Blood Group-specific Antibodies Research Articles

The Blood Group Rhc Protein from Human Erythrocyte Membranes as an Immunogen for Producing Antibodies in Mice

Background: Various methods have been used to isolate red blood cell (RBC) membrane antigens. In this regard, obtaining the antigen and preserving its structure is of special importance. However, limited studies have been conducted to purify cellular membrane antigens such as Rh proteins.
 Materials and Methods: In this experimental study, Rhc antigens of the RBC membrane was purified. Here, the RBC membrane was solubilized through the lysis buffer. Next, dialysis and affinity chromatography were performed using polyclonal anti-human RhCcEe antibody to isolate Rhc/e antigens from the RBCs with the following blood group characteristics: Rhc+, RhC-, Rhe+, and RhE-. The purified proteins were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot methods. The immunization process was performed in Balb/c mice using the Rhc antigen as an immunogen. After the last injection, the mouse serum was used to titrate antibodies.
 Results: Protein bands of the purified antigen were observed in the silver-stained SDS-PAGE gel (region of 25-35 kDa). The OD405nm = 0.56 ± 0.05 results showed the reactivity with Rhc antibody. The specificity of the purified protein was evaluated using the dot blot assay. The anti-sera titration was greater than 1/10,000 against Rhc-coated microwells. Rh antigens can be isolated from the RBC membrane using the non-ionic NP-40 detergent and affinity chromatography.
 Conclusion: The Rh antigen can be isolated from the RBC membrane with proper purity by solubilization with the non-ionic NP-40 detergent and purification by affinity chromatography. It seems that the membrane antigen maintains its antigenicity and structure. As a result, it can be detected by blood group-specific antibodies used in the hemagglutination method. Purified antigens may be used to generate antibodies or to study the protein structure.

The Differences in the Binding of IgM and IgG Antibodies with Erythrocytes and Epithelial Cells

Background:: The studies of agglutinating and adsorbing abilities of IgM and IgG antibodies towards the red blood cells (RBCs) and tissue cells are scarce. background: The studies of agglutinating and adsorbing abilities of IgM and IgG antibodies towards the red blood cells (RBCs) and tissue cells are scarce. Objectives:: The study aimed to estimate the differences in the avidity of blood group-specific IgM and IgG antibodies to RBCs and epithelial cells. Methods:: The reaction of hemagglutination, adsorption, mixed agglutination reaction and saliva inhibition test were used. Anti-B 2-54 monoclonal antibody, polyclonal citrated plasma and the heated plasma were used for investigation of IgM and IgG antibodies. Results:: IgM antibodies showed high adsorbing ability to RBCs and epithelial cells in an alkaline medium. On the contrary, the heated plasma containing IgG antibodies showed high adsorbing ability to RBCs and epithelial cells in the acid medium as compared to the alkaline medium. Complete adsorption of IgG antibodies was observed by epithelial cells as compared to erythrocytes. A mixed agglutination reaction confirmed the strong binding of anti-B IgG antibodies with group B epithelial cells in an acid medium. Conclusion:: The binding of polyclonal IgM and IgG group-specific antibodies with red blood cells and epithelial cells depends on the opposite values of pH of the medium. IgG antibodies completely adsorb on epithelial cells contrary to IgM antibodies. Highlights:: Blood group-specific IgG antibodies showed high avidity to epithelial cells as compared to red blood cells. IgG antibodies demonstrated high agglutinating ability in alkaline medium and strong adsorbing ability in acid medium contrary to IgM antibodies, demonstrating high adsorption properties in alkaline medium.

The role of acidification of the medium on the erythrocyte agglutinating and adsorbing properties of blood group-specific monoclonal antibodies.

