A comparison of in vitro tests for predicting the severity of haemolytic disease of the fetus and newborn.

Haemolytic disease of the newborn (HDN) is characterized by the presence of IgG antibodies in the maternal circulation which cause haemolysis in the fetus by crossing the placenta and sensitizing red cells for destruction by macrophages in the fetal spleen. Numerous serological, quantitative and cellular assays have been developed to predict the severity of HDN. These assays all measure and/or characterize alloantibodies in the maternal circulation. Quantitative assays which accurately measure antibody levels correlate with disease severity better than serological assays which are inherently less precise. Nevertheless, high antibody levels are found in some cases of mild HDN and relatively low antibody levels are found in some severe cases. This suggests that disease severity is influenced by factors in addition to antibody concentration. These factors remain to be fully elucidated but may include the subclass and glycosylation of maternal antibodies, the structure, site density, maturational development and tissue distribution of blood group antigens, the efficiency of IgG transport to the fetus, the functional maturity of the fetal spleen, polymorphisms which affect Fc receptor function, and the presence of HLA-related inhibitory antibodies. Cellular assays which are sensitive to factors affecting antibody function have therefore been developed in an attempt to improve the prediction of disease severity. Although these assays are cumbersome, there are now sufficient data to suggest that some cellular assays, when used as part of a structured approach to diagnostic testing, may provide clinically-useful information to complement serological and quantitative assays.

Antigen topography is critical for interaction of IgG2 anti-red-cell antibodies with Fc gamma receptors.

IgG antibodies to the Rh D polypeptide on red cells are normally IgG1 or IgG3, whereas antibodies produced in response to carbohydrate antigens such as the A and B blood groups are predominantly IgG2. The consequences of this isotype restriction for the immune destruction of red cells were investigated. Human IgG2 anti-D and IgG2 anti-A were isolated by affinity purification from an unusual anti-D serum (DEL) and anti-A sera, respectively. These antibodies were compared with IgG1 and IgG3 monoclonal anti-D in in vitro functional assays of the interaction between IgG-coated red cells (EA-IgG) and cells bearing IgG Fc receptors (Fc gamma R). Dimeric IgG2 anti-D bound efficiently to cell lines transfected with Fc gamma RIIa-H131, an allotypic form of Fc gamma RIIa which readily interacts with IgG2, IgG1 and IgG3. Unexpectedly, however, -D- phenotype red cells coated with IgG2 anti-D did not form rosettes with these cells, whereas EA-IgG2 anti-A and EA-IgG1 and EA-IgG3 anti-D effectively formed rosettes with these transfectants at the same sensitization level (100,000 molecules IgG/red cell). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, lysis of EA-IgG2 anti-A was mediated via Fc gamma RIIa, whereas lysis of EA-IgG1 and EA-IgG3 anti-D was mediated via Fc gamma RI or Fc gamma RIII; EA-IgG2 anti-D was inactive in all functional assays. These experiments suggest that both IgG subclass and antigen structure affect functional IgG-Fc gamma R interactions. The topography of the Rh D antigen, an integral membrane protein, ensures that anti-D is bound near the lipid bilayer surrounded by the glycocalyx. This may sterically hinder access of Fc gamma RIIa-H131 to the Fc gamma R recognition site on the relatively inflexible IgG2 anti-D, but not to that of IgG1 or IgG3 anti-D. In contrast, IgG2 bound to the A antigen on glycoproteins is not so constrained. The topography of the D and A antigens may thus determine whether functional interactions of red-cell-bound IgG2 anti-D and IgG2 anti-A with cells bearing Fc gamma receptors can occur.

Quantitative and functional asessment of anti-RhD: a comparative study of non-invasive methods in antenatal prediction of Rh hemolytic disease.

STUDY OBJECTIVE: To investigate if the prediction of hemolytic disease of the newborn (HDN) in infants of RhD immunized women has been improved by introduction of AutoAnalyzer quantitation and may be further improved by the use of a functional assay. METHODS: Manual antibody titration, AutoAnalyzer quantitation and chemiluminescence test (CLT) were compared by testing 42 sera from 38 RhD immunized mothers. The sera were also screened for the presence of monocyte-reactive antibodies which have the potential to protect the unborn. RESULTS: Among the 42 infants there were 19 unaffected by HDN and 23 affected by HDN. Manual titration correctly predicted the occurrence of HDN in 29/42 (69%), AutoAnalyzer was correct in 28/42 (67%) and CLT showed correct predictions with 30/42 (71%). In babies born without signs of HDN, maternal monocyte-reactive antibodies were found in 13/18 cases. The majority (9/13) were HLA class I-specific, the remaining four antibodies were either HLA class II or monocyte-specific. In affected HDN group, 8/16 monocyte-reactive anti- bodies were HLA class II or monocyte-specific. CONCLUSIONS: AutoAnalyzer and CLT improve the ability to discriminate unaffected babies from those affected by RhD HDN, when compared to manual titration. A protocol for the laboratory management of RhD immunized women is proposed that includes these tests to further improve the prediction of HDN. This study has also highlighted the need for more investigations into the protective role of monocyte-reactive antibodies in HDN.

