Use of LOR-15C9 monoclonal anti-D to differentiate erythrocytes with the partial DvI antigen from those with either partial D antigens or weak D antigens

Historically, red blood cells (RBCs) with partial D antigens have been defined serologically by their pattern of reactivity with polyclonal and monoclonal anti-D. Although numerous variants have been described in tests with well-characterized monoclonal anti-D, definition remains difficult to ascertain serologically. RBCs of known partial D type were tested with LOR-15C9 (a monoclonal anti-D) and commercial anti-D by the tube indirect antiglobulin test (IAT), by micro typing system IgG gel cards, and by immunoblotting. By IAT, LOR-15C9 reacted strongly with DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs in addition to RBCs with common D antigens; weakly with DII, DNU, and DIIIb RBCs; and not at all with DIVa, DIVb, DBT, or R0 Har RBCs. Reactivity was variable (1+ to 4+), with RBCs classified as weak D (Du). As expected, the commercial anti-D agglutinated all D variants and weak D RBC samples by the IAT and by using IgG gel cards; however, the reactivity with DVI RBCs was weaker than with LOR- 15C9. By immunoblotting, LOR-15C9 detected a band with an apparent molecular mass of approximate Mr 30,000-34,000 in membranes prepared from D-positive, DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs and an additional band of Mr 20,000-22,000 in membranes prepared from DVI RBCs. No band(s) was detected in membranes from DII, DNU, DIIIb, DIVa, DIVb, DBT, R0 Har, weak D, or D-negative samples. LOR-15C9 provides a useful tool to identify positively DVI samples and thereby differentiate this partial D from other D variants and from weak D samples.     

Monoclonal reagents for rhesus-D typing of Irish patients and donors: a review

Polyclonal anti-D reagents have been largely replaced by monoclonal reagents; however, although generally very potent, they can exhibit some variation in activity, particularly in the detection of weak Rhesus (Rh) D-positive cells. Hence, although patients and donors previously typed as Rh-D-negative (Du positive) are now being typed as Rh-D-positive, it is likely that two monoclonal reagents selected for Rh-D typing may give anomalous results. This has led to confusion in testing patients and donors in Irish laboratories. To address this problem, 29 commercially available monoclonal and monoclonal/polyclonal anti-D reagents were evaluated for reactivity with partial D and efficiency of weak-D detection, using the manual tube technique. The anti-D reagents used in column technologies (Biovue and Diamed) also were evaluated. Testing was limited by the availability of partial-D cells. The reagents directly agglutinated the majority of partial-D cells, with the exception of DVI, which required the indirect antiglobulin test for detection. Totem (Diagast) and Z039 (Scottish National Blood Transfusion Service) both directly agglutinated DVI. The weak-D detection rate for a number of immunoglobulin M (IgM) reagents was improved by incubation of initially negative tests. Sedimentation techniques, recommended for a number of reagents, were unsuitable for weak-D or partial-D detection. There were indications that IgM/IgG reagents gave lower direct-agglutination weak-D detection rates than did IgM reagents, although Totem and Novaclone produced results matching those of the IgM reagents. Laboratories should be aware of the guidelines for Rh-D typing of patients and donors, and select anti-D reagents accordingly.     

Typing of normal and variant red cells with ABO, Rh, and Kell typing reagents using a gel typing system

In 1989 Lapierre et al. described a novel method of detecting agglutination reactions by the use of a Sephadex (DiaMed ID Typing System) gel held in a microtube. This report examines the use of gels containing ABO, Rh, and Kell system specific antibodies. The anti-A and -B were monoclonal reagents; anti-A,B, and those for the Rh and Kell systems were polyclonal. Five hundred and fifty-one tests performed for the ABO system detected all but the most weakly reacting variants, a detection rate superior to most commercially available reagents. Five hundred and thirty samples were typed for Rh antigens. One hundred and twenty-seven of these were of various D category III through VII types (Dcats) and 154 were Du>s. The gel system detected all but seven DVI variants and seven Dus. The seven DVI variants, from individuals with no anti-D in their sera, gave reactions identical to the seven Dus when tested against a panel of over 50 monoclonal IgG and IgM anti-Ds. The 554 samples tested for the K1 antigen gave correct results.     

Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy

Rh discrepancies are a problem during routine testing because of partial D or weak D phenotypes. Panels of monoclonal antibodies (MoAb) are being developed to identify D variants such as partial D and weak D when there are anomalous D typing results; however, molecular characterization offers a more specific classification of weak and partial D. The weak D and partial D phenotypes are caused by many different RHD alleles encoding aberrant D proteins, resulting in distinct serologic phenotypes and the possibility of anti-D immunization. We evaluated currently used serologic methods and reagents to detect and identify D variants and correlated the results with molecular analyses. A total of 306 blood samples from Brazilian blood donors and patients with discrepant results in routine D typing were analyzed. In total, 166 (54.2%) weak D, 136 (44.4%) partial D, 3 (1%) DEL, and 1 (0.3%) DHAR variants were identified. Among weak D samples, 76 weak D type 1 (45.8%), 75 weak D type 2 (45.2%), 13 weak D type 3 (7.8%), and 2 weak D type 5 (1.2%) alleles were found. Among the partial D samples, 49 type 4.0 weak partial D (36%), 9 DAR (6.6%), 24 DFR (17.6%), 6 DBT (4.4%), 1 DHMi (0.73%), 26 DVI (19%), 14 DVa (10.3%), 5 DIVb (3.7%), and 2 DVII (1.5%) were observed. Two samples identified as DEL by adsorption-elution were characterized by molecular analyses as RHD(IVS5–38DEL4) and one sample was characterized as RHD(K409K). One sample was characterized as DHAR, a CE variant positive with some monoclonal anti-D. Our results showed that the use of different methods and anti-D reagents in the serologic routine analysis revealed D variants that can be further investigated. Molecular methods can help to differentiate between partial D and weak D and to characterize the weak D types, providing additional information of value in the determination of D phenotypes. This distinction is important for optimized management of D– RBC units and for the prevention of anti-D–related hemolytic disease of the fetus and newborn.     

Monoclonal antibodies directed against human Rh antigens in tests with red cells of non-human primates

Human anti-D (Rh_o) monoclonal antibodies (Mabs) of the IgG (70) and IgM (27) classes were tested with red blood cells (RBCs) of various non-human primates, from anthropoid apes to New World monkeys. Significant differences in reactivity were observed among antibodies of two classes depending on taxonomic position of primate animals. Only IgM Mabs gave positive reactions (9 out of 18 Mabs) with blood of Old World monkeys. Allotypic reactions with RBCs of African apes were produced by a majority of IgG Mabs but by very few IgM reagents, most of the latter reacting with RBCs of all chimpanzees and all gorillas tested. Eight out of 70 IgG anti-D defined chimpanzee polymorphisms related to chimpanzee R^c antigen which is the chimpanzee counterpart of human D antigen. Most of IgG anti-D Mabs (61/70) were found specific of D^gor antigen (gorilla counterpart of human antigen D). Most of anti-D which were found negative with all chimpanzee RBCs were also negative with human D^IVb RBCs and most of anti-D which agglutinated human D^IVb RBCs were positive with some or all chimpanzee blood samples. Differences among Mabs evidenced in tests with non-human primate RBCs reflect the complexity of the immune reactions to the human D antigen. The results obtained with anti-Rh Mabs of specificities other than D confirmed that chimpanzee, gorilla and gibbon express c-like epitopes and that antigens C, E, e are absent in non-human primates.     

Anti-D auto-immunization in a patient with weak D type 4.0

We report the case of a 56-year-old patient with blood group O+C?c+E?e+K?, followed for a myelodysplasic syndrome and treated by regular pheno-identical and compatible (RBCs) transfusion since December 2007. In June 2009, a positive crossmatch was found with 2 RBCs O+C?c+E?e+K?. A positive anti-body screening with a positive autocontrol was detected and anti-D was unidentified in the patient's serum. The DAT was positive (IgG) and elution identified an anti-D. The following assumptions were then made: it could be a partial D phenotype with anti-D alloantibodies or RH: 1 phenotype with an anti-D auto-antibodies. Molecular analysis by multiplex PCR and sequencing have depisted a weak D type 4.0 phenotype. In October 2009, over three months of RH:?1 RBC transfusion, the antibody screening and DAT (IgG) remained positive, and an eluate made from the patient's erythrocytes contained an anti-D. All these funding confirmed the autoimmune nature of the anti-D. This case report illustrates the importance of a well-conducted and immunohematological laboratories test in order to distinguish between auto- or allo-immune of anti-D in a RH: 1 poly-transfused patients. This distinction is of great importance for transfusion support.     