Studies on the specificity of ABH antigen-antibody interactions at different pH values are rare. Therefore, the aim of this study was to estimate the effect of acidification of the reacting medium on the agglutinating ability of anti-A monoclonal antibodies (mABs) and their inhibition by glycoconjugates of red blood cell membranes. Anti-A mABs were obtained from the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens (on July 19-20, 2002, in Paris, France). The glycoconjugates were isolated from erythrocytes' membranes. The inhibition of the lipid and protein isotypes of the blood group A antigen was assessed. The acidic medium decreased the agglutinating ability of acid immunoglobulin M (IgM) anti-A mABs, in contrast to alkaline immunoglobulin G (IgG) mABs. Meanwhile, at pH 6.5, IgM antibodies exhibited a high adsorption capacity, while IgG antibodies demonstrated a strong adsorption capacity at an alkaline pH. mABs 2-19, 2-28, 2-22, and 2-8 were inhibited by the acidic lipid and protein glycotopes of erythrocyte membranes. However, mAB 2-8 was inhibited by both acidic and alkaline glycotope variants. The agglutinating and adsorbing abilities of mABs, which are revealed at opposing pH values, should be taken into account during studies of antigen-antibody interactions.

Biochemical Characterization of a New 10% IVIG Preparation [IgG Next Generation (BT595)/Yimmugo®] Obtained from a Manufacturing Process Preserving IgA/IgM Potential of Human Plasma.

Human plasma is used for the generation of several life-saving drugs and contains valuable antibodies from the immunoglobulin classes IgG, IgM and IgA. Purified intravenous IgG solutions (IVIGs) form the majority of plasma-derived medicine to treat patients with various forms of immunodeficiencies. In conventional IVIG manufacturing processes, immunoglobulin classes IgM and IgA are often discarded as contaminants, but these antibody classes have been proven to be effective for the treatment of acute bacterial infections. Considering the increase in demand for human plasma-derived products and the ethical value of the raw material, a more resource-saving usage of human plasma is needed. Intensive research over the last decades showed that adverse reactions to IVIGs depend on the presence of thrombogenic factors, partially unfolded proteins, non-specific activation of the complement system, and blood group specific antibodies. Therefore, new IVIG preparations with reduced risks of adverse reactions are desirable. A new manufacturing process that yields two biologics was established and quality attributes of the new IVIG solution (Yimmugo®) obtained from this process are presented. Here, we provide a biochemical characterization of Yimmugo®, a new 10%IVIG preparation. It is derived from human blood plasma by a combined manufacturing process, where IgM and IgA are retained for the production of a new biologic (trimodulin, currently under investigation in phase III clinical trials). Several improvements have been implemented in the manufacturing of Yimmugo® to reduce the risk of adverse reactions. Gentle and efficient mixing by vibration (called "vibromixing") during a process step where proteins are at risk to aggregate was implemented to potentially minimize protein damage. In addition, a dedicated process step for the removal of the complement system activator properdin was implemented, which resulted in very low anticomplementary activity levels. The absence of measurable thrombogenic activity in combination with a very high degree of functional monomeric antibodies predict excellent efficacy and tolerability. Yimmugo® constitutes a new high quality IVIG preparation derived from a novel manufacturing process that takes advantage of the full therapeutic immunoglobulin potential of human plasma.

Type O blood group associates with higher anti-JC polyomavirus antibody levels.

BackgroundPatients with multiple sclerosis (MS) and high anti‐JC polyomavirus (JCPyV) antibodies in blood have an increased risk for the development of progressive multifocal leukoencephalopathy (PML) when treated for MS. To test the hypothesis that type O blood group associates with anti‐JCPyV antibody levels and the risk of developing PML, we characterized ABO blood group antigen on blood samples of 62 patients with PML, and 64 MS controls without PML.MethodsMonocentric retrospective cohort study. Anti‐JCPyV antibody levels in arbitrary units (AU) were determined in sera using an ELISA‐based method, and blood group specific antibodies using standardised test erythrocytes.ResultsAnti‐JCPyV antibody levels were higher in individuals with blood group O compared with all other blood groups (O: median AU: 129; not O: median AU: 53; p = .005). This association was not observed for the closely related BK virus. Of the 62 patients with PML, 29 (47%, 95% confidence interval (CI) 35%–59%) were of blood group O, which showed a nonsignificant trend to differ from the expected distribution in the German population (41%), and the MS controls studied (36%, 95% CI 25%–48%).ConclusionThe ABO blood group O antigen associates with higher anti‐JCPyV antibody levels and may impact the risk of the later development of PML. The overrepresentation of blood group O in cases with PML was in line with a previous publication. Larger studies are warranted to assess a potential value of host genetic markers, such as the ABO status, for PML risk prediction during immunotherapy.

