ITs to ≤ 1:4, while MP reduced 50% of ITs to ≤ 1:4. Additionally, reduction of anti-HLA class II antibody to mean fluorescence intensity (MFI) <3000 was achieved with both MP and CP. At 2 h of apheresis, CP reduced MFI by 2–3.5 fold and MP reduced MFI by 1.7–2.5 fold. Both demonstrated similar hemolytic and thrombotic profiles. Conclusions: In this in vitro plasmapheresis model of IT and anti-HLA antibody reduction, both MP and CP incorporated into the EC circuit can be used quickly and effectively to reduce circulating antibodies. While CP may have some greater efficiency, further study is necessary to verify this in vivo.

Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B.

Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re-examined the association between ABO antigens and GPA/GPB. Band 3/GPA-associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N-glycans and O-glycans. Serological anti-A (BIRMA 1) and anti-B IgM (GAMA 110) could be used for western blot (WB); however, only the anti-B IgM showed significant reactivity for the immunoprecipitates isolated by anti-band 3. The expression of the B antigen in un-deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared. Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N- and O-glycans. Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non-trivial expression of the B antigen on GPA and GPB on the erythrocyte surface.

High titers of anti-A1 and anti-B antibodies among Peruvian group O platelet donors

Critical antibody titers have been described as factors associated with hemolysis in ABO plasma-incompatible platelet (PLT) transfusions. This study was carried out to describe the frequency of high-titers anti-A and antiB IgM and IgG antibodies in group O apheresis platelet donors, and to explore differences according to the donor characteristics. A cross-sectional study was carried out at the Blood Bank of a National Hospital in Peru from January to March 2019. IgM and IgG antibodies against A1 and B antigens were quantified in 339 platelet donors using the direct hemagglutination technique and the solid-phase adherence technique, respectively. For analysis purposes, two cut-off points; ≥128 and ≥64, were used to define a critical titer for IgM due to a lack of consensus. An IgG titer of ≥256 was also defined as critical. Of the donors, 22.1 % had critical IgM titers when the cut-off point was defined as ≥128. However, when the IgM cut-off was ≥64, the frequency of platelet donors with critical titers increased to 54.0 %. The frequency of donors with critical IgG titers was 23.5 %. Higher IgG titers were associated with female donors while higher IgM titers were negative associated with age. One in two or three platelet donors, depending on the cutoff point used to define a critical IgM titer, had at least one critical titer of anti-A or anti-B antibodies. Early identification of platelet donors with critical antibody titers could prevent passive transfusion of ABO antibodies to non-isogroup recipients.

Assessment of severity and mortality of COVID‐19 with anti‐A1 and anti‐B IgM isohaemagglutinins, a reflection of the innate immune status

AimsThe relationship between the innate immune system that creates the polysaccharide antibody response and COVID‐19 is not fully understood. In this study, it was aimed to determine the predictive values of isohaemagglutinins in COVID‐19 severity/mortality.MethodsApproximately 15 440 patients diagnosed with COVID‐19 were examined, and a total of 286 patients with anti‐B and anti‐A1 IgM isohaemagglutinins test results were randomly enrolled in the study. These patients were stratified into two groups according to anti‐A1 (n: 138 blood type B or O) and anti‐B (n: 148 blood type A) IgM isohaemagglutinins. Anti‐A1 or/and anti‐B IgM, biochemical parameters, symptoms, chronic diseases, hospitalisation status, intubation status, admission to intensive care unit (ICU) and exitus status were recorded and evaluated for all patients.ResultsAnti‐A1 IgM and anti‐B IgM were significantly lower in ICU patients (7.5 ± 9.9 vs 18.0 ± 20.4 and 5.5 ± 6.3 vs 19.3 ± 33.6 titres, respectively; P < .01) and in exitus patients (3.8 ± 3.6 vs 16.7 ± 18.7 and 3.5 ± 4.7 vs 16.9 ± 29.6 titres respectively; P < .01). In the ROC analysis performed to differentiate between exitus and discharge within groups, the sensitivity of anti‐B IgM and anti‐A1 IgM at cut‐off ≤4 was 88.9% and 79.6%, specificity 66.0% and 73.4%, and AUC 0.831 and 0.861, respectively (P < .01). Anti‐A1 IgM decreased the mortality risk 0.811 times per unit while anti‐B IgM decreased 0.717 times (P < .01).ConclusionAnti‐B and anti‐A1 isohaemagglutinins, which are an expression of the innate immune system, can be used to predict the severity and mortality of COVID‐19 disease.

