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The effect of high-dose intravenous gammaglobulin (IVG) therapy with a CLB preparation was studied in 42 patients: 8 patients had acute and 26 patients had chronic idiopathic thrombocytopenic purpura (ITP); 5 patients had thrombocytopenia accompanied by various diseases such as systemic lupus erythematosus, auto-immune haemolytic anaemia and neutropenia; 3 patients had hypoplastic anaemia and 1 patient had neutropenia and rheumatoid arthritis. After treatment, a rise in platelet count occurred in about 75% of the patients with ITP, although there was no sustained response in any of the patients. There was no correlation between the strength of platelet antibodies as detected by the direct immunofluorescence test before infusion and the pattern of response to the infusion. In most cases of ITP, no immune complexes, as measured by Clq-binding assay, were observed. Furthermore, we found no relationship between the amount of Clq-binding activity of patients' sera and the reaction pattern after infusion of IVG. Splenectomy of the patient had no influence on the outcome of IVG therapy.

The immunofluorescence test on paraformaldehyde-fixed cells was used for the detection of antibodies bound to the platelets and granulocytes and present in the sera of 24 patients with Evans syndrome and a further 29 patients with both idiopathic thrombocytopenia (ITP) and idiopathic neutropenia (INP), but without autoimmune haemolytic anaemia (AIHA). The direct immunofluorescence test on platelets and/or on granulocytes was positive in all patients with a cytopenia, but the sera of only 17 patients with the Evans syndrome and 15 of the other patients contained platelet- or granulocyte-specific autoantibodies. From absorption and elution experiments, it appeared that the autoantibodies were directed against antigens specific for the various peripheral blood cells, i.e. erythrocytes, platelets and granulocytes and that they were not cross-reacting.

A patient is described who, notwithstanding a strongly positive direct antiglobulin test with anti-IgG serum, apparently did not suffer from haemolytic anaemia. The survival of the patient's red cells, measured with 51Cr, was only slightly decreased. In vitro, the sensitized cells of the patient showed only a minimal tendency to adhere to monocytes. The patient's spleen functioned normally, since 51Cr-labelled donor erythrocytes, either sensitized with IgG-anti-D or damaged by heating, were eliminated rapidly from the circulation and sequestered in the spleen. These apparently contradictory findings could be explained by the fact that the patient's red cells were sensitized with autoantibodies, mainly belonging to the IgG4 subclass. Only weak IgG1 and IgG3 autoantibodies were detectable. Since previously the patient had suffered from a severe haemolytic anaemia, it is postulated that a switch has occurred from 'active' to 'inactive' IgG autoantibodies, perhaps induced by prednisone therapy.

SUMMARY An analysis of the HLA data of 100 myasthenia gravis (MG) patients and their first degree relatives and spouses is presented. The family studies confirmed the excess of B8 in the myasthenia gravis patients and showed that the excess was not limited to the A1, B8 haplotype. An excess of B8 homozygous patients was found from the matings of two B8 heterozygous parents, suggesting the existence of a dominant B8 linked MG susceptibility. B8 homozygous individuals run a 4.7 times higher risk of acquiring myasthenia gravis than B8 heterozygous individuals.

Monocytes and macrophages are capable of a number of effector functions such as phagocytosis, killing of bacteria, and cyto-toxicity. Of these effector functions, the mechanism of cyto-toxicity has been the least studied.

Replicate blind AHG tests with weak IgG anti-D sensitised red cells revealed that 32% of workers caused 5 per cent or more false negative errors by using excessive agitation in reading techniques. The common quality control procedure of adding strongly sensitised cells to all negative AHG tests cannot reveal this type of error. Furthermore, strongly sensitised control cells may create an illusion of safety because AHG giving a false negative test with weak antibody in a serum sample may still show a reassuringly strong positive in the control test. "In-house" assessment of all staff and automatic cell-washers by blind replicate tests is recommended as an effective way of improving AHG test performance, thus reducing many of the errors involving false negative AHG tests seen year after year in External Proficiency Trials.

Summary We studied the lysis in vitro of group A red cells by IgG anti-A. IgG anti-A, which strongly lysed A red cells from adults, did not lyse A red cells from cord blood, if fresh cord serum from a child with blood group AB was used as a source of complement. In cases of haemolytic disease of the newborn due to A-O or B-O antagonism with a positive direct antiglobulin test with anti-IgG serum, the red cells did not react with anti-complement sera. Apparently, complement is also not activated in vivo in case of A-O haemolytic disease of the newborn.