Treatment-associated hemolysis in Kawasaki disease: association with blood-group antibody titers in IVIG products

AbstractHemolytic anemia resulting from IV Immunoglobulin (IVIG) treatment can be a serious complication, especially for those with underlying conditions with a high level of inflammation and after administration of high IVIG dosages, such as Kawasaki disease (KD), a multisystem vasculitis affecting young children. This hemolysis is caused by antibodies against blood groups A and B, but the precise mechanism for hemolysis is not known. We performed a single center, partly retrospective, partly prospective study of a cohort of 581 patients who received IVIG for treatment of KD from 2006 to 2013. Factors associated with hemolysis were identified through univariable and multivariable logistic regression. Six IVIG preparations were assayed for their hemolytic effect with serological and cellular assays to clarify the mechanism of red cell destruction. During the study period, a sudden increase in the incidence of hemolysis was observed, which coincided with the introduction of new IVIG preparations in North America that contained relatively high titers of anti-A and anti-B. These blood-group–specific antibodies were of the immunoglobulin G2 (IgG2) subclass and resulted in phagocytosis by monocyte-derived macrophages in an FcγRIIa-dependent manner. Phagocytosis was increased in the presence of proinflammatory mediators that mimicked the inflammatory state of KD. An increased frequency of severe hemolysis following IVIG administration was caused by ABO blood-group–specific IgG2 antibodies leading to FcγRIIa-dependent clearance of erythrocytes. This increase in adverse events necessitates a reconsideration of the criteria for maximum titer (1:64) of anti-A and anti-B in IVIG preparations.

Multiplex blood group typing by cellular surface plasmon resonance imaging.

Blood-group typing of donors and patients is essential to avoid incompatible transfusions. Transfusion of incompatible RBCs may result in alloimmunization complicating future transfusions or in the presence of antibodies in adverse reactions. With more than 300 blood group antigens identified, it is difficult to provide fully compatible blood. Currently, standard practice is to match for the most immunogenic antigens. While the current agglutination-based RBC-typing methods are reliable for testing a selected number of antigens, they are not easily adaptable for high-throughput multiplex blood typing beyond the current standard. Surface plasmon resonance (SPR) is a label-free method to follow molecular-and, very recently, also cellular-interactions in real time. Demonstration of binding of RBCs to blood group antigen-specific antibodies by SPR has already been achieved. Here, we demonstrate the generation of an SPR array equipped with clinically relevant blood group antibodies (A, B, and Rh blood groups). To validate this method, we blindly compared typing of 946 blood donors with results of current diagnostic agglutination-based methods. RBC typing was achieved by monitoring RBC binding to blood group-specific antibodies on the sensor simultaneously within 5 minutes per sample. Regeneration of the chip was robust, allowing for typing of at least 100 samples. The typing results gave a 100% match with classical serology with all antibodies tested besides anti-E/e monoclonals, which gave inconsistent results due to low antibody specificity. This study demonstrates that SPR-based RBC typing for multiple antigens can be realized simultaneously with high-quality antibodies, enabling reduced hands-on time and possibly improving cost efficiency.