A Comparison of Intra-Operative Isohemagglutinin Removal Techniques in Pediatric Heart Transplantation

ABO-incompatible (ABO-I) heart transplantation (HT) is used in children <2 years of age to reduce waitlist times and mortality with outcomes comparable to ABO-compatible HT. This strategy is made safer by reducing isohemagglutinin titers (IT). Limited data exists comparing techniques for IT reduction. This study's aim was to compare the in vitro magnitude of IT removal and time required between membrane-based plasmapheresis (MP) and centrifuge-based plasmapheresis (CP) incorporated into the cardiopulmonary bypass (CPB) circuit. Two MP (Prismaflex) and two CP (Spectra Optia, Terumo BCT) circuits were incorporated into four separate CBP circuits primed with high titer type O donor whole blood. Assays were performed to determine baseline isohemagglutinin titers and then at 30 minute increments until completion of the run (two plasma volume exchanges) at two hours. There was a decrease in anti-A and anti-B IgM and IgG titers with both MP and CP. Mean anti-A and anti-B titer reduction was 4.625 titers (93.7% change) and 4.375 titers (93.8% change) using MP and CP, respectively. At 2 hours of apheresis, CP reduced 62.5% of all ITs to ≤ 1:4, while MP reduced 50% of ITs to ≤1:4. Both demonstrated similar hemolytic and thrombotic profiles. In this in vitro plasmapheresis model of IT reduction, both MP and CP incorporated into the CPB circuit can be used quickly and effectively to reduce circulating IT. While CP may have some greater efficiency, further study is necessary to verify this in vivo.

Evaluation of anti-A and anti-B IgM and anti-AB IgG antibody titers in Group O blood Iraqi donors

BACKGROUND: Blood group O is transfused as a universal blood group in emergencies. The red cells of blood Group O possess no major ABO antigens, while the blood Group O plasma contains naturally occurring IgM anti-A and anti-B and cross-reacting IgG anti-AB antibodies. Thus, the transfusion of blood Group O units may cause marked destruction of the recipients' A or B red cells manifested as severe acute hemolytic transfusion reactions owing to potent ABO hemolytic antibodies.OBJECTIVES: This study was done to estimate the titers of anti-ABO antibodies in Group O blood donors and to suggest a possible scheme to introduce as a routine testing for high ABO antibodies' titers in blood Group O donors in Iraqi blood transfusion centers.SUBJECTS AND METHODS: Two hundred blood Group O Iraqi healthy blood donors (192 males and 8 females, age range: 18–59) were chosen to evaluate the titer of ABO antibodies (IgM and IgG) in their serums using tube test.RESULTS: The prevalence of high-titer anti-A and anti-B IgM antibody at 1:50 dilution in this study was 98.5% and 97.5%, respectively (P < 0.05). The prevalence of high-titer anti-AB IgG measured by dithiothreitol and 2-mercaptoethanol method of at least 50 was 63% and 74%, respectively (P < 0.05).CONCLUSIONS: There is a high prevalence of high-titer anti-A and anti-B IgM and IgG antibodies among blood Group O Iraqi donors. The detection of IgG antibodies is more practical by using dithiothreitol method as it needs much shorter incubation time; however, 2-ME is more sensitive. Detection of high-titer anti-A IgM antibody in dangerous universal donors, by immediate spin at 1:50 dilution, can be used alone in practice, replacing the need to use all portfolio of tests of anti-ABO antibodies.

Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors

Background and Objectives: Severe hemolytic transfusion reactions due to minor incompatibility are usually associated with the so-called “High titred” antibodies in the plasma of donors especially the O group as it is generally regarded as universal donors. Critical titers of 1:64 or higher was considered high titered antibodies for IgM anti-A and anti-B. Critical titers of 1:256 or higher was considered high titered antibodies for IgG anti-A and anti-B. The objective of this study was to see the prevalence of such high titered antibodies among our donor population and to observe for an association if any with the age, gender, and diet type and probiotic intake of the donors. Methodology: This was a cross-sectional analytical study conducted over 20 months from September 2015 to May 2017. About 560 donors were chosen randomly after screening and counseling and titration of anti-A and anti-B antibodies was done by the double dilution tube agglutination technique. The information on age, gender, diet type, and probiotic intake was gathered from the donor history questionnaire and an association between the factors described and the distribution of critical titers of anti-A and anti-B was elicited. Results: In the present study, the prevalence of high titer anti-A and anti-B IgM antibodies was 36% and 32%, respectively. The prevalence of high titer anti-A and anti-B IgG antibodies was 9.6% and 5.5%, respectively. The median IgM antibody titers reduced with the increasing age of the donors starting 18 years. There was a statistically significant association between a vegetarian diet and high titers. Conclusion: There are a considerable proportion of O group donors with high titers of antibodies and hence a routine pretransfusion screening for such antibodies can prevent the development of hemolytic reactions by segregating units into “high titer” and “low titer” antibodies and issuing only low titer products for out of group transfusions.

A novel method for ABO-incompatible heart transplantation

Since 1996, ABO-incompatible heart transplantation has been undertaken by performing whole-body plasma exchange to remove isohemagglutinins using the cardiopulmonary bypass (CPB) circuit at the time of transplantation. This requires large volumes of donated blood and blood products, causes hemodynamic instability during the exchange transfusion, and limits practical use to small children. We sought to determine the efficacy of anti-A/B immunoadsorption within the CPB circuit on removal of isohemagglutinins in an ex vivo setting before its use clinically. An anti-A/B immunoadsorption column was placed into a CPB circuit mimicking a typical ABO-incompatible transplant patient, which had been primed with type O whole human blood. Samples were taken for determination of isohemagglutinin titers following each plasma volume pass through the anti-A/B immunoadsorption column. There was a linear decrease of at least 1 dilution seen in both anti-A and anti-B IgG and IgM antibodies with each plasma volume pass through the column. This predictable removal allowed the formulation of selection criteria for ABO-incompatible heart transplantation given the reciprocal of titer and patient weight. This degree of predictability allowed us to use it successfully in the clinical setting, reducing antibodies to an undetectable level during ABO-incompatible heart transplantation. The incorporation of an anti-A/B immunoadsorption column into the extracorporeal circuit reduces allogeneic blood product requirement for ABO-incompatible heart transplantation, while providing efficacious removal of anti-A and anti-B isohemagglutinins. This can be undertaken within the time period of CPB before graft reperfusion and expands the potential recipient pool to larger patients with higher isohemagglutinin titers.

Dangerous universal donors: the reality of the Hemocentro in Belo Horizonte, Minas Gerais.

BackgroundThe term dangerous universal blood donor refers to potential agglutination of the erythrocytes of non-O recipients due to plasma of an O blood group donor, which contains high titers of anti-A and/or anti-B hemagglutinins. Thus, prior titration of anti-A and anti-B hemagglutinins is recommended to prevent transfusion reactions.ObjectiveThe aim of this study was to estimate the frequency of dangerous universal donors in the blood bank of Belo Horizonte (Fundação Central de Imuno-Hematologia – Fundação Hemominas – Minas Gerais) by determining the titers of anti-A and anti-B hemagglutinins in O blood group donors.MethodA total of 400 O blood group donors were randomly selected, from March 2014 to January 2015. The titers of anti-A and anti-B hemagglutinins (IgM and IgG classes) were obtained using the tube titration technique. Dangerous donors were those whose titers of anti-A or anti-B IgM were ≥128 and/or the titers of anti-A or anti-B IgG were ≥256. Donors were characterized according to gender, age and ethnicity. The hemagglutinins were characterized by specificity (anti-A and anti-B) and antibody class (IgG and IgM).ResultsAlmost one-third (30.5%) of the O blood group donors were universal dangerous. The frequency among women was higher than that of men (p-value = 0.019; odds ratio: 1.66; 95% confidence interval: 1.08–2.56) and among young donors (18–29 years old) it was higher than for donors between 49 and 59 years old (p-value = 0.015; odds ratio: 3.05; 95% confidence interval: 1.22–7.69). There was no significant association between dangerous universal donors and ethnicity, agglutinin specificity or antibody class.ConclusionEspecially platelet concentrates obtained by apheresis (that contain a substantial volume of plasma), coming from dangerous universal donors should be transfused in isogroup recipients whenever possible in order to prevent the occurrence of transfusion reactions.

