The quantification of C3 fragments on erythrocytes: estimation of C3 fragments on normal cells, acquired haemolytic anaemia cases and correlation with agglutination of sensitized cells

A sensitive method is described for the quantification of C3 fragments on erythrocytes. A radiolabelled monoclonal antibody, was used which was directed against a C3d determinant on all forms of cell bound C3. The number of C3 molecules on normal erythrocytes was estimated to be 420 +/- 140. The strength of the antiglobulin test increased from negative to 5+ over a range of only 850 C3 molecules (400-1250). A blood donor with a positive direct antiglobulin test was found to have 4800 molecules per cell whereas three cases of cold haemagglutinin disease with active haemolysis had from 16 000 to 52 020 C3 molecules per cell. This test has an application in the testing of acquired haemolytic anaemia cases with a positive direct antiglobulin test with C3 bound to the cells and in the standardization of sensitized cells used for testing antiglobulin reagents by various serological techniques.     

Autoimmune Hemolytic Anemia Associated with Autoanti-Ge

The first reported example of autoimmune hemolytic anemia due to an autoanti-Gerbich is described. The patient's red blood cells exhibited a strongly positive direct antiglobulin test with both IgG and complement antiglobulin reagents. The serum contained a potent antibody which produced agglutination of red blood cells as well as a positive indirect antiglobulin test. Treatment of the serum with 2-mercaptoethanol demonstrated that the antibody contained both IgG and IgM components. The serum antibody and the antibody eluted from the patient's red blood cells had anti-Gerbich specificity. The patient's cells typed as Gerbich-positive with saline-agglutinating anti-Gerbich sera. Of great interest was the fact that the patient's mother also has acquired immune hemolytic anemia, but the IgG antibody in her serum and eluted from her red blood cells had anti-pdl specificity.     

Functional characteristics of human T-cell subpopulations distinguished by a monoclonal antibody

In animals and in man, diverse immunologic functions are mediated by specialized T-cell (thymus-derived lymphocyte) subsets that are distinguishable from one another on the basis of differences in cell surface determinants. Unfortunately, in humans, subset-specific antibodies have been difficult to generate. In this study, production of a murine monoclonal antibody specific for a subset of human T cells was achieved by fusing a sensitized B cell (bone marrow-derived cell) with a myeloma cell and isolating the antibody secreted by the resultant hybrid clone. This antibody binds 30-35% of peripheral T lymphocytes (T_a⁺ cells) but fails to bind remaining T lymphocytes (T_a^- cells), B lymphocytes, or monocytes. T_a⁺ and T_a^- subpopulations were separated with a fluorescence-activated cell sorter and their in vitro responses to various stimuli were assessed. T_a⁺ and T_a^- cells respond equally well to soluble antigens, allogeneic B cells, and autologous B cells, but only T_a⁺ cells respond to concanavalin A. T_a⁺ cells cultured in the presence of concanavalin A gradually lose the T_a marker, an effect not observed after stimulation with phytohemagglutinin, soluble antigens, or alloantigens. These results suggest that the functional subpopulation of T cells defined by T_a does not correspond to any previously described human T cell subset. Furthermore, somatic cell hybridization has been shown to be a feasible method for production of monoclonal antibodies specific for subpopulations of human lymphocytes.     

Complement-Binding Anti-Jka Not Detectable by DiaMed Gels

Background: An anti-Jk^a was not detectable by the DiaMed-ID Micro Typing System but was strongly reactive when tested using other indirect antiglobulin test (IAT) methods. However, it was detectable in the DiaMed-ID system when the recommended diluent ID-Diluent 2, was replaced by other low ionic Strength saline (LISS) solutions. This led us to believe that the problem lay with the ID-Diluent 2. Methods: The anti-Jk^a was investigated using various low ionic strength diluents in the IAT against both polyspecific and monospecific antiglobulin reagents. We tested the ID-Diluent 2 for its ability to inhibit complement activation, and bind calcium. Results: The anti-Jk^a was shown to be a ‘complement-only’ binding antibody that reacted in all IATs performed except when tested with ID-Diluent 2. The ID-Diluent was shown to prevent haemolysis of ABO lytic antibodies, and further testing shows that it binds calcium. Conclusion: The use of a diluent which prevents haemolysis and binds calcium in an IAT system that contains anti-complement as well as anti-IgG must be questioned.     