Polymethyl methacrylate (PMMA) point of care for ABO-Rh(D) blood typing

Polymethyl methacrylate (PMMA) based point of care (POC) for ABO-Rh(D) blood grouping by the naked eye was investigated. PMMA substrate was covalently linked with carboxyl methyl dextran (CMD) and coupled to the blood group specific antibodies anti A, anti B, and anti D, which were patterned into “A”, “B”, and “+”. The red read out color originated from the color of hemoglobin in the RBCs. The major human blood group, ABO-Rh(D), was successfully sorted using these patterns. PMMA strip-based assays for blood grouping by the naked eye resulted in correct blood group identification of all 74 samples in comparison to slide agglutination test. The determination of captured RBC Rh(D) required a longer reaction time with immobilized anti D antibody on the PMMA substrate than other blood groups to produce sufficient color for unambiguous visual identification. The proper RBC concentration was found to be higher than 1% v/v for naked eye detection. The dilution of blood sample is playing a crucial role in reducing interference from blood proteins during detection. A used of undiluted blood sample for the testing should be avoided. The proper blood dilution was found to be 10% or 1:10 dilution. The proposed technique presents a quick, cheap, simple, and almost instantaneous assay for blood grouping.

Immunoserology and RHD Genotype Analysis of DVI Type 3 Genotype Pregnant Women with Anti-D

To performe the immuneserological and RHD Genotype analyses for DVI type 3 genotype pregnemt women with anti-D. RhD blood type of this pregnant women was identified by common serological methods, then the blood group specific antibodies was screened and identified; the polymerase chain reaction-sequence specific primer(PCR-SSP) was used to identify the pregnant women's RHD genotype; RhD blood group for the pregnant women, her spouse and daughter was genogrouped and genetically analyzed by multiplex ligation-dependent probe amplification(MLPA). The heredity of this family was analyzed finally. The titer of IgG anti-D in the pregnant woman serum was 1:8; the PCR-SSP showed that the 3rd to 6th exons of RHD gene were missing in the pregnant woman. the genotype of pregnant woman was identified as DVI type 3; the MLPA analysis showed that this pregnant women owned only one RHD allele with 3rd to 6th exons missed, and her genotype was identified as CDVIe/cde; her spouse was identified as CDe/CDe homozygous genotype, and her daughter as CDe/CDVIe. Accurate identification of RhD blood type is of great significance for a safe and effective clinical blood transfusion strategy, and for taking appropriate measures to prevent hemolytic disease of newborn (HDN) at women childbearing age.

Effect of intravenous immunoglobulin administration on erythrocyte and leucocyte parameters.

Intravenous immunoglobulins (IVIG) are an attractive therapeutic tool for therapy of toxic epidermal necrolysis and severe forms of certain autoimmune diseases, including dermatomyositis, autoimmune blistering diseases, systemic vasculitis and lupus erythematodes. Prompted by a case of IVIG-associated haemolytic anaemia, the effects of IVIG administrations on haematological parameters in patients with dermatological conditions were investigated. Erythrocyte and leucocyte parameters were retrospectively analysed in 16 patients who had received IVIG at doses from 1 to 3 g/kg bodyweight (n = 35 cycles). The influence of IVIG on leucocyte survival was determined in vitro. Decreased absolute erythrocyte numbers, haemoglobin and haematocrit levels and a case of haemolytic anaemia were linked to transfusion of high-, but not low-dose IVIG. In contrast, leucopenia post-IVIG occurred in the vast majority of the recipients, unrelated to the administered IVIG amounts. In vitro investigations revealed a dose-dependent impairment of cell survival by IVIG in the neutrophil and monocyte, but not in the lymphocyte subpopulations. In several IVIG preparations, substantial amounts of blood group anti-A/anti-B antibodies were detected which could have accounted for the observed changes in the haematological parameters in our study cohort. IVIG products should be administered strictly according to indications. Commercially available IVIG products can contain blood group-specific antibodies that may induce haemolysis in some recipients. Monitoring of blood counts during applied IVIG therapy, especially when high doses are administered, is recommended.