PTH-055 ABO Blood Group-Specific Immunoglobulins in Crohn’s Disease

Introduction The pathogenesis of Crohn’s disease (CD) is complex and believed to be interplayed by genetic susceptibility, enteric microflora and tissue injury mediated by abnormal immune response. Several studies have described the association between ABO blood group, enteric microflora composition and predisposition to gastrointestinal (GI) infections. The mucosal layer of the GI tract forms the first layer of immune defenses with mucus abundant in secretory immunoglobulins, and studies have shown that non-O blood group and FUT2-determined ABO antigen non-secretor were linked to more severe CD. It was suggested that ABO antibodies are induced via an enteric route by cross-reactivity from microflora-derived molecules . However, little is known about the role of ABO antibodies in the context of CD. Therefore we assessed whether blood group-specific antibodies are implicated in CD pathogenesis. Methods We compared the immunoglobulin class profile of blood group-specific antibodies in blood group A, B and O individuals. Plasma samples were collected from CD cohort (n = 45, recruited from the FaMIsHED study) with age- and gender-matched healthy donors (n = 89 data from service development study). Medications of CD cohort include none (n = 15), 5 ASA (n = 21), immunosuppressant (n = 12) and biologics (n = 10). A1rr and Brr cells were used, anti-A1 and anti-B IgG, IgA and IgM levels were measured by Relative Median Fluorescence using flow cytometry. Mann-Whitney U test was used to assess statistical significance of different antibody levels between CD and healthy cohorts. Results Blood group O CD patients (n = 20) have significantly lower anti-A1 IgG, IgA and IgM compared to the healthy blood group O cohort with p value of 0.0001, 0.0009 and Conclusion CD patients were found to have lower than baseline ABO antibodies. However, it is not known whether these findings represent the cause or effect of disturbed immune modulations, or their roles in modulating intestinal microflora. Further research into the difference in ABO antibodies class profile in a larger cohort, and comparison of ABO antibodies class profile of CD patients in remission and relapse are required. Disclosure of Interest None Declared