Selection of Monoclonal Antibodies for the Identification of D Variants: Ability to Detect Weak D and to Split epD2, epD5 and epD6/7

Red cells from known D variant donors were tested with 41 monoclonal anti-D reagents, 26 IgG and 15 IgM, with the view to selecting a panel to aid the identification of unusual D types. These antibodies gave reaction patterns which allowed the identification of most of the known D category cells, recognizing epD2, epD5, epD6/7, epD8 and epD9, but were unable to distinguish category III from normal D-positive cells. Reactivity with HMi, HMii, DFR, DBT and R_o^Har cells split epD2, epD5 and epD6/7 into two, three and eight groups, respectively. A panel comprising 15 monoclonal anti-D, 11 IgG and four IgM, was selected as representative of the antibodies tested. Reactivity of monoclonal anti-D was dependent on antibody concentration and antibody avidity. An antibody concentration of at least 12 pg/ml was required for optimum reactivity of the two monoclonal antibodies tested. A simple calculation of division of the titre by the antibody concentration provided a relatively simple means of establishing the reactivity performance of the antibody and correlated well with ability to detect weak D (D^u) cells. A characteristic variable reduction in reaction strength with all the IgG anti-D was observed with weak D cells. The IgM antibodies, except the high avidity RUM-1, T3D2T6, D9A4 and BS226, performed poorly in detecting weak D. The majority of the IgM antibodies tested reacted with R_o^Harr cells, while only one IgG antibody was positive.     

Receptor Sites on Human Monocytes for Complement: BINDING OF RED CELLS SENSITIZED BY COLD AUTOANTIBODIES

S ummary Receptor sites on isolated human monocytes were assessed by the demonstration of rosette formation by sensitized red cells around monocytes. This type of binding to monocytes occurred using red cells sensitized with a typical monoclonal IgM cold agglutinin but (in contrast to IgG antibodies) only if the red cells were also coated with complement (C) components. Prior treatment of monocytes with trypsin (1 mg/ml) inhibited the detection of the C receptor but not the detection of the IgG receptor. Although this reaction resembled the phenomenon of immune adherence to some extent, it required the presence of Mg²⁺ ions and was inhibited by EDTA. The effects of temperature variation on C-dependent rosette formation and on haemolysis were comparable. The binding of sensitized erythrocytes to monocytes, however, required significantly less antibody than for haemolysis. C-coated red cells remained reactive with mononuclear phagocytes after elution of the cold antibody as well as under conditions when C was bound to red cells without antibody.     

Delayed hemolytic transfusion reaction due to anti-Goa,an antibody against the low-prevalence Gonzales antigen

Go^a (D^Cor) is a low-frequency antigen in the Rh system found on red cells lacking part of the D mosaic (category IVa). Anti-Go^a has not been previously reported to cause hemolytic transfusion reactions. A 27-year-old African American male with sickle-cell disease, maintained on chronic transfusion, was noted to have dark plasma during an erythrocytapheresis, procedure, and the pretransfusion hemoglobin was noted to be 1 g/dl lower than 4 weeks before (with hyperbilirubinemia and a significantly increased LDH). Polyspecific direct antiglobulin test (DAT) was weakly positive (C3-weak, IgG-weak), and indirect antiglobulin tests (IATs) performed on the serum (pre- and posttransfusion reaction) and a red blood cell (RBC) eluate from the postreaction sample were negative. A segment from one of the four implicated units from the prior month's transfusion was strongly reactive at 37°C and using anti-human globulin (AHG) when crossmatched with the postreaction serum and the eluate. The postreaction serum, screened with a panel of red cells positive for low-prevalence antigens, reacted with three Go(a+) cells. The implicated unit was reactive with a previously identified anti-Go^a serum. © 1996 Wiley-Liss, Inc.     