1,820 units of leukocyte- and platelet-poor erythrocyte suspensions were prepared by filtration through cotton wool. On the average more than 98% of the leukocytes and 90-95% of the platelets could be removed. The red cell recovery was 96%. 97% of the units given to polytransfused patients did not cause febrile reactions. Serological follow-up of future transplantation recipients indicated that the immunization may be avoided by using erythrocyte suspensions of fresh, filtered blood.

Publisher Summary This chapter reviews the role of Fc-receptor (FcR)-possessing cells on the destruction of human erythrocytes (E) sensitized with iso- and auto-antibodies of the IgG class. It focuses on the mechanism of monocyte-FcR-mediated lysis (MFL) and phagocytosis of E sensitized with IgG iso-antibodies anti-D (D-anti-D system). The chapter describes an experiment in which preparation of monocytes from the peripheral blood of healthy human volunteers, and Cr-labeling and sensitizing of E were done by standard methods. Monocytes and E were incubated at a ratio of 1:1 for 16–20 hours at 37°C. Erythrocytolysis was measured as percentage 51 Cr release into the supernatant. Subsequently, erythrophagocytosis was determined by counting the radioactivity remaining in the monocyte pellet after water lysis. Concerning the mechanism of MFL, it is assumed that lysosomal enzyme release (LER) is an important feature of this process. For this reason, experiments were performed with cytochalasin B (CB) that has the ability, among other things, to enhance LER specifically. It is logical to perform experiments with an inhibitor of LER, and hence, colchicine was used for this experiment. At a molarity of 10 -2 , colchicine completely blocked MFL and CB effect on MFL. At 10 -3 M colchicines, there was partial restoration of MFL and the CB effect. However, full restoration of MFL and the CB effect on MFL occurred only at 10 -4 M colchicine. Remarkably, phagocytosis still inhibited 75% at the concentration of colchicine. This study concludes that there exists heterogeneity in FcR avidity among the monocytes of an individual. Only monocytes with a high FcR avidity are active in MFL,and MFL is dependent upon LER but is independent from phagocytosis.

The purpose of this study was to characterize the destruction of sensitized erythrocytes by human blood monocytes in vitro. The incubation in vitro of human monocytes with 51Cr-labelled human erythrocytes sensitized with IgG rhesus alloantibodies anti-D (EAIgG anti-D) resulted in release of 51Cr from the erythrocytes (lysis) as well as uptake of 51Cr-labelled erythrocytes by the monocytes (phagocytosis). The lysis of EAIgG anti-D by monocytes was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocortisone and colchicine inhibited lysis, but had no effect on phagocytosis. These agents did not affect binding of EAIgG anti-D to monocytes. The effect of these agents on lysis corresponded to their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was, likewise, enhanced by cytochalasin B and inhibited by hydrocortisone and colchicine. A significant correlation was found between lysosomal enzyme release and lysis. Together, these results strongly suggest that lysosomal enzymes, released by the monocytes when incubated with anti-D-sensitized erythrocytes, are responsible for the cytotoxic activity of these cells towards sensitized erythrocytes. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erythrocyte, because only erythrocytes that were bound to the monocytes were lysed. The finding of other investigators that removal of monocytes from suspensions of human mononuclear leucocytes results in a strong reduction in the cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro. was confirmed.

Monoclonal antibodies (McAbs) were produced against the IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1). Four out of the six McAbs described in this paper showed in the haemagglutination assay cross-reactivity with some or all IgG-coated cell samples. In the haemagglutination inhibition assay, all six McAbs are useful as typing reagent for the above allotypes. In this assay, two of the McAbs show two different specificities, which depend on the Ig-coated cell sample used. Five McAbs are useful for allotyping in a capture ELISA. The results with four of these are promising for the development of a quantitative determination of Gm allotypes.

The neutrophil-specific-NA antigen system is a biallelic system. It comprises the antigens NA1 and NA2, which in Caucasian populations show a phenotype frequency of 46% and 88%, respectively (1). It is clinically an important system because it may be involved in diseases such as neonatal alloimmune neutropenia and autoimmune neutropenia as well as in blood transfusion reactions such as rigors, fever, and respiratory distress (1,2).