Incidence of weak D in blood donors typed as D positive by the Olympus PK 7200

The incidence of weak D has been reported to be between 0.23 and 0.5 percent in Europe and 3.0 percent in the United States. All studies were performed before the introduction of monoclonal anti-D reagents. Using current commercial reagents, this study evaluated D+ samples for the presence of weak D. D+ donors, typed by the Olympus PK 7200, using diluted monoclonal blend anti-D and diluted polyclonal anti-D, were selected by sampling batches of 100 to 200 samples from the previous day's collection. Anti-D reagents used on the Olympus PK 7200 are required to detect RBCs with the weak D phenotype which do not agglutinate at immediate spin (IS) when tested with polyclonal anti-D by manual tube methods. More than 95 percent of donors tested were Caucasian. Using tube tests with two different monoclonal blend anti-D reagents and one polyclonal anti-D typing reagent, the presence or absence of the D antigen was evaluated after the IS reading. Donors found negative or weakly positive (< 2+) at IS were further typed for weak D by the IAT. The weak D samples were RHD genotyped by allele-specific PCR. Of 1,005 donors tested, 4 (0.4%) were classified as weak D by one or more anti-D reagents. Polyclonal anti-D reagent demonstrated weaker reactions when compared with the monoclonal blends. All weak D samples were found positive for exon 4, intron 4, and exon 10, a finding consistent with most D+ samples. The incidence of weak D found in this study is not significantly different from that found in earlier studies using polyclonal anti-D reagents.     

Heterogeneity in the ability of IgG1 monoclonal anti-D to promote lymphocyte-mediated red cell lysis

Thirty-four IgG anti-D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody-dependent cell-mediated cytotoxicity assays. Cell-bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non-lytic anti-D mAb. Greater sensitivity was achieved using fluid-phase antibody (as culture supernatants) in the assay than was obtained with pre-sensitized red cells. Minimum levels of 2000 anti-D molecules per cell were required for lysis using pre-sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti-E and anti-G) were lytic and two (anti-c and anti-Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.     

Discrepancies in Rh(D) typing of sensitized red blood cells using monoclonal/polyclonal anti-D reagents: case report and review

Instructions included with monoclonal Rh(D) typing reagents do not require routine use of an Rh control as immunoglobulin-coated red blood cells (RBCs) rarely yield falsely positive results with low protein reagents. However, the American Association of Blood Banks (AABB) Technical Manual recommends a concurrent control be performed on patients' RBCs that type as group AB, D(+). Proficiency testing surveys presented sensitized AB, D- RBCs, which resulted in a positive direct antiglobulin test and, in some samples, spontaneous agglutination in saline. One intent of the surveys was to monitor the accuracy of the reported Rh(D) type. On an initial survey, 19 of 115 (16.5%) participants reported the RBCs as D(+). Of these laboratories, 63.2 percent (12/19) had used a monoclonal/ polyclonal blend anti-D reagent. On a subsequent survey, after educational material had been distributed, only five of 113 (4.4%) participants reported the Rh type as D(+). Two of these five laboratories had used a monoclonal/polyclonal blend anti-D reagent. As RBCs coated with immunoglobulin may give unreliable results with Rh typing reagents, laboratories should follow the guidelines of the AABB Technical Manual. An appropriate control should be performed whenever RBCs from patients type as AB, D(+).     

