[Is the research of posttransfusional alloantibodies still relevant?] / Est-ce que la réalisation de la recherche d'anticorps antiérythrocytaires post­transfusionnelle est toujours pertinente ?

The decision of November 6th, 2006 defining the principles of best practices recommends that posttransfusional red cell alloantibodies research is performed after one to three months after. In the University hospital of Brest, the haemovigilance unit takes charge of sending the medical prescription within the required time and centralizing the results. We wished to estimate if the realization of this research still remains relevant. METHODS: A prospective analysis was performed in 2015. We evaluated the realization rate, the red cell alloantibodies rate and the recipient adverse reactions with the diagnostic category: alloimmunization (delayed serological transfusion reaction, DSTR). RESULTS: In 2015, 2162 prescriptions were sent to the 3271 transfused patients. One thousand and eighteen red cell alloantibodies research were done, i.e. a return rate of 61%. Among them, 12 alloantibodies appeared (0.9%) within an average of 56 days. Thirty-three other alloantibodies appeared and were discovered most frequently before a new transfusion. In 10 cases, a posttransfusional research was done that was negative. A survey was conducted among GHCOH members to describe the practices in these health institutions. Twelve questionnaires were analysed. Ten institutions performed a posttransfusional alloantibodies research by issuing a prescription at the patient's exit with a return rate between 0.14 and 16%; 1 institution has a centralized organization with a return rate of 68.3%; 1566 red cell alloantibodies research were performed and among them, 24 alloantibodies appeared (1.53%). CONCLUSION: These results indicate that to be effective, the management of this biological test must be centralized. Despite this, the red cell alloantibodies rate remains very low (0.9 and 1.53%) and raises the question of the relevance of this systematic testing after transfusion, which is in any case mandatory before a new transfusion of red blood cells.

Assessment of fusogenic properties of influenza virus hemagglutinin deacylated by site-directed mutagenesis and hydroxylamine treatment.

Influenza virus hemagglutinin (HA) subtype H7 expressed from a baculovirus vector in insect cells requires cysteine residues for palmitoylation. Mutant HA devoid of fatty acids shows hemagglutinating and hemolytic activities almost identical to those of the acylated wild-type HA (wt). Using a membrane mixing assay (R18), neither the kinetics nor the pH dependence of fusion induced by wt or mutant HA was significantly different from virus-induced fusion. HA-induced fusion of insect cells with human erythrocyte ghosts could also be demonstrated by a cytoplasmic content mixing assay. Both species of recombinant HA induced the flow of lucifer yellow from preloaded ghosts into the cytoplasm of HA-bearing cells. This indicates that membrane fusion mediated by wild-type and fatty-acid-free HA includes both leaflets of the lipid bilayers. Hydroxylamine treatment of wt HA (H7) and fatty-acid-free mutant HA present in lysates of insect cells led to the complete inhibition of hemolytic activity. Deacylation of spike proteins by NH2OH treatment of virus particles resulted in a block of hemolytic activity in influenza virus subtypes H7 and H10 as well as of that in the togaviruses Semliki Forest and Sindbis virus. However, the same treatment did not affect subtypes H2 and H3 or two vesicular stomatitis virus serotypes. With such a differential effect whether or not fatty acids are present in the spike proteins of the different virus particles, hydroxylamine must have other effects than just deacylation, and therefore seems unsuitable for the study of the biological functions of acylproteins.

An assessment of the immunoprecipitation of blood-group antigens using biotinylated red cells.

This study presents a simple protocol for labelling red cell membrane proteins and glycoproteins using biotin. The advantages include an increased capacity to label and process a large number of cell samples of different phenotypes without using large quantities of radionuclides, resulting in no hazardous waste by-products as in radiolabelling methods, and a fast exposure time for detection of the biotinylated component(s). Our assessment on biotinylated red cells was based on the retention of antigenic activity with 14 antibodies (13 monoclonals, one polyclonal), which have specificities for extracellular and cytoplasmic domains of various membrane components, by ELISA, haemagglutination tests and immunoprecipitation. A luminescent method was used to detect the immunoprecipitates on nitrocellulose membrane after SDS-polyacrylamide gel electrophoresis. The development time for band detection was between 5 s and 1 min. Free amino groups that constitute a part(s) of an antigenic determinant were also biotinylated. As a result antibody binding to the red cells was abrogated.

Biochemical characteristics of the heterophile transplantation antigen (HT-A).

The Heterophile Transplantation Antigen (HT-A) is a clinically important antigen which is found in some human kidneys and on the erythrocytes and in the serum of rats and some other mammals. Recent reports suggest the possibility that the anti-HT-A antibodies responsible for graft rejection may cross react with one or more HL-A specificities. The ideal way to investigate this possibility would be to perform careful serological and biochemical comparisons of purified HT-A and HL-A. We report here the first stage of these investigations, the examination of the biochemical properties of HT-A. The HT-A molecule of rat plasma is apparently a glycoprotein with a molecular weight greater than 1,000,000. The HT-A antigenic determinant site seems to reside in the carbohydrate portion of the glycoprotein. The native HT-A molecule is apparently susceptible to proteolytic enzymes of rat plasma and serum and is cleaved by these enzymes to produce a fragment with a lower apparent molecular weight and a larger fragment which coelutes with lipoproteins during Sepharose CL-4B chromatography. The data presented here should allow development of a rational purification scheme for the HT-A glycoprotein of rat plasma to be used in further studies in the relationship between HT-A and HL-A.

Mouse monoclonal anti-N. II. Physicochemical characterization and assessment for routine blood grouping.

The precise physicochemical conditions under which two examples of mouse monoclonal anti-N can be used as blood grouping reagents have been defined. The antibodies were shown to belong to the IgG1 and IgG2b subclasses respectively and both reacted within discrete ranges of temperature and pH with optimal reactions occurring at 20 degrees C or less and pH 8.5. Under these conditions and at concentrations of 2-3 micrograms/ml, the antibodies were used, in parallel with conventional polyclonal antisera, to type a series of random blood donors. The results obtained with one of the monoclonal antibodies and the polyclonal reagents were in perfect agreement and the monoclonal antibody was judged to have considerable potential as an N-grouping reagent.