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

Hemolysis After High Dose IVIG In Blood Group A Women With Alloimmune Thrombocytopenia

IntroductionHemolysis after IVIG infusion has been observed in clinical trials and case reports. Certain factors seem to place a patient receiving IVIG at higher risk for hemolytic anemia: 1) having blood type A, B, or AB and 2) receiving high doses of IVIG (≥ 2g/kg).IVIG contains isohemagglutinins that coat red blood cells and may cause red cell clearance by the RES (Table 1). Anti-A, Anti-B, and Anti-D titers have been detected in IVIG products at varying concentrations by different IVIG manufacturers. It has been suggested that Anti-A titers > 1:16 are more likely to cause clinically significant hemolysis and thus anemia in recipients.Table 1Anti-A Titer Levels by IVIG PreparationIVIG BrandAnti-A TiterGammagard liquid1:64Privigen 10%1:64Gamunex 10%1:32Octagam 10%1:8 - 1:32Gamimune-N1:8Octagam 5%1:2 - 1:8Carimune 9%1:4Table 2IVIG-Associated Hemolysis by Blood Type (BT)Blood TypePercent Experiencing HemolysisBlood Type Prevalence in USA61.9%40%B17.5%11%AB12.7%4%O4.8%45%15 summarized studies reported 63 patients who experienced hemolysis after IVIG infusion and showed the higher risk of non-group O recipients to hemolyze with IVIG:In the current study, we sought to explore the degree of anemia as related to ABO blood types in mothers treated with IVIG for fetal alloimmune thrombocytopenia (AIT). MethodsA retrospective chart review was conducted on 84 women who had received IVIG for AIT treatment during their pregnancy to increase the fetal platelet count. Women were randomized to receive IVIG 2g/kg/wk (arm A, n=43) or IVIG 1g/kg/wk + prednisone (arm B, n=41) starting at 20-30 weeks of gestation until delivery. One CBC per month was collected from each mother from the start of treatment until delivery. Hemoglobins and mean corpuscular volumes (MCVs) were tracked and development of anemia was compared among women with blood types (BT) A, B, AB and O in each treatment arm. Patients who non-randomly received IVIG 2g/kg/wk + prednisone together as rescuetreatment were excluded from this study. Results36 of the 84 women had hemoglobins less than 10 on at least one CBC during IVIG treatment. All 36 had MCV values in the normocytic range (80-100). The degree of anemia was significantly greater in Arm A (IVIG 2g/kg) than Arm B (IVIG 1g/kg + pred) (Fisher's exact test, p=0.016; table 3).Table 3Degree of Hemolysis among treatment armsArm A (IVIG 2g/kg/wk)Arm B (IVIG 1g/kg/wk + Prednisone 0.5 mg/kg/d)Hg<102412Hg≥101929Among women in arm A (IVIG 2g/kg), BT non-O had a significantly higher incidence of marked anemia (Hg<10) than those with BT O (Fisher's exact test, p=.001; table 4), with BT A having the highest number of women with Hg<10 (A=16, B=1, AB=1, O=4, unknown=2).There was no significant difference among women by blood type O (n=4) versus non-O (A=4,B=2,AB=0,unknown=2) in the incidence of anemia in arm B (IVIG 1g/kg + pred); both BT groups showed a low incidence of hemolysis (table 5).Table 4IVIG 2g/kg/wk (Arm A)Blood type OBlood type Non-OHg<10420Hg≥10136Table 5IVIG 1g/kg/wk + Pred (Arm B)Blood type OBlood type Non-OHg<1048Hg≥101415 ConclusionsPatients who were on 2g/kg/wk IVIG became substantially more anemic than those on the 1g/kg/wk IVIG + prednisone dose, which supports reported findings that higher dose (HD) IVIG is associated with a greater risk of anemia. Note: receiving IVIG 2g/kg/wk for multiple weeks is very high dose IVIG. In particular, those patients receiving HD IVIG who were BT A had a greater risk of developing a greater extent of anemia than those of blood type O. This finding is consistent with the hypothesis that blood-group specific antibodies in IVIG result in immune hemolysis primarily in BT A recipients because of the higher anti-A titers. Furthermore, while 1 g/kg/wk of IVIG is not “low dose”, the concomitant use of prednisone 0.5mg/kg/d may protect against development of a greater degree of anemia in these patients.IVIG is the standard of care treatment for AIT; however 1 g/kg/wk is known not sufficiently effective in severely thrombocytopenic fetuses (Berkowitz, OB-GYN 2006). Therefore we believe appropriate treatment of women whose previous babies did not have an ICH needs to be > 1g/kg/wk. Our study found that hemolytic anemia in non-O blood types may be an important side effect of 2g/kg/wk IVIG treatment, indicating that patient blood type should be considered when deciding which IVIG regimen to use. Disclosures:Bussel:Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; IgG of America: Research Funding; Genzyme: Research Funding; Cangene: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity's Board of Directors or advisory committees.

