Evidence that the c, D and E epitopes of the human Rh blood group system are on separate polypeptide molecules

The distribution of the epitopes, c, D, E and G of the human Rh system on red cells has been investigated using both 125I-labelled and fluorescently-labelled MAbs. There is a very wide range in the density of each epitope on individual red cells and the numbers of c, D and E epitopes on each cell are independent of each other. These observations taken together with the finding that there is no steric hindrance to binding between the antibodies, anti-c, anti-D and anti-E indicate that these epitopes are on separate Rh polypeptide molecules. The observations are taken to indicate that the total amount of Rh polypeptide on a single red cell is constant but that there is considerable heterogeneity between cells in the amount of each separate polypeptide carrying the different epitopes. In contrast, there was a mutual inhibition of binding of anti-G and anti-D monoclonals and direct positive correlation between the number of G and D sites on individual cells, indicating that the G and D epitopes are probably on the same Rh polypeptide.     

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.     

Epitopes on sialoglycoprotein alpha: evidence for heterogeneity in the molecule.

The binding of 15 125I-labelled mouse monoclonal antibodies to cell-surface sialoglycoprotein alpha (SGP alpha: synonym Glycophorin A) was studied using intact IgG and Fab fragments. It was estimated that the number of sialoglycoprotein alpha (SGP alpha) molecules per red cell is of the order of 1 x 10(6) and the number of sialoglycoprotein delta (SGP delta; synonym Glycophorin B) molecules per red cell is of the order of 1.7-2.5 x 10(5). Competitive binding assays showed that antibodies of the same blood group specificity (four anti-Ns reacting with SGP alpha and SGP delta (BRIC 33, BRIC 115, BRIC 120, BRIC 123) and four anti-Wrbs reacting with SGP alpha (R7, BRIC 14, BRIC 89, BRIC 93) inhibited binding of each other to red cells. Two antibodies (R1.3 reacting with SGP alpha and SGP delta and R18 reacting with SGP alpha) recognized distinct epitopes, and the remaining five antibodies (BRIC 116, BRIC 117, BRIC 119, BRIC 127, R10 reacting with SGP alpha) partially inhibited binding of each other to red cells. This latter observation and the finding that four of these antibodies (BRIC 116, BRIC 117, BRIC 119, BRIC 127) bind to a considerably smaller number of antigen sites (1.69-2.71 x 10(5) for intact IgG) than the maximum value obtained, suggests heterogeneity of glycosylation within SGP alpha molecules. The functional affinities of the IgG antibodies ranged from 1 x 10(5) to 4 x 10(7)M-1.     

Effect of Membrane Cholesterol Depletion on the Immunoreactivity of the D Antigen and IgG Anti-D

The Immunoreactivity of the main Rh antigen (D) and Its corresponding antibody, as determined by a IgG to IgG combining ratio, antibody dissociation and antigen accessibility to antibody, was examined In cholesterol depleted human red cells and ghost membranes. The anti-IgG reactivity of IgG anti-D bound to cholesterol depleted red cells and ghosts was demonstrably enhanced in vitro and in electron microscopy studies, particularly in ghosts. Dissociation of cell bound anti-D during buffer incubation was greater after cholesterol depletion, especially in ghosts. There was also reduced binding of anti-D to cholesterol-depleted eel is as previously reported. All these effects appeared to be independent of endogenous or exogenous proteolysis in either cholesterol-depleted membranes or controls as Judged from membrane electrophoretic analyses. A₂C, an agent which increases membrane fluidity, had no effect on anti-D binding or the antiglobulin reactivity of cell bound IgG. A reduction in anti-D binding also was observed In red cells depleted of cholesterol following Immobilization of membrane proteins by glutaraldehyde crosslinking. The findings show that cholesterol depletion not only affects the antigen but also Rh antibody reactivity. They also suggest that factors other than vertical antigen movement in a fluid bi layer may influence the behavior of the D antigen in cholesterol-modified erythrocytes.     