Comparison of the ability of red cells sensitized with a bispecific anti-D x anti-Fc gamma RIII Fab fragment to activate human K cells and peritoneal macrophages through Fc gamma RIII.

The functional activity of Fc gamma RIII on human K cells from peripheral blood was compared with that of Fc gamma RIII on peritoneal macrophages (PM) separated from the waste material of patients undergoing peritoneal dialysis. Fc gamma R function was assessed in vitro using human monoclonal IgG1 anti-D (AB5) or a bispecific antibody comprising Fab fragments of AB5 chemically linked to Fab fragments of monoclonal anti-Fc gamma RIII, 3G8 (AB5 x 3G8). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, K cells mediated the lysis of papainized red cells sensitized with the AB5 x 3G8 bispecific antibody but not with AB5. In contrast, red cell lysis by PM was not promoted by AB5 x 3G8 although AB5 was active. However, this lysis, being inhibited by monomeric IgG, was presumably mediated via Fc gamma RI. AB5 x 3G8 also failed to promote the binding and phagocytosis of both papainized and native red cells by PM although 99% of red cells and over 90% of peritoneal cells bound the bispecific antibody. In marked contrast to K cells therefore, Fc gamma RIII on PM was unable to mediate functional interactions with red cells sensitized with anti-D x anti-Fc gamma RIII bispecific antibody.

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.

The use of interferon-gamma-treated U937 cells in chemiluminescence assays to detect red cell, platelet and granulocyte antibodies of potential clinical significance.

The chemiluminescent (CL) response of interferon-gamma-treated U937 (IFN-U937) cells to sensitized target cells has been used to detect red cell, platelet and granulocyte antibodies. A clone of U937 cells was selected which expressed Fc receptor I (Fc gamma RI) and which, after incubation with IFN-gamma for 72 h, was capable of generating high levels of lucigenin-enhanced CL. The CL responses of IFN-U937 cells and peripheral blood human monocytes to sensitized red cells, platelets or granulocytes were then compared. Assays using monocytes or IFN-U937 cells were of comparable sensitivity for detection of antibodies against all three types of target cell. In addition, the use of IFN-U937 cells reduced interassay variation and simplified assay performance. The potential clinical usefulness of these CL assays was suggested by the ability of both monocytes and IFN-U937 cells to respond to red cells, platelets or granulocytes sensitized with sera from pregnant women whose babies had either haemolytic disease of the newborn (HDN), alloimmune thrombocytopenia or alloimmune neutropenia respectively. In addition, monocytes and IFN-U937 cells both responded to red cells sensitized with antibodies against a variety of specificities of assumed (although not documented) clinical significance for blood transfusion recipients. In contrast, monocytes and IFN-U937 cells responded only weakly to red cells sensitized with either anti-D in sera from mothers of babies unaffected by HDN, or with antisera containing high titre antibodies with specificities not normally associated with significantly reduced red cell survival.

Functional interactions of aglycosylated monoclonal anti-D with Fc gamma RI+ and Fc gamma RIII+ cells.

Four human monoclonal antibodies (mAb) to the Rh antigen D were produced in aglycosylated forms by culture of B-cell lines in medium containing tunicamycin (Tm-mAb). Erythrocytes sensitized with these or control mAb were compared in U937 rosette and monocyte chemiluminescence assays to determine Fc gamma receptor I (Fc gamma RI)-mediated functional activity, and in lymphocyte rosette and lymphocyte antibody-dependent cell-mediated cytotoxicity (ADCC) assays to study Fc gamma RIII-mediated binding and lysis. Fc gamma RI-mediated interactions with Tm-mAb were greatly reduced compared with control mAb. All Tm-mAb failed to promote ADCC, although lymphocyte rosette formation was unaltered. The anti-D titre of Tm-mAb and their interaction with mAb JL512 (recognizing an epitope in the CH2 domain) were unchanged. These data suggest that glycosylation of IgG is required for CH2 domain interactions with both Fc gamma RI and Fc gamma RIII, but not for CH3 domain interactions with Fc gamma RIII.

A rapid solid-phase rosette-inhibition assay for the detection of Fc receptor (FcRII)-blocking alloantibodies [corrected].