Chances and Risks in Living Donor Liver Transplantation: Blood-group Compatibility not Indispensable

With regard to living-donor liver transplantation, it should be added that blood-group compatibility of the donor and the recipient, though always desirable, is not an absolute sine qua non. There have been many reports, mainly from Asia, of living-donor liver transplantation where the donor and the recipient had different blood types. In the Gottingen Transplantation Center, three AB0-incompatible living-donor liver transplantations have been performed to date (report submitted for publication). The data from Asia show a higher rate of biliary and vascular complications after such procedures (3), resulting in a lower rate of transplant survival. Under favorable conditions, however, a three-year patient survival of 70% can be achieved in adults, and 85% in children (1). Intensive interdisciplinary collaboration and preparation of the recipient are essential for an optimal result of transplantation. Generally speaking, a special immunosuppression regimen is necessary, as well as a reduction of the serum titers of blood-group specific antibodies in the recipient. This can be achieved by plasmapheresis or by immunoadsorption (2, 3). Because of the lower rates of transplant and patient survival associated with AB0-incompatible living-donor liver transplantation, it remains a treatment option to be pursued only in individual cases in the face of the persisting donor shortage. The recent, promising advances in immune suppression and the ever-improving safety of liver donation surgery nonetheless justify the use of AB0-incompatible living-donor liver transplantation as a treatment option of last resort.

Detection of antibodies reacting with the antithetical duffy blood group antigens Fy a and Fy b using recombinant fusion proteins containing the duffy extracellular domain

Detecting blood group-specific antibodies in patient sera is essential to the management of blood transfusions or pregnancies. We produced the antithetical forms of the 65 amino acid extracellular domain (ECD) of the Duffy (Fy) blood group protein fused to glutathione sulfotransferase (GST): GST-Fy a; and GST-Fy b, differing only in Gly or Asp at position 44, respectively. The purified recombinant proteins were recognized more effectively by reference polyclonal or monoclonal antibodies than the antithetical Fy specificity by either ELISA or immunoblotting. Combined immunoblot and ELISA tests performed at 1:200 dilutions of sera using the recombinant proteins gave results in agreement with undiluted sera and agglutination for 17/19 alloimmunized patients. At 1:200, agglutination detected anti-Fy a or anti-Fy b in only three of 12 samples that were positive by ELISA. Recombinant ECD-Fy proteins are suitable and sensitive reagents for the detection of anti-Fy that use technology amenable to automation and/or miniaturization and avoid the need for intact red cells.

Potential implications of ABO blood group for vascular rejection in pig to human kidney xenotransplantation.