Coordinator's report: An assessment of the functional activity of human Rh monoclonal antibodies after their evaluation by nine laboratories

The functional activity of 55 anti-D and 26 MAbs of other Rh specificities was determined as part of the Third International Workshop and Symposium on Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens. Most MAbs were IgG1 (45 anti-D, 16 Rh). There was a large range of anti-D and IgG concentrations of the anti-D MAbs (0.4 – 1680 IU/ml, 1.5 – 400 μg/ml). Correlation between quantitative data from different laboratories was good.Six laboratories tested the anti-Ds in monocyte ADCC assays. Methods varied greatly, especially the effector cells used, the methods of red cell sensitisation, the effector to target cell ratio and the assay incubation times. There was some correlation of results between most laboratories, but results were better when similar assays were performed. The extent of monocyte-mediated haemolysis was related to the number of molecules of IgG anti-D bound to the red cells. Monocyte phagocytosis assays resulted in a greater variability in results between the four evaluating laboratories, though IgG3 MAbs were found more active than IgG1, and mostly promoted red cell adherence rather than phagocytosis.Two monocyte chemiluminescence assays found comparatively low activity of the IgG3 MAbs; the most active IgG1 anti-Ds were MAbs 93 and 95. Correlation between different monocyte-mediated assays was generally poor unless the assays were performed by the same laboratory. Results of a macrophage ADCC assay showed that the IgG3 anti-D's promoted greater haemolysis than most IgG1 MAbs. Only two IgG1 anti-D MAbs (70 and 104) mediated high haemolysis in this assay. Lymphocyte ADCC assays were utilised in four laboratories. The IgG3 antibodies exhibited low activity but one IgG1 anti-D (104) consistently mediated high haemolysis. There was no relationship between quantitation and haemolysis in this assay. A few MAbs exhibited low or no activity in most assays, mainly due to low quantitation. Most of the MAbs of non-D specificity mediated low functional activity which in most cases was not due to low quantitation. However two MAbs, 24 (anti-CcEe) and 25 (anti-CD), consistently showed good activity.This study highlighted the great variability in assay methodology between the nine participating laboratories, which resulted in some apparent differences in functional activity of some of the MAbs. The use of standardised methods as well as fewer MAbs tested and quantitated at different concentrations may help to determine the relevant factors contributing to IgG function.     

Equilibrium binding characteristics of monoclonal antibodies recognizing melanoma cell surface antigens

The equilibrium binding characteristics of a panel of six monoclonal antibodies (MAb) recognizing melanoma cell surface antigens (125 kdal cell surface melanoma associated glycoprotein antigen, 125kD-MAA; high molecular weight melanoma associated antigen, HMW-MAA; and a non-protein melanoma associated antigen, NP-MAA) were investigated using the cell lines SK-MEL-2, SK-MEL-5, and M21. The MAbs displayed equilibrium association constant (K) values ranging from 10(7) M-1 to 10(10) M-1 and maximum MAb binding values (Qmax) from 2 x 10(4) to 2 x 10(6) MAb molecules bound per cell. High trypsin concentrations were shown to have deleterious effects on Qmax values obtained for antibodies recognizing the 125kD-MAA, and even low trypsin concentrations affected Qmax values obtained for MAbs recognizing the HMW-MAA (although a complete linear recovery of HMW-MAA antigen was observed in 20-25 hours). Significant changes in Qmax were also noted for different cell passages. Except for MAb 43.2, little variation in K was observed when different cell lines were used. Linear Scatchard plots were obtained for all MAbs except 43.2 in which case concave down behavior was observed suggesting the existence of positive cooperativity between the binding sites of this MAb.     

Functional activity of human Rh monoclonal antibodies (MABS)

Anti-D and anti-c monoclonal antibodies (MABS) were found to be heterogeneous with respect to their activity in five cell-mediated functional assays. The two IgG3 MABS promoted greater rosette formation of red cells with monocytes than the IgG1 MABS. Discrepant results were obtained by different laboratories for the relative effectiveness of the MABS in promoting monocyte-mediated red cell phagocytosis and lysis, which may have been due to variations in the assay methods used. The IgG3 anti-D MAB promoted greater monocyte chemiluminescence than the IgG3 anti-c MAB, but of these two MABs only the anti-c mediated lysis of red cells in lymphocyte ADCC assays. The majority of the IgG1 anti-D MABS were ineffective at promoting red cell lysis by lymphocytes (K cells), but two were active even when diluted 1 in 300. There was no correlation of functional activity with red cell sensitization levels, IgG concentration or D epitope specificity of the MABS.     

Production and characterization of highly specific anti-methotrexate monoclonal antibodies

Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.     