Intravenous Rh immune globulin prevents alloimmunization in D- granulocyte recipients but obscures the detection of an allo-anti-K

Rh immune globulin (RhIG) has been used to prevent alloimmunization in D(-) recipients of apheresis platelet transfusions from D(+) donors that may contain up to 5 mL of D(+) red blood cells (RBCs). Granulocyte concentrates contain approximately 30 mL of RBCs and it has been necessary to give D(-) recipients granulocyte transfusions from D(+) donors. Intravenous RhIG has not yet been demonstrated to be effective in preventing D alloimmunization with granulocyte transfusions. Four D(-) recipients received multiple D(+) granulocyte transfusions from D(+) donors and multiple injections of intravenous RhIG at a standard dose of 600 microg for each D(+) transfusion. Two D(-) males with chronic granulomatous disease were given 32 and 13 daily granulocyte transfusions, 18 and 2 of which, respectively, were D(+). After the first dose of intravenous RhIG, both patients exhibited circulating anti-D that was undetectable 3 to 4 years later. Two patients with severe aplastic anemia were given 5 and 14 granulocyte transfusions, 4 and 7 of which, respectively, were D(+). Both patients died before the effectiveness of RhIG could be assessed. In one of these patients the indirect and direct antiglobulin tests became positive after the first dose of intravenous RhIG, which required that subsequent granulocyte transfusions from D(+) donors be crossmatched by immediate spin (IS) testing only. A delayed hemolytic reaction attributed to allo-anti-K occurred after granulocytes from a K(+) donor were given to this patient. These results suggest that intravenous RhIG can be used to prevent alloimmunization to D in D(-) patients receiving large quantities of RBCs from D(+) granulocyte transfusions. However, anti-D and other passive antibodies from RhIG prohibit the use of the antiglobulin crossmatch with antigen-positive granulocyte donor samples. It may be important to frequently collect new samples to screen for newly formed allo-antibodies when IS crossmatches are used in place of the antiglobulin crossmatch.     

Efficacy of murine monoclonal antibodies in RBC phenotyping of DAT-positive samples

Determining the phenotype of patient RBCs that are positive by the DAT may prove problematic. Antigen typing of RBCs coated with IgG requires direct agglutinating reagents or chemical treatment (such as chloroquine diphosphate [CDP] or citric acid) to remove sufficient IgG to permit testing with IAT-reactive reagents. The citric acid elution method is commonly used in the United States; however, antigens in the Kell system are altered to the extent that they may appear to be absent by this method. There are a limited number of direct agglutinating monoclonal antibodies available. Murine monoclonal antibodies provide an additional tool for typing RBCs with a positive DAT. Five murine monoclonal IgG antibodies (anti-K: MIMA-22, MIMA-23; anti-Kpa: MIMA-21, MIMA-27; anti-Fya: MIMA-19) were used in this study. Donor RBCs with known phenotypes were sensitized in vitro with alloanti-D, alloanti-c, and alloanti-K and with 20 autoantibodies (autoanti-D [n=3], autoanti-e [n=5], autoanti-Ce/e [n=5], autoanti-e+D+E [n=1], autoanti-I [n=1], and nonspecific [n=5]) to simulate a positive in vivo DAT. The sensitized RBCs were treated with CDP to remove IgG. To determine the efficacy of the murine monoclonal antibodies when testing DAT-positive samples, both sensitized and CDP-treated RBCs were tested with these monoclonal antibodies by the IAT using anti-mouse IgG. No discrepancies were noted with the unsensitized, sensitized, or CDP-treated RBCs. An exception was noted with a potent autoanti-I, where direct agglutination of the sensitized RBCs was obtained. This study demonstrates the value of using murine monoclonal antibodies to determine the phenotype of RBCs with a positive DAT caused by autoantibodies (e.g., in autoimmune hemolytic anemia) and supports previous studies showing that RBCs sensitized in vivo can be typed without chemical manipulation.     

Reactivity of 62 Rh MAbs with weak and variant antigens

We characterized serologically 5 anti-C (4 IgM and 1 IgG), 3 anti-c (2 IgM and 1 IgG), 4 anti-E (1 IgM and 3 IgG), 4 anti-e (3 IgM and 1 IgG) and 46 anti-D (16 IgM and 30 IgG) monoclonal antibodies, provided by the Rh Section of the Third International Workshop and Symposium on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (1996) for their ability to detect weak and variant antigens. The agglutination patterns were established using untreated and papain-treated red blood cells in a column agglutination technology system (BioVue, Ortho). Significant differences were found between the IgM and IgG antibodies. The papain treatment seemed to be important for IgM but not for IgG antibodies. Almost all of the IgM anti-D antibodies detected untreated D^IV samples and almost all of the IgG anti-D antibodies detected untreated weak D samples. Both IgM and IgG anti-D antibodies showed the highest number of negative reactions with D^VI and Rh 33 red blood cells. The C^WC^W sample was detected by only one of the 4 anti-C IgM MAbs using enzyme-treated red blood cells. All anti-c MAbs were able to detect treated C^x samples. Because of the small number of weakly expressed E and e samples, definitive conclusions cannot be drawn on the ability of these antibodies to detect these antigens.     