Antiidiotypic IgG crossreactive with Rh alloantibodies in red cell autoimmunity

IgG autoantibodies eluted from RBCs of antiglobulin positive normal blood donors contained at least two antibody populations, an IgG autoantibody (Ab 1), and an IgG population (Ab 2) that agglutinated RBCs coated with some Rh(D) alloantibodies. Eight of 24 autoantibody eluates tested agglutinated 3 of 10 anti-Rh(D) sensitized RBCs. The agglutinating activity was inhibited specifically by preincubation of the autoantibody eluate with the reactive anti-D. The reaction did not require the Fc domain of the anti-Rh(D), since autoantibody eluates agglutinated RBCs coated with F(ab')2 prepared from the reactive anti-D sera. These findings indicate that the RBCs of some antiglobulin- positive blood donors contain an immunoglobulin auto-antiidiotype (Ab 2) against the RBC autoantibody (Ab 1) which is demonstrable through its cross-reactivity with selected Rh(D) alloantibodies. Identification of auto-antiidiotypes in RBC autoimmunity lends support to the idiotype- antiidiotype network hypothesis of immune regulation and is consistent with the bizarre and complex serology of autoimmune hemolytic anemia. The absence of clinical hemolysis in antiglobulin-positive normal blood donors suggests that immunoglobulin idiotype-antiidiotype interactions may play a role in modulating the effects of RBC autoimmunity.     

Destruction of IgG-Sensitized Erythrocytes by Human Blood Monocytes: Modulation of Inhibition by IgG

The in vitro interaction between monocytes and erythrocytes sensitized with non-complement binding IgG antibodies (i.e. the Rh antibody anti-D:EAIgG anti-D) is completely inhibited by low concentrations of IgG (e.g. 30–100 μg/ml). However, the interaction between monocytes and erythrocytes sensitized with IgG anti-A (EAIgG anti-A) is not inhibited by IgG. The findings presented in this paper indicate that this difference is probably due to the difference in the number of IgG antibody molecules per EAIgG. Thus, the higher the number of IgG antibody molecules per EAIgG, the less the interaction between EAIgG and monocytes is inhibited by IgG.
A second factor which proved to have a strong influence on inhibition by IgG was the number of EAIgG per monocyte. When the number of EAIgG per monocyte was increased from 1 to 32, the percentage of inhibition by a fixed amount of IgG (50 μg/ml) decreased significantly. This in vitro effect is only evident when relatively weakly sensitized erythrocytes are used and, in vivo , destruction of these weakly sensitized red cells (e.g. EAIgG anti-D) is confined to the spleen. Since a considerable haemoconcentration occurs in this organ, it is conceivable that a high EAIgG:macrophage ratio is accomplished. The latter data are an indication that this high ratio may allow interaction between weakly sensitized erythrocytes and splenic macrophages despite the presence, in vivo , of a high concentration of IgG, and that, in this way, in the spleen, the inhibitory effect of IgG is overcome.     

An Example of a Naturally Occurring Anti-cE (Rh27) that Binds Complement

A serum sample from a nontransfused male containing anti-Rh27 and an additional weakly reacting antibody was investigated. One absorption of the serum with RZR1 cells left only anti-Rh27 while repeated absorption with R1R1 cells or autologous cells had no effect. Treatment of the serum with reducing agents destroyed all activity suggesting the antibody was predominantly IgM. Tests with monospecific antiglobulin reagents in the indirect antiglobulin test revealed very weak reactions with anti-IgM and anti-IgA, no reactions with anti-IgG, and strong reactions with anti-C3(C3c + C3d) and anti-C4. This is the first reported example of a naturally occurring complement binding anti-Rh27.     