Serologic and Phenotypic Analysis of Blood Types Via Silicon Nanophotonics

Tens of millions of donor and patient samples are tested yearly to establish blood type compatibility between donor and recipient and to protect recipients from blood-borne infectious diseases. Blood type testing, particularly donor testing, is traditionally based in centralized clinical laboratories. However, current blood typing methods are encumbered by reagent availability, cost, technical training requirements, and time, placing a costly burden on the medical system. To address practical needs in blood typing, we have developed a multiplexed blood analysis platform using a low-cost and scalable silicon photonic biochip. This study investigates the use of silicon microring sensors to capture, detect, and quantify specific red blood cell (RBC) membrane antigens and anti-blood type antibodies from blood.To validate ABO blood phenotyping, microring resonators were streptavidin coated and functionalized with biotinylated anti-A IgM or biotinylated anti-B IgM antibodies. First, the response of anti-A/B functionalized microring resonators to characterized RBC membranes (RBC ghosts, 108 cells/ml) were measured in real-time (Figure 1). The biosensor arrays also exhibited minimal non-specific adsorption of RBC membrane fragments to the sensor surface. Microring resonators were shown to be suitable for identifying RBC ABO phenotype from donor blood samples.For ABO serologic analysis, silicon chips were functionalized with synthetic multivalent polymeric blood group antigens to serve as capture elements for circulating anti-ABO antibodies. Each chip also had sensors functionalized with biotinylated Protein A (btn-ProtA) and a biotinylated polyacrylamide polymer scaffold (btn-paa) to serve as on-chip positive and negative controls, respectively. The multiplexed biosensor chips were exposed to 100mL of plasma, followed by an anti-human-IgM antibody to enhance detection and quantification of antibodies bound to the surface. The resonance shift in each microring resonator was monitored over time, and the sensor response of the polymeric A and B blood group antigens was normalized to the control sensors. Figure 2 illustrates the levels of bound anti-A and anti-B for a panel of donor blood samples with varying ABO blood type, expressed as a relative shift in sensor resonance wavelength. These results demonstrate the detection of the 'naturally occurring’ anti-A/B IgM antibodies for each respective ABO blood type.We have demonstrated that microring resonator biosensor arrays can quantitatively determine the donor ABO phenotypic and serologic status while incorporating on-chip controls for process standardization. Our work serves as proof-of-concept that a multiplexed silicon nanophotonics platform can rapidly detect both RBC antigens and anti-RBC antibodies in biological samples. This method has the potential for broad applicability in hematology and transfusion medicine for blood typing, quantitative monitoring of specific antibodies, and pathogen screening. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A preliminary study on the stabilization of blood typing antibodies sorbed into paper

This study investigated the stability of the primary blood typing antibodies (Anti-A, Anti-B and Anti-D IgM) on paper. This knowledge is critical to manufacture a new type of paper-based blood typing device where blood group antibodies must be kept active on paper for extended periods. Two strategies were explored. The first involved mixing additives such as polyvinylpyrrolidone (PVP), dextran and glycerol, with antibodies before sorption onto paper. While all the additives tested improved the antibody stability on paper, their protection for storage at room temperature was limited; dextran provided the longest protection, followed by PVP and then glycerol. The second strategy relied on freeze-drying to stabilize the antibodies in paper. Freeze dried antibodies sorbed into paper could be stored for long periods at ambient conditions without significantly loss of their activity. The thermal stability of antibodies in paper was also improved by freeze-drying. Our work shows that the use of additives and freeze-drying are effective approaches to retain the activities of IgM blood group antibodies on paper. These approaches will be further explored for the large scale development of a new generation of clinical and home-care blood testing devices.

The Characteristics of ABO Antibodies in Group O Thai Blood Donors

This study aimed to characterize anti-A and anti-B hemolysins, IgM, and IgG titers in Thai blood donors. Altogether, 300 serum samples from group O donors at the National Blood Centre, Thai Red Cross Society, were screened for anti-A and anti-B hemolysins and treated with 0.01 M dithiothreitol to characterize IgM and IgG titers by standard tube technique. Antibody titers were compared with hemolysis grade. Male and female ratio = 1:1.3 and ages ranged from 17 to 60 years. The overall prevalence of anti-A and anti-B hemolysins was 69%. Anti-A and anti-B hemolysins comprised 18.3% and 16.7%, respectively and 34% had both antibodies. High titers of anti-A hemolysins were associated with females (P< 0.05), and only anti-B IgM titers were associated with age (P< 0.05). Interestingly, the association of anti-A IgM titers, anti-A IgG titers, and hemolysin grade was demonstrated (P< 0.05). A significant association between hemolysin grade and anti-B IgM titers was found (P< 0.05). The prevalence of anti-A and anti-B hemolysins and high titers of IgM and IgG in Thais are high. Hemolysin grade showed significant associations with IgM titers; therefore, when providing ABO-incompatible platelet transfusion, especially for female plateletpheresis donors, IgM high titers of anti-A and anti-B screening is suggested.