Detection of an antigen on the inner surface of Rh negative erythrocytes which binds anti-D IgG

Previous investigation have demonstrated the presence of the Rho(D) antigen in Rh negative erythrocytes. The intact Rh negative cell, however, does not bind anti-D IgG. Presently we have shown that an anti-D binding antigen resides on the cytoplasmic surface of Rh negative erythrocyte membranes. Unsealed Rh negative membranes, in which both the inner and outer surface are exposed, bind anti-D IgG. Dicyclohexylcarbodiimide specifically blocked the binding of anti-D IgG to these membranes. Sealed Rh negative membranes which expose only their external surface, failed to bind anti-D antiserum. These results were confirmed by proteolytic digestion of membrane preparations and subsequent Rho(D) antigen purification. Only when protease had access to the inner surface of Rh negative erythrocyte membranes did degradation of this 'D' antigen occur. Thus, intact Rh negative erythrocytes contain an antigen which binds anti-D antibody but is located on the inner surface of the membrane. In contrast, Rh positive erythrocytes expose Rho(D) antigen on the external surface of the membrane.     

Differential exposure of epitopes on the human red cell antigen D (Rh).

The D polypeptide of the human Rh blood group system has a number of different epitopes on its surface. It is also known that there is considerable variation in the number of D antigen sites available to different human monoclonal anti-D antibodies. For instance, certain monoclonal antibodies recognise only a small number of sites on the red cell surface (about 9,000 sites/red cell on R1R2 cells) whereas other antibodies recognise a high number (about 20,000-30,000 sites/red cell). It has been found that cholesterol enrichment of the red cell membrane increases the number of sites available to those antibodies which recognize only a few sites but has no effect on those recognising many sites. The results are consistent with the view that access to some of the D epitopes is partially hindered by neighbouring molecules in the membrane and that alteration of the lipid content of the membrane changes it in such a way as to allow increased access to these obstructed epitopes.     

Rh血型抗体的检测及结果分析

目的:调查Rh血型抗体的检出率及其特异性分布特点.分析Rh血型抗体的临床意义及产生规律.方法:采用微柱凝胶抗球蛋白技术筛查和鉴定红细胞血型不规则抗体,对鉴定为Rh血型抗体者,采用单克隆抗-D、抗-C、抗-c、抗-E、抗-e鉴定红细胞Rh血型抗原,以确认抗体的准确性;检测抗体的效价、Ig类型及37℃反应性,以明确其临床意义;询问孕产史、输血史,如果为新生儿检测其母亲血浆中是否有相同特异性的抗体,以分析抗体产生的原因.结果:就诊者54000例,共检出Rh血型抗体47例,检出率为0.087%,其中有妊娠史者27例,有输血史者13例,既有妊娠史又有输血史者1例,抗体来自母体的新生儿6例;抗体的特异性为:抗-E 29例(61.70%)、抗-D 8例(17.02%)、抗-cE5例(10.64%)、抗-c 4例(8.51%)、抗-C 1例(2.13%);47例Rh血型抗体均为IgG或IgG IgM类,37℃均可与具有相应抗原的红细胞反应,抗体效价介于1～4096.结论:被检就诊者Rh血型抗体的检出率低于白种人;在检出的Rh血型抗体中,抗-E占绝对多数,而抗-D的检出率呈逐步减少的趋势;妊娠和输血引起的同种免疫是Rh血型抗体产生的原因,新生儿自母体被动获得的Rh血型抗体是Non-ABO-HDN最主要的致病抗体.     

Observations on the reactions between D-positive red cells and [125I]IgM anti-D molecules and subunits

The method of preparation of partially purified [¹²⁵I]IgM anti-D and of subunits of 180,000, 90,000 and 40,000 molecular weight with anti-D activity is described. It was found that approximately 11,500 whole IgM anti-D molecules would bind to each red cell of phenotype R₁R₂. On the other hand, approximately 31,000 molecules of both whole IgG anti-D and an IgM anti-D subunit of molecular weight 40,000 would bind to the same cell. The following possibilities are proposed to explain these findings: (1) IgM anti-D molecules bound to the red cell sterically hinder binding of other IgM antibody molecules to closely adjacent D-antigen sites and (2) some D-antigen sites are too far below the surface of the red cell to permit binding by the large IgM molecule.

The average values of the equilibrium constants (K₀) of two different examples of IgM anti-D were 1.7×10⁹ and 2.5×10⁹ litres/mole, and of the IgG antibodies obtained from the same plasmas were 9- and 32-fold lower respectively. The K₀ of the IgM subunits was on average 23-fold lower than that of the whole IgM and it is concluded that IgM anti-D binds bivalently to each red cell but that the binding energy of one of the sites is relatively small.