Significance of multiple types of antibodies on red blood cells of patients with positive direct antiglobulin test: a study of monospecific antiglobulin reactions in 85 patients

Blood samples from 85 patients with a positive direct antiglobulin test were tested with monospecific antiglobulin reagents: anti-IgG, anti-IgM, anti-IgA, and anti-C3. No typical pattern of antiglobulin reaction could be correlated with specific diseases except for the patients with methyldopa-induced positive direct antiglobulin test, all of whom had only IgG on their red blood cells. The presence of more than 1 type of antibody on red blood cells was associated with severe haemolysis. These patients responded less frequently to steroids, and in most of them no underlying disease could be found. Most patients with complement alone on red blood cells had no evidence of haemolysis, and when present it was never severe.     

Immunochemical Characterization of the Rh Cw Antigen Using Human Monoclonal Antibodies

Background and objectives: Human monoclonal antibodies have been produced against various Rh antigens. We report here the production of another one against Rh C and the first such antibody against Rh C^w. Materials and methods: Two heterohybridomas secreting IgG human monoclonal antibodies against Rh C^w and Rh C (MS-353 and MS-242 respectively) were produced from alloimmunized donors. Their specificity was confirmed by serological testing. Results: MS-353 reacted with all C^w-positive phenotypes but not with any C^w-negative phenotypes, whereas MS-242 reacted with all C^w-positive and C-positive phenotypes. Using ¹²⁵I-labelled antibodies, the average number of C^w sites/red cell was 32,000 (R₁^wR₁^w), 15,200 (R₁^wR₁), 19,800 (R₁^wR₂), 15,300 (R₁^wr), 26,200 (r′^wr), and the average number of C sites per red cell on equivalent C_w/C-positive phenotypes was similar: 22,400 (R₁R₁), 21,800 (R₁^wR₁), 12,300 (R₁R₂), 11,700 (R₁^wR₂), 13,400 (R₁r), 15,100 (R₁^wr), 21,100 (r′r), 18,700 (r′^wr). MS-353 and MS-242 were mutually inhibitory. Both antibodies specifically immunoprecipitated a 33- to 34-kD polypeptide from ¹²⁵I-surface-labelled cells. Conclusion: MS-353 is suitable for the serological detection of C^w-positive cells, and the immunochemical data are entirely consistent with the report that the C^w antigen is associated with a point mutation in the RHCE gene [Blood, 1995;86:1196–1201].     

Human lgG antigranulocyte antibodies: Comparison of detection by quantitative antiglobulin consumption and by binding of 125I staph protein A

The amount of IgG in the serum of patients with suspected immune neutropenia that binds to normal paraformaldehyde-fixed human granulocytes was measured simultaneously by a quantitative antiglobulin consumption assay and by binding of ¹²⁵I-staphylococcal protein A (SPA). There was a significant linear relationship between the results of these two assays for the sera of 42 different patients. However, SPA binding appeared more sensitive than the quantitative antiglobulin assay for determining IgG antigranulocyte antibodies in serum. In a patient with Felty's syndrome who underwent splenectomy, the results of both assays on sequential serum samples correlated with clinical improvement. Thus, SPA binding appears to be a sensitive and reliable technique for measuring antigranulocyte antibodies, and there is a close correlation between antibody measured by antiglobulin consumption and those detected by SPA binding.     

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

Summary When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group 0-test red cell panels at 37°C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.     

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr₂s₂) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r₂s₂). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r₂s₂ proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r₂s₂ contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r₂s₂. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