There are 3 varieties of idiopathic thrombocytopenic purpura (ITP) in children: the acute, the chronic and the recurrent forms. We have tested the platelets from 80 children with these forms of ITP by an immunofluorescence test. Platelet-bound immunoglobulins were detected in 19/22 acute, 41/48 chronic and 5/10 recurrent cases of ITP, i.e. in 65 of a total of 80 cases. A significance differences between acute and chronic ITP was found in the observation that only IgM was bound to the platelets of 7/16 patients with acute ITP. In all cases of chronic ITP with a positive direct immunofluorescence test, IgG was present on the platelet membrane. It is assumed because some patients with chronic ITP do not have elavated platelet-bound immunoglobulins, and show refractoriness to immunosuppressive therapy, that in these patients the disorder is not of a humoral autoimmune nature.

In view of the importance of the species of animals used fat the preparation of specific heteroantisera against human blood cells, comparative immunization experiments Were carried out in goats, rabbit, chickens, guinea pigs, and rats, using lysates leukocytes, granulocytes, lymphocytes, monocytes, and thrombocytes. The antisera obtained were tested by the indirect immunofluorescence technique. The strongest antisera were obtained in goats, but these antisera required extensive absorptions to make them specific for the immunizing cell, whereas rabbit antisera needed only few absorptions. Antisera from both of these animals could be rendered specific for lymphocytes, granulocytes and platelets. In the guinea pig only immunization with lymphocytes and granulocytes resulted in useful antisera, which were all specific for granulocytes after appropriate absorptions. Antisera obtained from rats could be rendered specific for granulocytes and lymphocytes by absorption. Antibodies against platelets could not be detected in any of the rat antisera. Chickens, however, produced strong anti-platelet anti-bodies. A specific anti-granulocyte antiserum could also be obtained in these animals. Specific anti-monocyte antisera could not be prepared from any of the animal sera. Immunizations with leukocyte lysate resulted in granulocyte-specific antisera in most animals but not in chickens. All antisera produced in chickens were specific for platelets. It can be concluded from these results that animals of different species may react very differently to the antigens of some human blood cells.

Sera of 22 patients with Felty's syndrome and 14 patients with rheumatoid arthritis were tested in assays routinely used for the detection of neutrophil antibodies (i.e. immunofluorescence, agglutination and cytotoxicity tests) as well as in the antibody-dependent lymphocyte-mediated granulocytotoxicity test. One or more of the routinely used assays were positive in a high percentage of the sera (77% and 64%, respectively). The antibody-dependent lymphocyte-mediated granulocytotoxicity test was only positive with sera of three patients with Felty's syndrome. Positive results in the immunofluorescence and agglutination tests could be attributed to the presence of immune complexes in the sera, whereas positive antibody-dependent lymphocyte-mediated granulocytotoxicity tests were probably due to the presence of HLA alloantibodies. It is concluded that serological tests routinely used for the detection of neutrophil autoantibodies should be interpreted with caution in patients with Felty's syndrome. Our results also indicate that the neutropenia in Felty's syndrome is rarely, if ever, due to neutrophil-specific autoantibodies.

By slowly lowering the surface tension of the aqueous medium through the admixture of 47.5% dimethyl sulfoxide (DMSO) and 0.1% bovine serum albumin (BSA) and by raising the pH to 9, complete elution of A, D, and K antibodies from sensitized erythrocytes (RBC) could be achieved. The eluates comprising the antibodies were subjected to dialysis to remove the DMSO and to neutralize the pH, and to ultrafiltration to remove the excess water. Recovery of a sizeable proportion of the eluted RBC (of blood groups A and K) proved possible by slow and careful removal of the DMSO with phosphate-buffered saline containing 2% BSA.

The HLA-Cw1 antigen was well defined in 1970 at the Fourth International Histocompatibility Workshop [1].

An indirect immunofluorescence technique for the detection of alloantibodies against granulocytes was developed. A major problem was the unspecific fluorescence of granulocytes, due to unspecific adherence of immunoglobulins to the cell membrane. This could be suppressed by the prefixation of the granulocytes with paraformaldehyde. The developed test proved to be specific and sensitive and to have a high reproducibility. Strongly positive reactions were obtained with antisera containing granulocyte-specific agglutinins or granulocyte-cytotoxins, but also with some sera that did not react in either the agglutination or the cytotoxicity test. HLA antisera also gave positive reactions, but strong anti-A, anti-B or anti-D sera did not react. With this technique leucocyte antibodies can be detected in a higher percentage of patients with febrile transfusion reactions.