Serologic aspects of treating immune thrombocytopenic purpura using intravenous Rh immune globulin

In patients with immune thrombocytopenic purpura (ITP), IgG autoantibody-coated platelets are phagocytized by mononuclear macrophages, primarily in the spleen. Intravenous Rh immune globulin (IV RhIG) has been used since 1983 to treat D(+), nonsplenectomized patients with ITP. The beneficial therapeutic effect of IV RhIG is attributed to competitive inhibition of phagocytosis of IgG-coated platelets by IgG anti-D-coated D(+) red blood cells (reticuloendothelial or Fc receptor blockade). Following infusions of IV RhIG in D(+) ITP patients, the direct and indirect antiglobulin tests become transiently positive, reflecting passively transferred anti-D and other alloantibodies that were present in the infused IV RhIG. These consistent and predictable serologic findings contrast with the inconsistent and weak anti-D reactivity observed when D(-) women are treated with relatively small doses of intramuscular RhIG for Rh immunoprophylaxis. The pathophysiology of ITP and the effect of infusing IV RhIG in patients with ITP are illustrated in this review, using computer-generated figures.     

Recombinant polymeric IgG anti-Rh: a novel strategy for development of direct agglutinating reagents

Anti-Rh alloantibodies are used in research and clinic laboratories to define the Rh antigenic profile of human blood samples. IgM anti-Rh antibodies directly agglutinate Rh-positive RBCs. Anti-Rh antibodies of the IgG isotype bind to Rh antigens with a higher intrinsic affinity than IgM and sensitize RBCs, but do not induce direct hemagglutination. The aim of this work was to produce IgG anti-Rh possessing direct hemagglutinating properties of IgM. To achieve this goal, recombinant antibody technology was used to construct genes encoding Ig light and heavy chains that will form polymers with anti-Rh specificity. Expression vectors and liposome-mediated DNA transfer were used to generate transfectomas secreting human recombinant IgG3 anti-Rh. ELISA, SDS-PAGE, and hemagglutination were used to identify and characterize the recombinant antibody produced. Thus, a recombinant polymeric IgM-like IgG3 anti-Rh antibody was produced that directly agglutinates RBCs with specificity identical to that of the parent non-agglutinating IgG. The results obtained suggest that the technology used here to generate polymeric IgM-like IgG3 anti-Rh antibodies can be applied to produce Rh blood typing reagents. This approach might also be used to develop reagents for which cell surface antigen binding and agglutination or aggregation is required.     

Update on HDFN: new information on long-standing controversies

Hemolytic disease of the fetus and newborn (HDFN) results from maternal IgG antibodies that cross the placenta to the fetal circulation during gestation and cause RBC destruction and complications before birth (HDF), or anemia and hyperbilirubinemia after birth (HDN), or both. In its most severe form,HDF produces hydrops fetalis, which is characterized by total body edema, hepatosplenomegaly, and heart failure and can lead to intrauterine death. Before discovery of Rh immunoglobulin (RhIG), HDFN from anti-D was a significant cause of perinatal mortality or long-term disability. Routine administration of RhIG to D- women during pregnancy and shortly after the birth of D+ infants effectively reduced the incidence of HDFN caused by anti-D. Maternal alloimmunization to other RBC antigens in the Rh, Kell, and other blood group systems can not be routinely prevented and these antibodies can also cause HDFN. Advances in prenatal care, noninvasive monitoring, and intrauterine transfusion have improved the outlook for affected pregnancies to the extent that even hydrops fetalis can be reversed and effectively treated in many cases. This review will provide an update on the current issues in prevention and treatment of HDFN, emphasizing recent insights into long-standing controversies regarding maternal weak D phenotypes and D alloimmunization, noninvasive fetal diagnosis and monitoring of affected pregnancies, and recent treatment guidelines.     