A substantial hurdle for successful xenotransplantation is to negate the effect of xenoreactive natural antibodies [mainly Galalpha1-3Galbeta1-4GlcNAc (alpha-Gal) specific] that cause hyperacute xenograft rejection. Galalpha1-3Gal molecules (alpha-Gal) have close structural homology with human ABO blood groups and therefore an individual's blood group might influence the formation of alpha-Gal specific antibodies. Genetic heterogeneity controlling alpha-Gal specific antibody formation could have important implications for future pig to human xenotransplantation clinical trials. We have investigated the relationship between ABO blood group and immunoglobulin M (IgM) and immunoglobulin G (IgG) alpha-Gal specific antibody titres in sera obtained from renal dialysis patients and healthy blood donors. Serially diluted sera (n = 166) obtained from renal dialysis patients awaiting kidney transplantation (n = 116) and healthy blood donors (n = 50) were tested for IgM and IgG alpha-Gal antibodies using an enzyme-linked immunosorbent assay (ELISA) specific for alpha-Gal. The study cohort comprised 62, 48, 36 and 20 sera obtained from blood group O, A, B and AB individuals, respectively. Reciprocal alpha-Gal specific antibody titres were calculated from ELISA titration curves and stratified by individual blood group. No significant heterogeneity was found in IgM alpha-Gal specific antibody titres across ABO blood groups. In contrast, marked heterogeneity was observed in IgG alpha-Gal specific antibody titres when stratified by blood group. IgG alpha-Gal specific antibody titres were higher in sera obtained from blood group O renal dialysis patients [median titre 40, interquartile range (IQR) 14 to 72], compared with blood group A (median titre 18, IQR 7 to 54, P = 0.05), blood group B (median titre 6, IQR 0 to 15, P < 0.001) and blood group AB patients (median titre 3.5, IQR 0 to 16, P = 0.002). A similar correlation was found for IgG alpha-Gal specific antibody titres in sera obtained from healthy blood donors with median titres of 20 (IQR 12 to 34), 37 (10 to 91), 9 (0 to 20), and 5.5 (0 to 12) in blood groups O, A, B and AB individuals, respectively. There was a strong interrelationship between alpha-Gal specific antibody class and blood group, with both IgM and IgG alpha-Gal specific antibodies found in 84% of the blood group O sera, 73% of blood group A sera, 50% of blood group B sera and 40% of blood group AB sera (P < 0.001). In a subgroup of 39 renal dialysis patients, IgM and IgG alpha-Gal specific antibody titres were measured in two serum samples obtained at different time-points (median time interval 581 days, range 42 to 4414), and showed a high degree of stability (correlation coefficient 0.88 and 0.90 for IgM and IgG, respectively). IgG alpha-Gal specific antibody titres are significantly higher in the sera of blood group O and A renal dialysis patients and healthy individuals compared with blood groups B and AB. These data indicate that future clinical trials of pig to human xenotransplantation may be more problematic for non-blood group B patients who are likely to have high levels of IgG alpha-Gal specific antibodies that are associated with acute vascular rejection.

Recombinant technology in transfusion medicine.

Recombinant technology in transfusion medicine has really only just begun to have large-scale impact. The preparation of blood products, determination of blood group phenotype, detection of blood group specific antibodies does not currently employ DNA-based methods for their preparation or detection. The detection of bloodborne viruses, production of blood grouping reagents and diagnosis of HLA polymorphism all include recombinant DNA-based technologies and are beginning to impact on routine laboratory life in Transfusion medicine. This review analyses the current use of recombinant technology in transfusion medicine, and indicates where there is likely to be significant development of this methodology (particularly in molecular diagnostics) oven the next decade or so. The impact of molecular medicine in the field of transfusion has already begun. Recent licensing of thrombopoietin for clinical use may have a profound effect on the very high current demand for platelet transfusions. Gene therapy protocols for the treatment of haemophilias and other coagulation disorders, and the production of recombinant blood products may reshape the demand for clotting factors from human plasma. I also consider the potential impact of the exciting technologies of DNA arraying and nucleic acid therapeutics in the fields of molecular diagnostics and the possible treatment of leukemia respectively.