Flow Cytometric Measurement of ABO Antibodies in ABO-Incompatible Living Donor Kidney Transplantation

Due to different detection methods, a comparison of anti-A/B antibody (Ab) levels among transplantation centers after living donor ABO-incompatible kidney transplantation is problematic. In the present study, anti-A/B Ab levels were determined prior to, and after, blood group A-to-O kidney transplantation using a recently established semiquantitative flow cytometry-based method, ABO fluorescence-activated cell sorting (ABO-FACS), and compared with standard agglutination titers and indirect antiglobulin testing. Pretransplant agglutination titers were reduced from 1:64 to 1:4, by a total of 14 Glycosorb A column immunoadsorptions (IADSs). Compared with the agglutination titers, antidonor immunoglobulin (Ig) M ABO-FACS mean fluorescence intensity ratios (MFIRs) decreased faster and remained low. No difference was observed using donor type or third-party A red blood cells (RBCs) for the ABO-FACS. Glycosorb A columns were not specific, also reducing anti-B and antiporcine IgM levels, which was confirmed by detecting anti-A/B and antiporcine Abs in the column eluates. In conclusion, analysis of pre- and posttransplant Abs from ABO-incompatible kidney transplant recipients by ABO-FACS allows a better understanding of Ab kinetics, which may improve the design of future IADS protocols.

ABO‐incompatible deceased donor liver transplantation with the use of antigen‐specific immunoadsorption and anti‐CD20 monoclonal antibody

In patients with fulminant liver failure requiring emergency liver transplantation, the only donor organ that becomes available may be ABO incompatible. The risk of graft failure because of antibody-mediated acute rejection is high, but can be reduced by various means. We reported a deceased donor ABO-incompatible liver allograft recipient who was treated with antigen-specific immunoadsorption in combination with anti-CD20 monoclonal antibody and conventional plasmapheresis and immunosuppression. The patient was a 33-yr-old male with blood group A who presented with subacute liver failure of unknown aetiology and received a blood group AB liver graft. Pretransplant he underwent plasmapheresis and received one dose of rituximab. The immunosuppressive regimen consisted of methylprednisolone, tacrolimus and mycophenolate mofetil. Despite regular post-operative plasmapheresis sessions, anti-B antibody titres increased. Antigen-specific immunoadsorption with depletion of anti-B antibodies was performed from day nine to day 17. Thereafter, anti-B IgM and IgG antibody titres remained low. After one month the patient was reoperated with hepaticojejunostomy because of bile duct necrosis and with reconstruction of a stenotic hepatic artery. A mild rejection was successfully treated with methylprednisolone four months post-transplant. At six months post-transplant there was a stricture of the biliary-enteric anastomosis, but the graft was well functioning. We conclude that antigen-specific immunoadsorption can be an important adjuvant therapy to control recipient anti-A/B antibody levels and prevent acute rejection in ABO-incompatible deceased donor liver transplantation.

ＡＢＯ型ｍｉｎｏｒ ｍｉｓｍａｔｃｈ生体肝移植後に抗Ｂ抗体が長期間遷延した１症例

We report a case of hemolytic anemia due to anti-B antibody after minor ABO-mismatched partial liver transplantation from a living donor. The patient was a 5-year-old girl with congenital biliary atresia, and group AB, Rh-positive blood. A partial liver transplant from her group A, D (Rh0) mother was performed. The recipient HLA was A2, 26; B54, 62; DR4, 15, and that of the donor was A2, -; B54, -; DR4, 14. This type of transplantation is the so-called one-way donor-recipient HLA matching. Her hemoglobin fell rapidly from 11.0g/dl to 6.0g/dl between days 10 and 15 after liver transplantation without evidence of bleeding. On day 14, anti-B IgM antibody was found in her serum. On day 16, two units of group O washed red blood cells were transfused, after which no further transfusions were required. This anti-B IgM antibody was thought to be produced from passenger B lymphocytes in the donor's liver. Although this type of antibody usually disappears within 4 weeks, it was continuously detected for about 4 months in this case. The long-term presence of anti-B antibody may be caused by one-way donor-recipient HLA matching.