Immunoprophylaxis using intravenous Rh immune globulin should be standard practice when selected D-negative patients are transfused with D-positive random donor platelets

A 67-year-old female developed excessive bleeding and thrombocytopenia following cardiovascular surgery. Her blood type was group A, D-. The only platelet products available in the transfusion service were random donor platelet concentrates from D+ donors. She was transfused with a pool of 6 D+ random donor platelet concentrates. Anti-D undetected in her pretransfusion serum by solid-phase antibody screen was present 11 days later. Retrospectively, the patient provided a history of having two pregnancies more than 40 years ago, prior to the availability of immunoprophylaxis by Rh immune globulin (RhIG). Although studies have shown that as many as 19 percent of D- people may develop anti-D following transfusion of platelets from D+ donors, there is no specific standard requiring immunoprophylaxis with RhIG to prevent Rh alloimmunization after transfusion of random donor platelet concentrates from D+ donors. In contrast, vigorous efforts are routine for preventing Rh alloimmunization in D- patients requiring red cell transfusions or D- females during pregnancy or after delivery of D+ newborns. The absence of a comparable practice standard for platelet transfusions is based, in part, on concern that intramuscular injections of conventional RhIG may cause local hemorrhage in thrombocytopenic persons. The recent availability of a Food and Drug Administration-approved preparation of intravenous RhIG makes Rh immunoprophylaxis in thrombocytopenic patients safe and practical. We recommend that intravenous RhIG be considered if it is necessary to transfuse random donor platelet concentrates from D+ donors to D- recipients. As a minimal standard, intravenous RhIG should be administered to all D- females of childbearing age who are recipients of pools of random donor platelet concentrates from D+ donors.     

Human monoclonal antibodies specific for blood group antigens demonstrate multispecific properties characteristic of natural autoantibodies.

A panel of 72 human monoclonal antibodies with specificities for blood group antigens, A, Rh D, Rh C, Rh c, Rh E, Rh e, Rh G, Jka and Jkb, has been established from the peripheral blood of deliberately immunized donors. Previous work has established that the antibodies are highly specific for their respective blood group antigens, and a number of them are in routine clinical use as blood grouping reagents. This panel was screened for reactivity against six unrelated foreign and autoantigens by ELISA, for rheumatoid factor activity by ELISA and agglutination techniques, and for reactivity with a number of different tissues by immunofluorescence. Binding of the monoclonal antibodies to unrelated exo- and autoantigens was commonly seen amongst the antibodies of the IgM class, and to a lesser degree amongst the IgG class, with reaction patterns similar to those given by natural autoantibodies. Only five of the IgM antibodies failed to demonstrate any unexpected cross-reactivities and these included 1/13 anti-D, 2/7 anti-E, 1/13 anti-c and 1/2 anti-A. We propose that rather than natural autoantibodies representing a distinct population of immunoglobulins, multispecificity (polyspecificity, or polyreactivity) may be a feature of antibodies produced in response to exogenous antigens. The implications of this for the study of autoantibodies are discussed.     

A method to detect McLeod phenotype red blood cells

It is important to identify the McLeod phenotype in order to differentiate the McLeod syndrome from other causes of acanthocytosis, e.g., chorea acanthocytosis. A proportion of males with the McLeod phenotype have X-linked chronic granulomatous disease. Because anti-Kx + -Km, which is needed for identification, is not readily available, detection of the McLeod phenotype relies on observed weakening of Kell antigens on the individual's red blood cells (RBCs). Identification of McLeod carrier females (obligate heterozygotes) is even more difficult because only a minor subpopulation of RBCs may express the weakened Kell phenotype. RBCs from 12 sets of mother/son or father/daughter pairs were tested by standard hemagglutination tube tests and by flow cytometry using both monoclonal and polyclonal Kell system antibodies. Monoclonal anti-K14 (G10) in tests with RBCs from McLeod males reacted +/- by hemagglutination (control cells 2+) and had a median fluorescence of 6-11 by flow cytometry (control cells 441). Monoclonal anti-k (F7) and human polyclonal anti-k (C30A-1) gave stronger reactions by hemagglutination with RBCs from McLeod males and were not appropriate to differentiate RBCs with the McLeod phenotype from RBCs with normal Kell antigen expression. Crisp mixed-field agglutination was obtained in tests with monoclonal anti-K14 versus RBCs from the 12 obligate carrier females. Flow cytometry using anti-K14 also clearly identified two RBC populations (median fluorescence of 6-11 and 229-382, respectively). Kell system antibodies may vary in their ability to detect a weakened expression of the corresponding antigen on RBCs with the McLeod phenotype by hemagglutination. Antibodies suitable for differentiating McLeod syndrome from other forms of acanthocytosis should be reagents that are preselected after extensive testing. In this study, flow cytometry clearly identified the two RBC populations in McLeod carrier females.     