Development and Application of Surface Plasmon Resonance-Based Biosensors for the Detection of Cell–Ligand Interactions

Surface plasmon resonance (SPR)-based biosensors were investigated with a view to providing a portable, inexpensive alternative to existing technologies for “real-time” biomolecular interaction analysis of whole cell–ligand interactions. A fiber optic SPR-based (FOSPR) biosensor, employing wavelength-dependent SPR, was constructed to enable continuous real-time data acquisition. In addition, a commercially available integrated angle-dependent SPR-based refractometer (ISPR) was modified to facilitate biosensing applications. Solid-phase detection of whole red blood cells (RBCs) using affinity-captured blood group specific antibodies was demonstrated using the BIACORE 1000, BIACORE Probe, FOSPR, and ISPR sensors. Nonspecific binding of RBCs to the hydrogel-based biointerface was negligible. However, the background noise level of the FOSPR-based biosensor was ∼25-fold higher than that of the widely used BIACORE 1000 system while that of the ISPR-based biosensor was over 100-fold higher. Nevertheless, the FOSPR biosensor was suitable for the analysis of macromolecular analytes contained in crude matrices.

BLOOD GROUP TYPING BY ULTRASOUND BACKSCATTERING—QUANTITATIVE MEASUREMENTS OF AGGLUTINATES AND THEIR SHEAR-DEPENDENT BEHAVIOR

A new method is presented to investigate the agglutination of red blood cells (RBC). In the present work, ultrasound backscattering was used to study erythrocyte suspensions prior to, and after, the reaction with blood group-specific antibodies. Scatter values allow for determination of the agglutination strength as well as for assessing the effect of external factors, such as shear stress, on the agglutination equilibrium. Based on the statistically significant increase of scattering values obtained from the test samples (when compared to the control samples), the blood group characteristics M, N, A1, AB, D and C could be determined reliably. It was demonstrated that a method is capable of providing quantitative data about the agglutination. It was possible to detect very small particles at an early stage of agglutination. Furthermore, the effect of shear stress on the agglutination equilibrium of RBC was estimated. Higher scatter values observed within the 50 s−1 to 170 s−1 range of shear level is interpreted as a strengthening of the agglutination resulting from the increase of shear stress. A decrease in agglutination was observed in all samples when shear levels exceeded 170 s−1. This effect was reached after anti-M and anti-N reaction, as well as after anti-A or anti-B reaction. Under conditions of increasing shear, control samples without added antibody or glutardialdehyde (GDA)-hardened RBC (with antibody treatment) exhibit no significant change in scatter values.

Covalent binding of poly(ethylene glycol) (PEG) to the surface of red blood cells inhibits aggregation and reduces low shear blood viscosity.

A simple method to coat human red blood cells (RBC) with PEG is described. Using a reactive derivative, monomethoxy-PEG (mPEG) was covalently attached to the surface of RBC in aqueous media under mild conditions. The PEG coating dramatically reduced aggregation and low shear viscosity of RBC resuspended in autologous plasma, and inhibited RBC agglutination by blood group-specific antibodies. Morphology and deformability of the PEG-treated cells were unaltered. The PEG coating of the RBC surface may be of significant benefit in the treatment of a variety of diseases characterized by vasoocclusion or impaired blood flow, e.g., myocardial infarction, sickle cell disease.

Detection of blood group antigens utilising immobilised antibodies and surface plasmon resonance

Surface plasmon resonance (SPR) detection using the BIAcore™ biosensing system was employed for the detection of blood group-associated antigens (BGAA) on whole erythrocytes. The quantitative detection of erythrocytes was accomplished by covalently immobilising blood group-specific antibodies (IgM) to a dextran matrix and monitoring the cell binding response. Non-specific binding of erythrocytes to the IgM coated surface was not detected. Relatively mild regeneration conditions (20 mM NaOH) were employed to elute bound erythrocytes, in order to preserve the activity of the immobilised antibody and allow the surface to be used repeatedly. Regeneration of the surface was particularly difficult when a high IgM immobilisation level was used and when the number of bound cells was high. Despite these considerations, a quantitative relationship between the cell binding response and erythrocyte concentration was confirmed. Erythrocyte preparations, diluted by a factor of ten as compared to physiological concentrations, were detectable. The occurrence of non-specific false positives appears to be minimal and allows the system to be used for blood typing. As a model study, the lectin concanavalin A (ConA) was covalently immobilised onto a hydrophilic dextran matrix and successfully used to support the capture of erythrocytes from suspension.