A procedure for the detection of FcR-blocking alloantibodies is described which uses human B lymphocytes immobilised on plastic by poly-L-lysine. Antibodies which inhibited rosette formation between B lymphocytes or Daudi cells and ox erythrocytes coated with rabbit antibodies (EA) were detected in 10 out of 10 sera containing anti-HLA A2 antibodies and 3 out of 3 sera containing anti-HLA class II antibodies. Inhibition of rosette formation (EAI activity) was mediated by protein G-separated IgG. Analysis of rosette formation using these 13 sera and lymphocytes from 39 donors revealed that the degree of inhibition was bimodal; most sera were either clearly inhibitory or non-inhibitory in the assay. However, there was no correlation between inhibition of rosette formation (EAI activity) and lymphocytotoxicity. Four pairs of sera showed similar patterns of reactivity (r greater than 0.6, p less than 0.01; 2 x 2 chi-square test), and cells from 2 donors showed antithetical reactions with 12 of 13 sera (r = -0.86, p less than 0.001). These data suggest that the solid-phase rosette inhibition assay is a rapid and reproducible means of detecting antibodies reactive with non-HLA class I or class II antigens on human B cells.

Functional interactions of red cells sensitized by IgG1 and IgG3 human monoclonal anti-D with enzyme-modified human monocytes and FcR-bearing cell lines.

Human monoclonal IgG1 and IgG3 antibodies specific for the Rh antigen D (anti-D) were tested for their ability to promote the binding of D-positive red cells to peripheral blood monocytes and Fc receptor (FcR)-bearing cell lines (U937, K562 and Daudi). Monocyte-mediated antibody-dependent cell-mediated cytotoxicity and metabolic (chemiluminescent) responses were also determined. By comparing the activity of different cell lines in rosette assays, and by using murine myeloma IgG2a and IgG1 to block FcRI and FcRII respectively, these functional interactions of sensitized red cells (E-IgG1 and E-IgG3) with monocytes or cell lines were shown to be mediated predominantly and perhaps solely by FcRI. E-IgG3 bound to human monocytes and cell lines to a greater extent than E-IgG1. Rosette formation by E-IgG3 was relatively less susceptible to inhibition by fluid-phase murine IgG2a than was rosette formation by E-IgG1. These findings may be due to the long hinge region of IgG3 which enables it to bridge the gap between two negatively charged cells more efficiently than IgG1. Consistent with this hypothesis was the greatly increased rosette formation achieved by treating monocytes or U937 cells with neuraminidase or bromelain, procedures shown to reduce the zeta potential of these cells. The lytic and metabolic activities of untreated human monocytes were also greater towards E-IgG3 than E-IgG1, red cell binding being a prerequisite for these responses. However, after pretreatment of monocytes with neuraminidase, these responses were greater with E-IgG1 than with E-IgG3. Further, the addition of polybrene to non-specifically enhance cell to cell binding also resulted in greater lysis and chemiluminescence with E-IgG1 than with E-IgG3. These results indicate that, although E-IgG3 are more effective than E-IgG1 in promoting red cell binding to monocytes, E-IgG1 are more efficient at activating the lytic and metabolic processes providing the steric disadvantages of the shorter hinge region of cell-bound IgG1 are circumvented.

Synergistic effect of blending IgG1 and IgG3 monoclonal anti-D in promoting the metabolic response of monocytes to sensitized red cells.

Monoclonal antibodies specific for the Rh antigen D were used to sensitize red cells for use in a series of cellular assays. IgG3 anti-D was more efficient than IgG1 anti-D in promoting the attachment and lysis of red cells by human monocytes. In contrast, IgG1 anti-D was more efficient at mediating phagocytosis. The metabolic response of monocytes, measured by chemiluminescence (CL), was greater towards IgG3-sensitized red cells than IgG1-sensitized cells; however, the CL response was further increased when red cells were sensitized in antibody mixtures comprising both subclasses. Using monoclonal antibodies from five IgG1-secreting cell lines and from three IgG3-secreting cell lines, this synergistic increase was seen with 0/4 IgG1/IgG1 combinations, 0/2 IgG3/IgG3 combinations and 8/8 IgG1/IgG3 combinations. Synergism was observed only when both subclasses were present on the same red cells; mixing of IgG1-sensitized red cells with IgG3-sensitized red cells before addition to monocytes did not increase CL generation. The binding and phagocytosis of red cells by monocytes and the lysis of red cells by monocytes or lymphocytes were not greater when red cells were sensitized with IgG1 and IgG3 antibodies together compared to red cells coated with single subclasses.