anti-Rh Sera Research Articles

Karl landsteiner and his major contributions to haematology

Karl landsteiner and his major contributions to haematology

Antibodies for trypsinized human red blood cells in human sera.

During studies of agglutination of trypsinized human red blood cells (RBC) by anti-Rh sera, it was noticed that some human sera without Rh antibodies agglutinated these erythrocytes. Of 933 normal and pathological sera subsequently screened, 116 produced such agglutination. The agglutinins were of IgM nature and were directed against an antigen exposed or created by tryptic digestion of RBC. They reacted equally well with autologous as with allogeneic trypsinized RBC and were different from T agglutinins and from Paul-Bunnell antibodies. The mode of formation of these antibodies remains unknown.

Monoclonal antibodies directed against human Rh antigens in tests with the red cells of nonhuman primates

Monoclonal antibodies against Rh related antigens on human red cells often crossreact with the red cells of the highest subhuman primate species. Depending on specificity of antibody, the species tested, and technique used, these reactions can be either species-specific or type specific. In tests with chimpanzee red cells, some of the latter type reactions have specificities related to the R antigen of the R-C-E-F blood group system of chimpanzee; specificities of some others seem to be unrelated to any known chimpanzee blood groups. Monoclonal anti-D reagents that give uniformly positive reactions with human D-positive (common and rare types) red cells, display wide individual differences in tests with chimpanzee blood. This indicates that there are minute structural variations of antibody molecules from one monoclonal anti-D antibodies apparently have no bearing on recognition of the D combining site on the human red cells, but come into play when in contact with chimpanzee rbcs. Some of the monoclonal antibodies directed against Rh and LW molecules are distinguished by unusually strong reactions with the red cells of the Old World monkeys (macaques and baboons), which is in contrast with negative or weak reactions of the same antibodies with the red cells of anthropoid apes and human bloods. One may recall, that polyclonal anti-Rh sera do not react with the blood of rhesus monkeys, the phenomenon that was the source of controversy surrounding the discovery of the rhesus factor of the human blood.

Results with the group 3 antibodies

This chapter focuses on the LW Factor. The LW factor is a blood factor found in more than 99% of persons, which is detected by antisera prepared by immunizing guinea-pigs or rabbits with rhesus-monkey or baboon red cells, and then absorbing out all other antibodies. Those who have the factor are LW positive and those who lack it are LW negative. The anti-rhesus rabbit and guinea-pig sera contain additional fractions of antibodies having specificities other than anti-M and anti-Rh. Antibodies are reactive for rhesus monkey and baboon red cells, and are also cross-reactive for all human red cells. The role of Rh sensitization, in intragroup hemolytic transfusion reactions and in the pathogenesis of erythroblastosis fetalis, creates a demand for large quantities of diagnostic anti-Rh sera. The animal anti-rhesus sera strongly agglutinate red cells from all newborn human babies, whether Rh negative or Rh positive, and to higher titers than Rh-positive blood from adults.

Absence of two membrane proteins containing extracellular thiol groups in Rhnull human erythrocytes.

Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.

Importance of IgG subclasses of anti-Rh antibodies for the detection of Fc-receptor-bearing human lymphocytes.

13 anti-Rh sera were compared for their usefulness in the detection of Fc-receptor-bearing lymphocytes (EAhum test). IgG subclasses of anti-Rh antibodies were determined by the antiglobulin test with monospecific sera and by the detection of Gm allotypic markers in the haemagglutination inhibition test. Six sera with IgG1 + IgG3 or IgG1 + IgG2 + IgG3 antibodies and one with pure IgG3 antibodies were found to be useful, whereas six other sera with only IgG1 were unsuitable for the EAhum test. G3m markers were detected only on the anti-Rh antibodies which were capable of forming rosettes with lymphocytes. The data show that human peripheral lymphocytes possess Fc receptors for IgG3 immunoglobulins.

Increases of leukocyte tissue factor activity stimulated by red cells sensitized with human blood group alloantibodies

Human Rh+ and A+ red cells sensitized with human anti-Rh and anti-A blood group alloantibodies are shown to stimulate large increases of tissue factor activity as compared to controls after incubation with leukocytes for 3–18 hours. As little as a 1 1280 dilution of human anti-Rh serum was stimulatory. Immunospecific effects in experiments with 5 different antibodies exclude possible artifacts resulting from contaminants such as endotoxin. These results suggest that production of leukocyte tissue factor may be similarly stimulated by sensitized red cells during immune hemolysis and serve as an activator of coagulation.

Importance of IgG Subclasses of Anti-Rh Antibodies for the Detection of Fc-Receptor-Bearing Human Lymphocytes

13 anti-Rh sera were compared for their usefulness in the detection of Fcreceptor- bearing lymphocytes (EA(hum) test). IgG subclasses of anti-Rh antibodies were determined by the antiglobulin test with monospecific sera and by the detection of Gm allotypic markers in the haemagglutination inhibition test. Six sera with IgG(1) + IgG(3) or IgG(1) + IgG(2) + IgG(3) antibodies and one with pure IgG(3) antibodies were found to be useful, whereas six other sera with only IgG(1) were unsuitable for the EA(hum) test. G3m markers were detected only on the anti-Rh antibodies which were capable of forming rosettes with lymphocytes. The data show that human peripheral lymphocytes possess Fc receptors for IgG(3) immunoglobulins.

