Serological studies of monoclonal RH antibodies with RH1 (D), RH2 (C), RH3 (E) and RH5 (e) variant RBCs.

One hundred and forty five Mabs against RH antigens were tested. In this paper, we chose to detail reactivity of MoAbs directed against variant RBCs of the CNRGS collection for which we studied the molecular background. Because we developed procedures to identify variants of the RhD, RhC, RhE and Rhe antigens, we were especially interested in finding new monoclonal antibodies that could help us to characterize more accurately these variants. Therefore, we drew parallels between our procedures and results obtained with the 2001 workshop antibodies.

Two new alleles of the RHCE gene in Black individuals: the RHce allele ceMO and the RHcE allele cEMI.

Six unrelated individuals of Afro-Caribbean origin, whose red cells have a marked reduction of the Rhe antigen expression, have been identified. All exhibited the same serological profile with anti-e monoclonal antibodies and lacked expression of the high frequency e-related antigen hrS. Transcripts and genomic analysis showed that these phenotypes resulted from the presence of two new RHCE alleles, ceMO and cEMI. The ceMO allele corresponded to a RHce gene carrying a G667T mutation (exon 5) and was detected at the homozygous state in sample 1 and at the heterozygous state in samples 2-6. The G667T mutation resulted in a Val223Phe substitution on the Rhce polypeptide, in close proximity to Ala226 (e-antigen polymorphism), which might account for the altered expression of e. The ceMO allele is also associated with the lack of expression of the hrS antigen. The absence of the hrS antigen expression may have implications in transfusion as hrS-negative individuals may develop clinically significant antibodies. The cEMI allele corresponded to a silent RHE allele carrying a nine nucleotide deletion within exon 3 and was detected at the heterozygous state in sample 2. This deletion resulted in a shortened polypeptide of 414 residues (instead of 417) that was absent (or severely reduced) at the red cell surface, as the E antigen was undetectable using serology and Western blot analysis with anti-E reagents. In DNA-based polymerase chain reaction genotyping for RHE determination, the cEMI allele provided a false positive result as the cells carrying this allele are serologically phenotyped as E-negative. The incidence of this allele in the Black population is unknown but, as shown already for D genotyping, one must exercise caution when genotyping is performed to detect the e/E polymorphism.

[Platelet phenotyping and study of alloantibodies using flow cytometry]. / Phénotypage plaquettaire et recherche d'allo-anticorps en cytométrie de flux.

In this study, we describe a flow cytometric technic for the detection and characterization of platelet allo antibodies and for platelets grouping in the platelet group PLA. This new technique is a variant of the platelet suspension immuno fluorescence test. It is rapid, simple, sensitive and specific. So it is very useful in the cases of neonatal thrombocytopenia and posttransfusion purpura. Moreover, anti-HLA antibodies don't obstruct the detection of anti-PLA1 antibodies.

[Immunization against the ZWa (PLA1) platelet antigen: group at risk, prevention of complications. Apropos of 132 cases]. / L'immunisation contre l'antigène plaquettaire ZWa (PLA1): groupe à risque, prévention des complications. A propos de 132 cas.

The identification of anti-ZWa (-PLA1) alloimmunisation is not very frequent. It can be observed in most perinatal alloimmune thrombocytopenias (PAT) and rare post transfusional purpuras (PTP). On the other hand, the clinical consequences of these immunisations are often dramatic, particularly for the foetuses for which there has been no prevention so far. The retrospective study of 132 cases, 123 PAT and 9 PTP, shows the possible irreversible complications for 18% of the newborns with PAT, but especially for 10% of the foetuses which will show PAT at birth. HLA markers are very useful to detect the people who are likely to develop an anti-PLA1 immunization for they are PLA1 negative and HLA DR3. Then, it becomes possible to prevent the complications of these immunisations. It is what we tried to do through the diagnosis and the treatment of PAT in 3 foetuses.

[Neonatal alloimmune thrombopenia]. / Les thrombopénies néonatales alloimmunes.

88 families in which 84 cases of neonatal alloimmune thrombocytopenia (NAT) occurred, were studied. In 84 families, the NAT was the consequence of an incompatibility in the PLA system. Furthermore, the phenotype HLA-DR3 increases greatly the risk of immunisation (RR: 76,5). The importance of the risk of neurological sequellae was shown by the clinical study (about 25% of the surviving neonates). The occurrence of the accident at the first birth of a PLA1 positive child in a sibship was frequent (59%). In addition, the NAT recurred at each birth of a PLA1 positive child with only five exceptions. All of them concern a female neonate and this might be meaningful. Therapeutical data are heterogeneous and difficult to interpret. However, it appears that the prevention of obstetrical traumatism by caesarean section and compatible platelet transfusions are useful. It is too early to evaluate the efficacy of prenatal transfusions of mother's washed platelets. However, in the two cases in which we use them, they gave a good and sustained platelet count increment. The prenatal diagnosis of NAT and the PLA grouping of the foetus has been proposed in three cases and are feasible at 20 weeks of pregnancy.