Summary. Determination of fetal red blood cell antigens in early pregnancy can be important in cases with a history of severe haemolytic disease of the newborn. From chorionic villus biopsies (CVB) between the 8th and 12th week of gestation a small number of fetal red blood cells was obtained, inevitably highly contaminated with maternal blood cells. Two techniques were used to demonstrate the minor (fetal) cell population with a blood group antigen differing from the major (maternal) cell population: (1) a solid-phase microfluor-escence technique (introduced in this paper) which was compared with (2) the mixed agglutination technique. In series of artificial mixtures of erythrocytes it was shown that with the microfluorescence technique the ABO and Rhesus phenotypes of minor cell populations could be determined at a ratio of 1 in 4000 erythrocytes of the major population, making this technique 4 times as sensitive as the mixed agglutination technique. We further investigated the reliability of the microfluorescence technique to demonstrate antagonistic fetal blood groups in the first trimester of pregnancy. Of 18 women undergoing CVB prior to therapeutic abortion, blood group antagonism (ABO and Rhesus systems) was demonstrate in all 11 cases in which it was present. Therefore, it seems that CVB can be reliably used for the prenatal diagnosis of (recurrent) blood group antagonism.

The lymphocytes of 52 patients with the clinical diagnosis of idiopathic thrombocytopenic purpura (ITP) were typed for the HLA-A, -B and -C antigens, and of 27 of those patients also for the DR antigens. ITP proved not to be significantly associated with any of the HLA-A, -C or -DR antigens tested for. On the platelets of 35 of these 52 patients autoantibodies were detected in the direct immunofluorescence test. In these 35 patients with autoimmune thrombocytopenia (AITP), an increased frequency of HLA-Bw38 was found (14.3% versus 2.6% in controls). The frequency of none of the HLA antigens was significantly increased in the group of 17 ITP patients without demonstrable autoantibodies. The difference in association with HLA-Bw38 between AITP and ITP without demonstrable autoantibodies was not significant.

The granulocytes of 23 patients with chronic granulocytic leukaemia were examined in the indirect immunofluorescence test and some also in the microleucoagglutination test, with specific alloantisera against the NA1, NA2, NB1 and ND1 antigens and a specific granulocyte xenoantiserum. In 10 cases a complete loss of alloantigens was detected. In one patient a partial loss was found. A significant correlation was established between this loss and an accelerated activity (impending blast crisis) of the disease.

A family is described in which the mother developed platelet-specific antibodies, not directed against the antigens of the Zw(PlA) or the Ko system. The antibodies were only detectable in the immumofluorescence test and the radioactive antiglobulin test on platelets, and proved to be mainly IgG1 antibodies, although weak IgM antibodies of the same specificity were also detected. By cross-absorption studies, investigation of the family and a small-scale population study, it appeared that the antibodies were directed against a new antigen which we have called Baka. The frequency of the Baka phenotype in the Dutch population was 90.76%, the calculated genotype frequency 0.696. The Baka phenotype was neither sex-linked nor, so far as evaluable, closely linked to other platelet, red-cell, granulocyte, or HLA groups. The first child of this mother died of neonatal thrombocytopenia, the second child was unaffected. During the second pregnancy, the titre of the antibodies did not rise. The platelets of this child were found not to carry the Baka antigen.

The Fifth International Histocompatibility Workshop resulted in the designation of HLA-A25 and HLA-A26 as two subtypes of HLA-A10 [1]. Aw34, which belongs to the A10 cross-reacting group, was defined during the Sixth Workshop [2]. The existence of A26 was confirmed in various races, such as Caucasians, Orientals, and Negroes, at the Sixth (1975) and Seventh (1977) Workshops [2, 3]. During the Eighth Workshop (1980) [4] A26 was well defined. The gene frequency observed in Caucasians was 3.2%; in Israeli Jews, 10.5% (1980); in Orientals, 7.2%; and in Negroes, 4.5%. In the last population the suggestion of a short variant was put forward. A26 was found to be in linkage disequilibrium with B38 in several races.

Immunofluorescence tests on platelets have always been hampered by nonspecific fluorescence caused by non-immunological binding of plasma proteins to the platelet membrane. It was found that this could be easily overcome by fixation of the cells with paraformaldehyde (PFA). By using PFA-fixed platelets, a simple method for the detection of platelet antibodies, the platelet suspension immunofluorescence test (PSIFT) was developed. PFA fixation did not alter or inactivate the platelet antigens tested. Platelet-reactive antibodies detected specifically with the PSIFT included platelet-specific agglutinins of the IgM class, non-agglutinating platelet-specific antibodies of the IgG class, drug-dependent platelet antibodies, HLA antibodies, as well as anti-A and anti-B antibodies. The sensitivity of the new test was satisfactory, as was its reproducibility. Measurement of platelet immunofluorescence was possible in a continuous flow microfluorometer, making a principle, quantitation of platelet antibodies and antigens possible.