Ripley-Like Serum and other Anti-Rh Sera in Detection of Fc Receptor-Bearing Human Lymphocytes

Three anti-Rh sera useful for the EA(hum) test were found (one comparable to Riserum). It was shown that the usefulness of anti-Rh sera for the EA(hum) test depends on the amount of anti-Rh antibodies absorbed onto red cells demonstrated in the manual antiglobulin test. It does not depend on the quality of antibodies and the ability of complement binding.

Blocking of Certain Antigenic Sites in the F(ab) Region by Combination of Univalent Fragments of Rh Antibodies with Red Cell Antigens

Evidence was obtained in the present study substantiating the fact that divalent Rh antibodies are bound to antigenic sites on the red cells through only one of the two combining sites. Univalent F(ab) fragments bound to the red cell lacked such free-combining sites and showed a blockage of various idiotypic-like antigens which were readily detected when divalent antibodies or divalent fragments were used. The univalent fragments contained the antigens because they completely inhibited the divalent system. These antigens were detected with idiotypic antisera made to a variety of monoclonal Ig through the use of certain very heterogeneous anti-Rh sera. As is the case with many other idiotypic systems, combination with antigen which blocked the antibody-combining site eliminated the reactivity. Advantage was taken of these differences between divalent and univalent antibodies to determine antigens in the F(ab) portion of Rh antibodies which are or are not blocked by such combination with the red cell. A variety of subclass and genetic markers found in the constant regions of the F(ab) units were not affected. However, in addition to the idiotype-related antigens a series of V region markers, particularly those determining various V region subgroups, were not detected after combination.

Haemaccel® — An improved diluent for incomplete enzyme treated anti-Rh serum

Stable and avid anti-Rh sera readily produced by diluting Bromelin treated sera in Haemaccel®, a colloidal polymerizate of degraded gelatin. This simple technique makes it possible to convert anti-Rh sera of comparatively low titre into suitable reagents for diagnostic use.

Characterization of Rh Antibodies Formed after Incompatible Pregnancies and after Repeated Booster Injections

Indirect hemagglutination studies were performed on 10 incomplete anti-Rh sera from mothers after incompatible pregnancies, on 19 incomplete anti-Rh plasma samples from preimmunized and repeatedly boosted individuals and on 3 anti-Rh plasma pools. Group O Rh-positive red blood cells were agglutinated by antisera to immunoglobulins. Anti-Rh antibodies after incompatible pregnancies were found to belong to the immunoglobulin class IgG and to the subclasses IgGl, IgG3 and IgG4 only. In most anti-Rh samples from preimmunized and repeatedly boosted subjects, IgG and IgA were present. In six anti-Rh samples of this group, the subclass IgG2 could be demonstrated.

Immunology of pregnancy.

The presentation made by Dr. Verbickij is important in several respects. First, it is an excellent piece of work on the immunology of pregnancy. Next, it shows how a model can be established in a non-human primate to facilitate research on clinical problems. There has been a great amount of interest in the immunology of pregnancy in recent years. The stimulus to much of the early research was the development of methods to prevent rhesus haemolytic disease in human neonates. We are all familiar with the great progress being made in these studies following the introduction of the use of anti-Rh serum (Clarke 1968). There has been a significant reduction in the incidence of afflicted children, and this progress seems bound to continue as more experience is gained on the best ways of using the antiserum.

Toxoplasmosis as a cause of foetal death.

Four of 523 mothers with stillborn infants were positive in the toxoplasma complement fixation test and had a dye test titre of 256 or higher. In 1 instance, the autopsy findings were suggestive of intrauterine toxo­ plasma infection, whereas in the other cases the available data did not support this diagnosis. The activity of the maternal health service in Fin­ land is directed to a considerable extent to the prevention of congenital syphilis and isoimmune haemolytic disease. However, the occurrence of infectious syphilis among pregnant women is quite low (5), and the rate of maternal isoimmunization will undoubtedly be reduced by the anti-Rh serum prophylaxis. The organization of the maternal health ser­ vice provides the opportunity to make use of it for other purposes. One possibility in this respect is presented by congenital toxoplasmosis. It results at least in most instances from a toxoplasma in­ fection acquired during the second half of preg­ nancy. An early infection may give rise to abor­ tion, but there is no clear evidence that it will cause developmental anomalies. Congenital trans­ mission from chronically infected mothers is ob­ viously very rare, if it takes place at all. Auto­ mated complement fixation systems are available for mass screening of serum specimens, and there is at least some treatment for the disease. About one fourth of the Finnish female popula­ tion of childbearing age have toxoplasma antibo­ dies as measured by the dye test (2). Since residual titres persist for many years, if not throughout the whole life, the incidence of toxoplasma infec­ tion during pregnancy may be of the order of 5 per 1 000. However, there is no information on the frequency of congenital toxoplasmosis in Fin­ land. The aim of the present work was to evaluate the role of toxoplasmosis as a cause of foetal death.