In vivo studies of monoclonal anti-D and the mechanism of immune suppression.

Administration of anti-D immunoglobulin to D- women after delivery of a D+ infant has dramatically reduced the number of immunised women and cases of haemolytic disease of the fetus and newborn. The use of monoclonal anti-D might alleviate some of the pressures on maintaining adequate supplies of plasma sourced anti-D. Two human monoclonal antibodies, BRAD-3 (IgG1) and BRAD-5 (IgG3), with proven activity in in vitro functional (immunological) assays with cells bearing IgG Fc receptors (Fc gamma R) were selected for clinical studies. They were prepared by purification of IgG secreted by culture of the Epstein-Barr virus-transformed B cell lines in hollow fibre bioreactors. The clearance of D+ red cells injected into D- subjects was accelerated by prior injection of the monoclonal antibodies, both individually and blended (3:1, BRAD-5: BRAD-3). The subjects were protected from Rh D immunisation. A large multicentre study evaluated the BRAD-3/5 blend for its ability to prevent Rh D immunisation in 95 D- subjects given 400 micrograms i.m. 24 hours after injection of 5 ml D+ red cells. Challenge injections of D+ red cells alone were given 24 and 36 weeks later, and blood samples were taken every 4 weeks from the subjects throughout the study for detection of anti-D responses. There was one definite and one possible failure of protection; in one subject the plasma anti-D level rose from week 12 onwards, and in another individual rapid seroconversion was observed at week 28. Considering the relatively large dose of red cells and the number of subjects studied, it was concluded that the failure rate was much lower than in routine Rh D prophylaxis. The responder rate was 13% by week 36 and 24% by week 48. The low percentage of responders and the modest levels of endogenous anti-D produced suggested that administration of monoclonal anti-D had induced long-term specific suppression of anti-D responses in these subjects. The most likely mechanism of action was considered to be inhibition of B cells resulting from co-crosslinking antigen receptors with inhibitory Fc gamma R when the B cells contacted red cells that had bound passive anti-D.     

A new assay uses monoclonal anti-Rh(D) antibodies to determine rheumatoid factor specificity: reactivity to a monoclonal antibody of the Gm allotype G3m(21) is more frequent in rheumatoid patients negative for G3m(21)

A new method has been developed to determine the specificities of polyclonal rheumatoid factors (naturally occurring antibodies which react with human Fc gamma) (RF) found in sera from patients with rheumatoid arthritis. In this method, monoclonal anti-Rh(D) antibodies of known IgG isotype and allotype are bound to erythrocytes and then act as the target IgG antigen for RF in a direct haemagglutination test. Using two monoclonal anti-D antibodies of the IgG3 isotype and G3m(21) allotype, which were cloned from different donors, we found that a large number of rheumatoid sera reacted with both these G3m(21) proteins. In contrast reactivity of rheumatoid sera with polyclonal anti-D of the G3m(21) allotype in the direct haemagglutination test was rare. A strong correlation was found between reactivities to both G3m(21) monoclonal anti-D antibodies but not with a monoclonal anti-D antibody carrying the alternative allele, namely G3m(5). Haemagglutination inhibition experiments using human paraproteins of known IgG isotype and allotype provided some additional evidence that this method can detect RF with specificity for the G3m(21) allotypic determinant or a related allotypic determinant in polyclonal rheumatoid sera. When each patient's autoantibody response was related to their Gm phenotype, we found that the frequency of reactivity for G3m(21) monoclonal anti-D antibodies was significantly greater in patients negative for G3m(21) than in patients positive for the G3m(21) allotype. IgM preparations from patients' sera were dissociated at acid pH but no 'hidden' antibodies were found. We suggest trans-placental sensitization as one of several possible interpretations of this finding.