[Neonatal alloimmune thrombopenia. Clinical and biological study of 84 cases]. / Thrombopénies néo-natales allo-immunes. Etude clinique et biologique de 84 cas.

Eighty-four patients with neonatal alloimmune thrombocytopenia (NAT) were investigated clinically and by biological laboratory methods. The condition appeared at birth, usually as an isolated thrombocytopenic purpura, but in about 20% of the neonates the haemorrhagic syndrome was associated with signs of infection or with jaundice and hepatosplenomegaly. Considerable variations were observed in the severity of the purpura; in 3 cases the thrombocytopenia was clinically silent. Haemorrhagic brain lesions were present in 7% of the neonates, and severe neurological sequelae in 14 of the 59 children on long-term follow-up. The overall mortality rate was 9.2%. The PLA1 system was involved in 56 of the 59 families studied, with PLA1-negative mothers developing immunization against the foetus' PLA1 antigen. In 20% of these mothers the antibody was not demonstrable, and the diagnosis relied on the mother's phenotype and on a history of previous NAT. The strong association demonstrated between the HLA-DR3 antigen and the ability to develop anti-PLA1 antibodies is of extreme importance. It may be helpful to confirm the diagnosis in mothers without detectable anti-PLA1 antibodies and to identify mothers at risk of alloimmunization. Neurological sequelae, which were due to post-natal haemorrhage in at least 70% of the cases, could now be avoided by an early diagnosis, modern transfusional techniques and caesarian section. However, antenatal lesions cannot be avoided, except by preventive measures, yet to be developed, against alloimmunization or the cytopenic effect of the platelet antibody.

[Activation of oxidative metabolism of granulocytes and monocytes by IGG-coated platelets from patients with thrombocytopenia]. / Activation du métabolisme oxydatif des granulocytes et des monocytes par des plaquettes recouvertes d'IgG provenant de patients porteurs de thrombopénies.

We have previously shown that monoclonal anti-T cell antibodies bound to their specific targets can trigger the activation of monocyte/macrophage oxidative metabolism through an Fc receptor-mediated interaction. The present study demonstrates that IgG coated platelets from patients with thrombocytopenia-associated diseases can induce a similar respiratory burst activation in polymorphonuclear and mononuclear phagocytes from normal individuals. The intensity of the oxidative reaction as measured by luminol-dependent chemiluminescence is in close correlation with the level of surface-bound IgG molecules as determined by a radioactive anti-immunoglobulin assay. This new methodology to evaluating IgG fixed on human platelets by their capacity to trigger the generation of highly reactive oxygen species by granulocytes and monocytes has also suggested a new mechanism in the genesis of thrombocytopenia associated with autoimmune diseases.

An immune response gene linked to MHC in man.

With a view to finding a relationship between immune response and MHC in man, 83 D negative mothers with allo-into-D antibodies and 26 PLA1 negative mothers with allo-anti-PLA1 antibodies were investigated as regards their HLA-A, B and DR antigens. We have found that there is a highly significant relation between the DR3 antigen and an immune response to the PLA1 antigen, but none to the D antigen.

[Estimation of platelet-bound IgG by means of a test using radioactive antiglobulin]. / Evaluation des IgG fixées sur les plaquettes à l'aide d'un test utilisant une antiglobuline radio-active.

A method of evaluation of platelet bound IgG (PA IgG) is described. This method rely upon the fixation of a radio-labelled goat anti-human IgG on platelets. Each new batch of labelling of the antiglobulin is tested with a reference system using a semi-purified anti-D IgG and O R1R1 and O rr erythrocytes. This system is used for the standardization and the validation of the antiglobulin, and it allows to make correspond an amount of antiglobulin to a known quantity of IgG molecules. The normal level of PA IgG in 98 healthy controls was found to be 351 +/- 252 antiglobulin combining sites (ACS) per platelet. The upper limit (99% confident limit) in the controls is 999 ACS per platelet. 412 thrombocytopenic patients have been tested, 78.7% of ITP had an elevated PA IgG. However positive results are also frequent in other thrombocytopenia even when an immune mechanism cannot be evoked. These positive results raise the problem of the signification of PA IgG and of their exact nature. Further dates are necessary to ascertain the role of auto-antibodies, immune complexes or of platelet membrane modifications in PA IgG increase.

[Neonatal thrombopenia caused by antiPLA1 and HLA-DR3 antigen alloimmunization]. / Thrombopénies néonatales par allo-immunisation anti PLA1 et antigène HLA-DR3.