Blood Groups in Man.

This is certainly the most often cited book on blood groups. Unfortunately, its popularity appears to have been based more on fluent and persuasive style of writing than scientific reliability. This new edition has been improved by elimination of some of the misinformation of former editions. Namely, the chapter on blood groups and disease, little d, and the so-called shorthand notations are gone. Unfortunately, the deleted material has been replaced with other misinformation. An outstanding example is the so-called LW factor (where the authors rediscover a fact pointed out by Landsteiner and Wiener more than 25 years ago). This is that guineapig and rabbit antirhesus sera and human anti-Rh sera are not identical in specificity even though they both detect the same agglutinogen Rh. (This is comparable to a profile and front view of a face, which are different but still show the same face.) Race and Sanger mistake this

An evaluation of the effects of polyvinylpyrrolidone on blood typing with anti-Rh serum: importance of albumin concentration.

Discussion and ConclusionsThe present work, in agreement with the results of others (14, 15), indicated that the titer can vary as a function of PVP concentration (Fig. 1). In addition, however, the present experiments showed the importance of the relationship between bovine albumin and PVP concentrations in systems utilizing both of these polymers. For example, the order of titers obtained with various levels of bovine albumin could be reversed (i.e., from 18.3% > 10% > 5% to 5% > 10% > 18.3%) simply by changing the concentration of K-90 PVP from 0 to 1% (Fig. 2).There is no specific requirement for the level of protein in commercial typing sera (12), and there is appreciable variation in the albumin content of such antisera. It is therefore apparent that if an investigator intends to use a system involving PVP and commercial anti-Rh serum, different levels of PVP may be necessary to achieve optimal results with different antisera. Furthermore, the possibility of false results in Rh typing in the presenc...

Immunochemical and serological studies of enzymatically fragmented human IgG globulins—II: Hydrolysis with subtilisin, elastase, trypsin, and chymotrypsin

Fragmentation of the IgG globulins of a single anti-Rh serum by trypsin, chymotrypsin, subtilisin and elastase is compared to previous studies using papain, pepsin, ficin and bromelin. Natural antibodies in human sera are able to differentiate the Fab fragments produced by the different enzymes.

EVOLUTION OF THE ANTI-GM SUBSTANCES AND OF THE REACTIVITY OF RHEUMATOID SERA FOR HUMAN RED CELLS SENSITIZED WITH 3 DIFFERENT ANTI-RH SERA OR IMMUNE RABBIT SERA AND FOR F. II LATEX.

AbstractIn rheumatoid arthritis (R.A.) in addition to the rheumatoid factors detected by the Waaler‐Rose and latex F. II tests,Henceforth we will use instead of “rheumatoid factors detected by the Waaler‐Rose and latex F II tests,” the abbrevation “rheumatoid factor.” anti‐Gm substances and agglutinins for human red cells sensitized with 3 different anti‐Rh sera appear, mainly in cases of severe disease of long duration. Usually, during the evolution of the disease, their titer does not parallel that of the rheumatoid factors; this fact is in favor of the individuality of the anti‐Gm substances.

A comparative histo- and immunopathological study of the placenta in diabetes mellitus and in erythroblastosis fetalis

A study has been carried out comparing the normal placenta with that in diabetes and erythroblastosis. A proliferative vascular lesion has been demonstrated, which binds fluorescent insulin specifically in the diabetic placenta and fluorescent anti-Rh serum in the erythroblastotic placenta. In both diseases there is an increase in the syncytial knot/villus ratio. Neither the vascular endothelium nor the syncytial knots bind the fluorescent reagents; the antigen-antibody complex evidently diffuses through these cells and is trapped in basement membrane structures.Serological studies indicate that in both disease states maternal antibodies cross the placental barrier and enter the fetal circulation. However, the binding of fluorescent reagent by placental tissue in cases with negative titers indicates the presence of fixed tissue antibody. Pathological and clinical evidence suggests that the latter is more directly related to the occurrence of placental lesions than is circulation antibody. In the case of diabetic subjects who have never received insulin, the binding of fluorescent insulin may be indicative of an autoimmune phenomenon.

The Specificity of the Nonspecific

Summary Although plant hemagglutinins, which have been known nearly as long as specific immune agglutinins, were long thought to be nonspecific, some have been found to have a blood-group specificity which is quite sharp. Some of these agglutinins, such as the lectins of Dolichos biflorus and Ulex europeus, have found important practical applications in blood group techniques. Other group specificities have been found, and even some of the plant extracts which agglutinate all human erythrocytes probably have a measure of specificity, since they are specificially inhibited by certain carbohydrates. The information that may be gained in this way concerning reactive structures in the red cell is discussed. A study of such reactions led Mäkelä to classify all aldopyranoses into four groups, depending on their configuration at carbon atoms 3 and 4. The inhibition reactions of human anti-Rh sera suggest that this classification may be of significance in this connection also.