Antibodies are important mediators of the human complement response, which offers critical protection against microbial infections and damaged host cells (1). In order to initiate a complement response, an antibody molecule first needs to bind antigens on the target cell via its antigen-binding (Fab) domains (2–5). Subsequently, the antibody’s constant (Fc) domain recruits the first complement protein complex, C1, to the cell surface (SI Appendix, Fig. S1A). The large C1 complex (also denoted as C1qr₂s₂, 766 kDa) consists of the recognition protein C1q (410 kDa) and a heterotetramer of serine proteases C1r and C1s (denoted C1r₂s₂, 356 kDa) (SI Appendix, Fig. S1B). While C1q is responsible for antibody recognition, its attached proteases C1r₂s₂ induce the activation of downstream enzymatic complexes (i.e., C3 convertases [C4b2b (6)]) that catalyze the covalent deposition of C3-derived molecules (e.g., C3b and its degradation product iC3b) onto the target cell surface (SI Appendix, Fig. S1A) (7, 8). C3b opsonizes the target cell surface and can induce formation of lytic pores (membrane attack complex [MAC]) in the target cell membrane (9–11). In contrast to human cells and gram-negative bacteria, gram-positive bacteria are not susceptible to the MAC due to their thick cell wall (12). On these bacteria, efficient decoration with C3b and iC3b is essential for triggering effective phagocytic uptake of target cells via complement receptors (CR) expressed on phagocytes of which the integrin CR3 (also denoted CD11b/CD18) is considered most important (13, 14).In recent years, our insights into IgG-dependent complement activation have increased significantly. A combination of structural, biophysical, and functional studies revealed that surface-bound IgG molecules (after Fab-mediated antigen binding) require organization into higher-ordered structures, namely hexamers, to induce complement activation most effectively (15–19). Hexamerized IgGs are being held together by noncovalent Fc–Fc interactions and form an optimal platform for C1q docking (SI Appendix, Fig. S1A). C1q has a “bunch of tulips–” like structure, consisting of six collagen arms that each end in a globular (gC1q) domain (SI Appendix, Fig. S1B) that binds the Fc region of an IgG. As the affinity of C1q for a single IgG is very weak (20, 21), avidity achieved through simultaneous binding of its globular domains to six oligomerized IgG molecules is paramount for a strong response (15, 17–19). Furthermore, it was found that IgG hexamerization could be manipulated by specific point mutations in the Fc–Fc contact region that enhance such oligomerization (15, 18, 22). While these hexamer-enhancing mutations in IgG potentiate the efficacy of MAC-dependent cytotoxicity on tumor cells and gram-negative bacteria (15, 23), their effect on complement-dependent phagocytosis is not known.Because complement is an important effector mechanism to kill bacteria and tumor cells, development of complement-enhancing antibodies represents an attractive strategy for immune therapies (1, 24). Immunotherapy based on human monoclonal antibodies is not yet available for bacterial infections (25–28). Such developments are mainly hampered by the fact that little is known about the basic mechanisms of complement activation on bacterial cells. For instance, we do not understand why certain antibodies induce complement activation on bacteria and others do not. In this study, we set out to investigate how antibacterial IgGs induce an effective complement response. By surprise, we noticed that C1q–IgG stability differs between human IgG subclasses. More detailed molecular investigations revealed that C1r₂s₂ proteases are important for generating stable C1q–IgG complexes on various target surfaces. Furthermore, we demonstrate that C1q–IgG stability is influenced by antibody oligomerization. These molecular insights into C1q binding to surface-bound IgGs may pave the way for optimal design of antibody therapies.     

Historical Note: Past, Present and Future of the Antiglobulin Test

This is a review of two invited talks on the antiglobulin reaction on the occasion of the 50th Anniversaries of the International Blood Group Reference Laboratory, now at Bristol, and the National Blood Service, London, and coincidentally the 51st anniversary of the antiglobulin test! The first talk (as specially requested) is a very personal reminiscence of the discovery, with Rob Race and Arthur Mourant, of the antiglobulin test in blood group serology. The second talk traces developments in antiglobulin testing in general immunology using, of course, isotype-specific antiglobulin reagents as and when they became available. Special emphasis was given to: (1) testing for poorly agglutinating bacterial antibodies; (2) special procedures for measuring reaginic (IgE) antibodies to various allergens; (3) the incorporation of an antiglobulin step or stage in many of the routine automated immunoassay procedures; (4) the special experimental procedures in the form of mixed antiglobulin reactions to reveal antibodies to the surface antigens on tissue cells, and, finally, (5) by chemically coupling antiglobulin to red blood cells, a distinctive integrated immunoassay system was developed, enabling reverse passive haemagglutination as an assay for immunoglobulins, rosetting reactions to reveal native IgG receptors on lymphocytes or antibody immunoglobulin reacting with CD marker antigens; this same reagent, namely red-cell-labelled antiglobulin, can be used to detect antibodies reacting with either bacteria or antigens adsorbed on the walls of wells of microtitre plates. The need for improved stabilization and preservation of the antiglobulin-linked red cells to prolong their ‘shelf-life’ is stressed.     