     

Generation of species-specific, rat monoclonal antibodies that bind to the kappa chain of mouse immunoglobulin.

Large numbers of mouse monoclonal antibodies (MAbs) with exquisite binding specificities have become available. However the study of these primary antibodies in biological fluids by second, anti-mouse antibody techniques, has been confounded by the large quantity of other animal immunoglobulin which is often present in these fluids. To overcome this problem, we have derived high affinity rat MAbs which bind specifically to the antigen binding fragments (Fab) of mouse antibody and display minimal or no crossreactivity with human or rabbit immunoglobulin at the concentrations which are usually found in plasma. Equilibrium binding studies demonstrated that these antibodies had binding affinity constants for mouse Fab that ranged from 4.3 x 10(8) to 4.1 x 10(9) M-1. All of these rat MAbs bound to the kappa chain of mouse immunoglobulin, though competition binding studies amongst these MAbs indicated that they probably recognized three different epitopes. Because of their specificity and affinity, these rat anti-mouse kappa MAbs may be useful in a wide variety of experimental biological systems both in vitro and in vivo.     

Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies.

Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.     

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.     

Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system.

An automated biosensor system for measuring molecular interactions has been used to study the kinetics of monoclonal antibody-antigen reactions. The system combines a microfluidic unit in contact with a sensor surface for surface plasmon resonance detection. The specificity of the surface is determined by the operator. Antibody or antigen is immobilised in a dextran matrix attached to the sensor surface. The interaction of matrix bound antibody or antigen with the corresponding partner in solution is monitored in real time. None of the interacting molecules needs to be labelled and it is not necessary to determine the concentration of the the matrix bound component in advance. Two systems were studied: matrix bound monoclonal antibodies (MAbs) interacting with HIV-1 core protein p24 and immobilised aminotheophylline reacting with MAbs. Control of the amount of immobilised ligand and reusable sensor surfaces permits the comparison of different MAbs reacting with antigen under almost identical conditions. Differences in affinity and reaction rates are immediately apparent. The calculated association rate constants for p24 MAbs ranged from 3 x 10(4) - 7.4 x 10(5) M-1 s-1 and for theophylline MAbs association rate constants as high as 1 x 10(6) M-1 s-1 were encountered. The calculated dissociation rate constants were in the region 2 x 10(-4) s-1 to 2 x 10(-2) s-1.     

Detection of tube agglutination 37 degrees C-only antibodies by solid-phase red cell adherence

There are no published data on the detection of tube agglutination (TA) 37 degrees C-only antibodies by solid-phase (SP) red cell adherence assays using anti-IgG-coated indicator red cells. Thirteen examples of TA 37 degrees C-only antibodies were tested by conventional SP methods. Four TA 37 degrees C-only antibodies failed to react by SP. Three were anti- Lea, considered clinically insignificant, and one was anti-E, an antibody of potential clinical significance. The remaining nine TA 37 degrees C-only antibodies reacted by SP, including three anti-c, two anti- D, two anti-E, one anti-N, and one anti-M. The anti-M reacted with indicator red cells that lacked the red cell antigen and failed to react with IgG-coated indicator red cells whose anti-IgG component had been neutralized, indicating the antibody contained an IgG component. Two anti-D and one anti-c continued to react in an SP test using neutralized anti-IgG antigen-positive indicator red cells, i.e., indicator binding independent of antiglobulin, suggesting an IgM nature to these antibodies. Therefore, many TA 37 degrees C-only antibodies can be detected by SP either through detection of an IgG component by the anti-IgG of the indicator red cells, or through IgM crosslinking of antigen-positive indicator red cells to antigen-positive SP reagent red cell membranes.     