Summary The determination of the subclasses of IgG antibodies against blood groups A and B is important in order to improve our understanding and predict haemolytic disease of the newborn due to IgG anti-A or -B. We describe two techniques that circumvent the problem of the agglutination of A and B red cells by the corresponding IgG antibodies in saline: an antiglobulin consumption test and a modified solid-phase micro-immunofluorescence test. The results of the two techniques are compared with the results obtained in the indirect antiglobulin test beyond the saline agglutination titre in a microplate technique. The solid-phase micro-immunofluorescence test was the most sensitive for the determination of the subclasses of IgG anti-A and -B. Usually sera contained IgG2 anti-A, B in a higher titre than antibodies of other subclasses.

Before discussing the mechanisms involved in alloimmunization against platelets and granulocytes, it is important to realize which categories of allo-antigens are present on these cells. The presence on platelets of antigens, shared with red cells, has been clearly established i.e. the ABH, Lewis, Ii and P antigens. There is no evidence that alloimmunization against these antigens may follow transfusion of incompatible platelets. However, once a patient has antibodies against these antigens part of a population of transfused incompatible platelets will have a shortened survival time. That only part of the platelets are destroyed is due to a heterogeneous distribution of these antigens over the platelet population [1]. These antigens are also heterogeneously distributed over the megakaryocytes in the bone marrow [2]. However the progeny of an individual megakaryocyte carry an equal number of these antigens. Important for alloimmunization against platelets are the HLA-A, B and C antigens (class I) and platelet-specific antigens. Although it has been thought that granulocytes also carry alloantigens shared with red cells, this has not been confirmed in recent studies with more reliable techniques [1,3]. Alloantigens present on granulocytes are either shared with monocytes and lymphocytes or monocytes alone. In addition the HLA-A, B and C antigen and antigens specific for granulocytes are present on granulocytes.

Allotypes of immunoglobulins form the most polymorphic genetic system of human serum proteins. Allotypes have been shown on the heavy chains of IgG1, IgG2, IgG3, IgA2 and IgE and on kappa light chains, which are coded by the γ1, γ2, γ3, α2, e and κ genes respectively. They are called G1m, G2m, G3m, A2m, Em and Km allotypes (Table 1). Most allotypes have originated from the genes by mutation of only one or a few nucleotides resulting in the difference of one or two amino acids. Until now 24 allotypes have been described: four G1m, one G2m, thirteen G3m, two A2m, one Em and three Km allotypes (ref 1).

Six alloantisera contained anti-Cw2: 9w455–9w460. In the combined analysis, serum 9w458 had the lowest number of extra reactions and the fewest false negatives, whereas the other sera tended to give some extra reactions, usually associated with Cw7, B27, and/or B7. In our experience, sera 9w457, 9w458, and 9w460 behaved perfectly well as anti-Cw2 reagents, while 9w455, 9w456, and 9w459 were difficult to read because of turbidity. There was no evidence that Cw2 may be split.

The mechanisms by which red cells are destroyed under the influence of antibodies with different immunochemical and biological characteristics are described. It is shown that the interaction of antibody with the red cell per se does not lead to a disturbance of red cell function. Activation of the whole complement system leads to direct lysis of the erythrocyte (complement hemolysis). The fixation of red cells coated with activated C3:C3 receptors on phagocytic cells is another mechanism which leads to red cell destruction. The hypothesis that adherence to the Fc-receptors of phagocytic cells is essential for the destruction of red cells under the influence of noncomplement-binding antibodies is discussed. Arguments in favour of this theory are correlation between the subclass of IgG red cell autoantibodies and the absence or presence of increased hemolysis in the patient and a correlation between the degree to which red cells of patients with this kind of antibody adhere to monocytes in vitro and the degree of hemolysis in the patient. It is shown by in vitro experiments how this adherence process can take place in vivo in the presence of normal plasma IgG although the latter completely inhibits the adherence phenomenon in vitro.

In the present study, we show, on the basis of serology, absorption experiments, and one-dimensional isoelectric focusing, that the well-defined HLA-Aw33 antigen can be separated into two subtypes, provisionally called Aw33.1 and Aw33.2, which differ in race distribution and linkage disequilibrium.