The HLA typing of mothers alloimmunised against the PLA1 antigen of their thrombocytopenic neonate shows that the A1, B8, DR3 haplotype is involved. The very strong association found with DR3 (20/21) suggests that an immune response gene located in the D region of the major histocompatibility complex might be responsible for the ability to develop an anti PLA1 antibody.

Studies of platelet antibodies in patients with thrombocytopenic purpura by two different techniques.

The direct antiglobulin consumption (DAC) test, used for the detection of platelet autoantibodies, was found to be positive in 86% of a group of 150 patients with thrombocytopenic purpura. The platelet radioactive Coombs' (DRC) test was performed in 30 patients and the correlation between the two tests was statistically significant (r = 0.48, p less than 0.01). The two tests were used in an indirect mode for the detection of antiplatelet antibodies in serum. The results of the two tests were in good agreement (r = 0.06, p less than 0.001). There was not a statistically significant correlation between the level of platelet-associated IgG and the presence of platelet antibodies in the serum. These results indicate that the DAC test or DRC test can be used interchangeably for the detection of antibody in serum or of antibody bound to platelets, and that only the direct test, using the patients' platelets, is useful in the diagnosis of autoimmune thrombocytopenic purpura.

Posttransfusional immunologic thrombocytopenia. A case report.

A case of posttransfusional immunologic thrombocytopenia is reported in a 75-year-od PlA1-negative woman. This was the second episode of postoperative and posttransfusion thrombocytopenia in the same patient who had had only one pregnancy. Both thrombocytopenic episodes were subclinical and discovered by systematical hematologic study. A potent anti-PlA1-antibody (titer 1/128) was demonstrated by the platelet-indirect radioactive Coombs test which appeared more sensitive than other platelet immunological assays used. The very long duration of the thrombocytopenia is discussed as well as the therapeutic possibilities in such cases.

[Glanzmann thrombasthenia, PLA1 antigen and anti-Glanzmann antibody]. / Thrombasthénie de Glanzmann antigène PLA1 et anticorps anti-Glanzmann.

Six cases of Glanzmann's thrombasthenia were studied using a platelet indirect radioactive Coombs (PIRC). In serum of two among six patients, an antibody was found, which reacted positively with all platelets except those of thrombasthenic patients. Such "anti-public" antibody which shortens the life of transfused platelets is a very serious complication of Glanzmann's thrombasthenia. Attempts to define the PLA group of the six Glanzmann patients, with human allo antisera recognizing PLA1 antigen, gave negative results. Three hypothesis were discussed: (1) Interference in the test of the aggregation defect of thrombasthenic platelets. However, anti-HLA antibodies were normally fixed in the PIRC test. (2) PLA2 gene and Glanzmann gene have a strong gametic association and all Glanzmann's patients are PLA1 negative. (3) Glanzmann gene is coding for the PLA gene substrate. In such case, thrombasthenic patients might be genetically PLA1 positive and phenotypically PLA1 negative. Moreover they would also be PLA2 negative, this could not be tested due to the lack of anti-PLA2 antiserum.

[Typing and detection of antibodies in the PLA system (platelet). Application to the study of neonatal thrombopenia by feto-maternal PLA allo-immunisation].

A platelet indirect radio-active Coombs test has been described. The technique for purification and labelling the antiglobulin has been precised. This test allows the typing of platelets in the PLA system and the study of sera from mothers of thrombocytopenic child. As examples, four families of neonatal thrombocytopenia are reported, with PLA1 negative mother. In the serum of three of these mothers, we could demonstrate anti-PLA antibodies in spite of a negative platelet complement fixation. This test has many advantages compared to other tests such as platelet complement fixation, assay for blocking antibodies or antiglobulin consumption: it gives objective and quantitative results and is highly reproducible, anticomplementary serum may be tested. It has enabled us to select PLA1 negative donors for exsanguino-transfusions of thrombocytopenic children born from PLA1 negative mothers.

Platelet indirect radioactive Coombs test. Its utilization for PLA1 grouping.

A platelet indirect radioactive Coombs test (PIRC) has been described. The technique for purification and labelling the antiglobulin has been precised. This test allowed the typing of platelets in the PLA system by using an absorbed serum from a mother of a thrombocytopenic child. Six other families of neonatal thrombocytopenias were tested. In three of them, the mothers were found PLA1 negative (PLA2, PLA2). Among a panel of 93 platelets, two (0.022) were found PLA1, negative. This PIRC test has many advantages compared to other tests such as platelet complement fixation, assay for blocking antibodies or antiglobulin consumption: it gives objective and quantitative results and is highly reproducible; anticomplementary serum may be tested.