Rh antigen immunoreactivity after histidine modification

125I-anti-D IgG and unlabeled blood group allo-antisera in combination with 125I-protein A were employed in assessing antibody binding to red cells (RBC) treated with histidine reagents. The acylating reagent diethylpyrocarbonate (DEP), the alkylating reagent p-bromophenacyl bromide (pBPB) and the photosensitizer dye Rose Bengal (RB) were used under conditions that usually result in the selective modification of histidine in isolated proteins. Progressive apparent inactivation of the D antigen in ghost membranes occurred with increasing DEP concns, which was not demonstrably reversible by hydroxylamine since this reagent itself inactivated the D antigen. Exposure of red cells to 5 mM p BPB resulted in a 50% decrease in binding of 125I-anti-D IgG. Photo-oxidation of RBC in the presence of Rose Bengal apparently inactivated all the major Rh antigens as detected either by labeled anti-D IgG binding, IgG agglutinating serological reagents, or the binding of 125I-labeled protein A following the sensitization of cells with unlabeled antisera. Under conditions of RB treatment, where hemolysis was absent or minimal, 125I anti-D IgG binding decreased to 38-49% of the level seen in controls. Rose Bengal treatment of R1r RBC revealed varying inactivation of all the Rh antigens, i.e. D 15%, C 89%, c 73%, e 54% inactivated, whereas antibody binding activity of the Fya and Fyb antigens present in the same cell was unaffected. Previous reports as well as the pH profile of anti-D binding have implicated the participation of histidine in Rh antigen expression. Our results are consistent with histidine involvement in Rh activity. Whether Rh antigens have essential histidine(s) involved directly in epitope structure, or instead depend on a critical histidine(s) at the lipid-protein interface that modulates antigen expression remains to be determined.     

IgG subclass distribution of anti-Rh,anti-Kell and anti-Duffy antibodies measured by sensitive haemagglutination assays.

The IgG subclass distribution of anti Rh antibodies (anti-D, ''anti-Du'', anti-c, anti-E), anti-Kell and anti-Duffy (anti-Fya) antibodies was measured by two haemagglutination techniques on microtitre plates. The first technique involved rabbit subclass specific antisera which were used to agglutinate red cells previously reacted with the patients'' antibodies at high concentration. The second, which was more sensitive, had an additional step by introducing sheep anti-rabbit antibodies (sandwich technique). By the sensitive sandwich technique we revealed, for anti-D antibodies: IgG1 8/19, IgG3 1/19, IgG1/IgG3 8/19, IgG1/IgG2/IgG3/IgG 41/19, IgG1/IgG4 1/19; for the Du reactive anti-D antibodies: IgG1 1/8, IgG1/IgG3 1/8, IgG1/IgG3/IgG4 6/8; for the anti-E antibodies: IgG1/IgG2/IgG4 2/3, IgG1/IgG2/IgG3/IgG4 1/3; for the anti-c antibodies: IgG1 2/5, IgG3 1/5, IgG1/IgG3 1/5; for the anti-Kell antibodies: IgG1 9/20, IgG1/IgG3 1/20, IgG1/IgG4 8/20, IgG1/IgG3/IgG4 2/20; and for anti-Duffy antibodies: IgG1 1/8, IgG1/IgG4 7/8. These results are partly at variance with previously published results.     

On a much higher than reported incidence of anti-c in R1R1 patients with anti-E

A previous study involving tube IATs, untreated RBCs, and a low ionic-strength additive reagent revealed that approximately one-third of R(1)R(1) patients with anti-E have a concomitant anti-c. However, the current study finds a much higher incidence of anti-c in such patients, using gel technology in conjunction with ficin-pretreated RBCs. Results of antibody identification studies and transfusion records of 82 R(1)R(1) patients with anti-E were reviewed. Serologic test methods included a LISS wash solution for tube IATs (15 min at 37 degrees C, anti-IgG), ficin-tube IATs (30 min at 37 degrees C, anti-IgG + anti-C3), and gel IATs (untreated or ficin-treated RBCs or both, anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealed anti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreated RBCs were used, 21 additional patients with anti-E were found to have anti-c. In samples from 26 R(1)R(1) patients with anti-E, anti-c was not demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel tests were inconclusive. In five cases in which E- RBCs not tested for c antigen were transfused to patients found by ficin-gel IAT to be without anti-c, all subsequently performed crossmatches with E-, c-untested RBCs were compatible. The incidence of anti-c in R(1)R(1) patients with anti-E in this study was 32 of 82 (39%) with untreated RBCs and 53 of 82 (65%) when the ficin gel data were included. The latter is significantly higher than the 32 percent incidence previously reported (p = 0.0001). Accordingly, all patients at our facility with an Rh antibody are now tested for those additional Rh antibodies they can make, as predicted from their Rh phenotype. The data from this study strongly support the selection of R(1)R(1) RBCs for all c- patients with anti-E.