Characterization of Blood‐Group‐ABO(H)‐Active Glycosphingolipids in Type‐AB Human Erythrocytes

Neutral glycolipids in Folch's upper phase were isolated from human erythrocyte membranes of 22 individuals with blood type AB. On immunostaining by TLC with anti-A IgG, all reactive glycolipids in type A corresponded to reactive glycolipids in type-AB erythrocytes. With anti-B IgM, all reactive glycolipids in type-B erythrocytes also corresponded to reactive glycolipids in type-AB erythrocytes. By comparison of the reactivity to that of the anti-A and anti-B antibodies, it was found that, in type-AB erythrocytes, all glycolipids reactive with either one of the anti-A or anti-B antibodies were detected in both type-A and type-B erythrocytes, and that A-active glycolipids had higher Rf values than B-active glycolipids on TLC plates. A series of glycolipids reactive with both antibodies were purified from the Folch's upper neutral glycolipid fraction of erythrocyte membranes by column chromatography, and was characterized by TLC-immunostaining and negative secondary-ion mass spectrometry. The results strongly suggested that A-active and B-active carbohydrate chain epitopes existed separately as glycolipid molecules in blood-type-AB erythrocytes. It was also confirmed that these phenotypes observed in erythrocyte membranes were exhibited by blood-group-active glycosphingolipids in the small intestine of blood-type-AB individuals. Furthermore, upon treatment of fractions obtained from silicic acid column chromatography with alpha-N-acetylhexosaminidase or alpha-galactosidase, a branched hybrid-type molecule with both A and B determinants was not detected.

Red-cell bound anti-A is more efficient than anti-B in competition for fluid phase complement

Lysis of group A and B erythrocytes by human complement was studied by an anti-A (BRIC.131) and an anti-B (BRIC.30) IgM monoclonal antibody in a 51Cr-release assay. The relative concentration of membrane-bound immunoglobulins was detected by flow cytometric analysis, and the amount of C1q and C3 bound to the sensitized red cells was measured by using purified, 125I-labelled molecules. The direct haemolysis was identical with both reagents in the presence of excess and suboptimal complement over a wide range of antibody concentration (between 50 and 7000 ng/ml). The indirect effect of membrane-bound antibody, i.e. its influence on complement binding by sensitized bystander cells, was examined in a cold target competition assay in which sensitized, nonlabelled cells are present when complement is incubated with sensitized labelled cells. We have found that the competitive capacity of sensitized erythrocytes correlated with the amount of membrane-bound immunoglobulins. In accordance with our earlier findings, an equal level of target and competitor cell lysis was obtained only if the fluid phase anti-B antibody concentration was 2 to 4 times higher than that of the anti-A antibodies. We demonstrate in this paper that the different competitive activity of IgM anti-A and anti-B monoclonal antibodies might be accounted for by differences in their C1q and C3 binding capacities.

Interactions in the complement-mediated lysis of blood group AB erythrocytes sensitized simultaneously with anti-A and anti-B monoclonal antibodies

The lysis of group AB erythrocytes by human complement was studied by different anti-A and anti-B IgM monoclonal antibodies (mabs) in a 51Cr-release assay. The concentration of membrane-bound immunoglobulin was detected by ELISA, and the amount of C1q and C3 bound to sensitized red cells was measured by using purified, 125I-labelled molecules. We have demonstrated that there is an exponential relationship between the concentration of the sensitizing IgM mabs and C1q binding to the sensitized AB cell. The efficiency of binding was related to the number of antibodies bound; thus, anti-A sensitized cells bound 3–6 times more C1q than anti-B sensitized cells did. AB cells, on the other hand, bound similar amounts of C3 whether anti-A or anti-B was present. The lytic efficiencies of the various IgM mabs during short incubation times were different, suggesting that the complement activation rates vary widely with different antibodies on the AB cell membrane. The binding of C1q to an antibody-sensitized target activates a cascade, whose components may migrate away from the sensitizing antibody; interactions between the activation processes generated by the anti-A and anti-B antibodies may thus occur. Choosing appropriate pairs of anti-A and anti-B mabs for the simultaneous sensitization of AB cells has indeed resulted in stimulation in some and inhibition in other combinations of mabs. It is suggested that stimulation is observed when the activated intermediates are produced in excess, whereas inhibition occurs when a shortage of activated intermediates prevents mutual utilization.