A new assay for IgG rheumatoid factor activity and its use to analyse rheumatoid factor reactivity with human IgG isotypes

Summary A new assay for IgGRF activity is described which employs human IgGFc as the antigen and a radiolabelled monoclonal antibody directed against human IgG (C_HI domain) as the developing antibody. Using this assay IgGRF activity against human IgG isotypes was measured and most sera from RA patients were shown to react predominantly with IgG₁ and IgG₂ but few reacted with IgG₃ and IgG₄. The same sera were tested for IgMRF to the IgG isotypes. IgG₂ was the best antigen, IgG₁ and IgG₄ were less so and reactivity with IgG₃ was the lowest. IgGRF without associated IgMRF was obtained, its specificity compared to that of IgMRF, and found to be broadly similar. With the new assay high levels of serum IgGRF were found in those RA patients with extraarticular disease but not in RA patients with synovitis alone.     

Role of anti-Naka antibody,monocytes and platelets in the development of transfusion-related acute lung injury

Background and Objectives Transfusion-related acute lung injury (TRALI) is one of the most serious side-effects of transfusion. We report here the first two cases of TRALI caused by anti-Nak^a (anti-CD36) antibody from a single blood donor. The aim of this study was to clarify the role of the anti-Nak^a antibody in TRALI development. Materials and Methods Human lung microvascular endothelial cells were co-cultured with Nak^a-positive monocytes and Nak^a-positive platelets together with serum prepared from blood products of a TRALI-caused anti-Nak^a antibody-carrying donor. Expressions of leukotriene B₄ (LTB₄) and tumour necrosis factor α (TNF-α) in the co-culture supernatants were determined. Results The expressions of LTB₄ and TNF-α were found to be markedly increased, particularly in the presence of both Nak^a-positive monocytes and platelets. The expressions of these mediators were almost completed within 4 h after the initiation of co-culture. Monocyte contribution seemed to be stronger than that of platelets. In the absence of human lung microvascular endothelial, no significant LTB₄ or TNF-α release was observed. Conclusion Anti-Nak^a antibody may be strongly implicated in lung microvascular endothelial dysfunctions that lead to TRALI in a monocyte- and platelet-dependent manner.     

Fc Receptors for IgG1 and IgG3 on Human Mononudear Cells -An Evaluation with Known Levels of Erythrocyte-Bound IgG

The Fc receptors on mononuclear cells were investigated by a rosette technique in which human erythrocytes were sensitized with a known number of molecules of anti-Rh antibodies (IgG1 or IgG3). The number of IgG molecules was quantitated by a radiometric antiglobulin test. The present quantitative evaluation reveals that (1) there is a logarithmic relationship between the proportion of rosettes and the amount of erythrocyte-bound immunoglobulin for both types of mononuclear cells, and for both subclasses; (2) similar percentage of rosettes can be obtained with fewer IgG3 than IgG1 molecules (about 1:4); (3) for a given number of erythrocyte-bound immunoglobulins a higher percentage of rosettes is observed with monocytes than with lymphocytes (ratios of about 3:1 for IgG1 and 5:1 for IgG3); (4) the minimum number of IgG3 molecules for adherence is 180-460 for monocytes, 520-1,300 for lymphocytes, whilst for IgG1 the numbers are 1,180-4,300 for monocytes and 3,400-14,200 for lymphocytes; (5) the minimum levels of sensitization by alloantibodies for adherence should be detectable by the antiglobulin test.     

Dipyrone-Induced Immune Haemolytic Anaemia

Abstract. We report a new case of immune haemolytic anaemia induced by dipyrone. The haemolytic crisis was followed by acute renal failure. The direct antiglobulin test on the red cells was positive. The responsible antibody was of the IgG class; it was complement binding and produced haemolysis in vitro. The clinical and serological findings suggest that the red cells became sensitized as the result of the adsorption to the cell membrane of drug-antibody immune complexes.