Showing papers in "Transfusion Clinique Et Biologique in 2002"

Recombinant monoclonal antibody technology.

Abstract: With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination.

Abstract: Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H + L) as a secondary antibody were recommended but not mandatory A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal 'standard RBC' throughout all experiments An RBC panel comprising two partial D and four weak D types was supplied as well The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC) The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2 Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation

Section 1A: Rh serology. Coordinator's report.

Abstract: One hundred forty-two Rh-specific monoclonal antibodies (Mabs) were evaluated by serology in 27 laboratories Evaluators were asked to test each Mab at three dilutions in specified serological techniques against normal positive and normal negative phenotype cells, and any Rh variant cells that they had available Raw data was submitted to the coordinator for overall analysis Results were analysed by expressing the sum of reaction grades for each Mab with each variant cell as a percentage of the sum of reaction grades of that Mab with normal phenotype cells Anti-D Mabs were sorted into 23 groups which had the same pattern of reactions with different partial D phenotype cells Eighteen of these corresponded to previously defined patterns; five were new patterns Combined with data from the previous workshop, this means that 30 different reaction patterns have been defined A new nomenclature is introduced for numbering the epitopes Reactions with new variants DNB, DNU and DAR indicated some further subsplits of these patterns Reactions with Category Va cells indicated that there were five different types of Va cells that could be distinguished serologically with monoclonal antibodies No patterns of reactivity corresponding to the epitope groups could be observed with the different types of weak D tested Anti-E Mabs were sorted into 14 groups, and the E variant cells into seven groups

In vivo studies of monoclonal anti-D and the mechanism of immune suppression.

Abstract: Administration of anti-D immunoglobulin to D– women after delivery of a D+ infant has dramatically reduced the number of immunised women and cases of haemolytic disease of the fetus and newborn. The use of monoclonal anti-D might alleviate some of the pressures on maintaining adequate supplies of plasma sourced anti-D. Two human monoclonal antibodies, BRAD-3 (IgG1) and BRAD-5 (IgG3), with proven activity in in vitro functional (immunological) assays with cells bearing IgG Fc receptors (FcγR) were selected for clinical studies. They were prepared by purification of IgG secreted by culture of the Epstein-Barr virus-transformed B cell lines in hollow fibre bioreactors. The clearance of D+ red cells injected into D– subjects was accelerated by prior injection of the monoclonal antibodies, both individually and blended (3:1, BRAD-5: BRAD-3). The subjects were protected from Rh D immunisation. A large multicentre study evaluated the BRAD-3/5 blend for its ability to prevent Rh D immunisation in 95 D– subjects given 400 μg i.m. 24 hours after injection of 5 ml D+ red cells. Challenge injections of D+ red cells alone were given 24 and 36 weeks later, and blood samples were taken every 4 weeks from the subjects throughout the study for detection of anti-D responses. There was one definite and one possible failure of protection; in one subject the plasma anti-D level rose from week 12 onwards, and in another individual rapid seroconversion was observed at week 28. Considering the relatively large dose of red cells and the number of subjects studied, it was concluded that the failure rate was much lower than in routine Rh D prophylaxis. The responder rate was 13% by week 36 and 24% by week 48. The low percentage of responders and the modest levels of endogenous anti-D produced suggested that administration of monoclonal anti-D had induced long-term specific suppression of anti-D responses in these subjects. The most likely mechanism of action was considered to be inhibition of B cells resulting from co-crosslinking antigen receptors with inhibitory FcγR when the B cells contacted red cells that had bound passive anti-D.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Abstract: Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (FcγR)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit FcγRI, FcγRII or FcγRIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be FcγRI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fcγ receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through FcγRI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through FcγRIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through FcγRIII. Haemolysis via FcγRIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only FcγRI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through FcγRI and FcγRIII while IgG2 was inhibited by Mabs to both FcγRII and FcγRIII, suggesting a variety of FcγR are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

Exploiting antibodies as catalysts: potential therapeutic applications

Abstract: In this review, we explore recent developments in the generation of catalytic antibodies and their potential in therapy.

Séroprévalence virale, transfusion et allo-immunisation chez des adultes drépanocytaires guadeloupéens

Abstract: Resume La prevalence des seropositivites (VIH 1 et 2, HTLV1, hepatites B et C), la couverture vaccinale anti-hepatite B, la frequence des patients transfuses et des allo-immunisations ont ete evaluees, par une etude retrospective, dans une population d’adultes drepanocytaires guadeloupeens. Les donnees ont ete extraites des dossiers de suivi longitudinal du centre caribeen de drepanocytose. Parmi les 331 patients etudies, on ne notait aucune contamination transfusionnelle par le VIH. Tous les patients positifs pour HTLV1 (n = 11 soit 3,3% de l’echantillon) et pour l’hepatite C (n = 9 : 2,7%) avaient beneficie d’une transfusion. Cinq sujets (1,5%) avaient une hepatite B active. Le vaccin contre l’hepatite B protegeait efficacement 247 patients (74,4%) et 57 (17,2%) etaient immunises post-hepatite B. Un antecedent transfusionnel etait retrouve chez 213 drepanocytaires (64%) avec une frequence plus elevee chez les SS (88%) que chez les SC (36%) 〚p

Recombinant forms of Gerbich blood group antigens: expression and purification.

Abstract: Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.

Prévalence des virus des hépatites B et C, du VIH et de l’HTLV chez les candidats à une transfusion autologue programmée en France, 1993–2000

Abstract: Resume La surveillance epidemiologique des candidats a une transfusion autologue a ete mise en place en France en 1993. Le nombre de candidats a augmente regulierement entre 1993 et 1997 et a diminue ensuite, passant a moins de 50 000 en l’an 2000. Le sex-ratio est stable au cours du temps (0,85 homme pour 1 femme). La population des candidats a une transfusion autologue a tendance a vieillir : la proportion des moins de 50 ans est passee de 29 % en 1993 a 18 % en 2000 alors que celle des 70 ans et plus est passee de 22 a 34 % sur la meme periode. Les taux de prevalence ont diminue entre 1993 et 2000, d’un facteur de 2,5 pour l’Ag HBs et d’un facteur de 5 pour le VHC. Pour le VIH, une legere tendance a la diminution est observee et pour l’HTLV, le taux est stable au cours du temps. En 2000, le taux de prevalence du VHC (0,23 %) etait 2 fois plus eleve que celui de l’Ag HBs (0,12 %), 15 fois plus eleve que celui de l’HTLV en France metropolitaine (0,015 %) et 100 fois plus eleve que celui du VIH (0,002 %). La prevalence est comparable chez les hommes et chez les femmes pour le VHC, environ 2 fois plus elevee chez les hommes que chez les femmes pour l’Ag HBs et 3 fois plus elevee pour le VIH. A contrario, pour l’HTLV, la prevalence est environ 2 fois plus elevee chez les femmes. Les taux de prevalence de l’Ag HBs et du VHC ont pu etre aussi calcules par groupe d’âge : pour l’Ag HBs, les taux de prevalence augmentent jusqu’a 30–39 ans chez les femmes et 40–49 ans chez les hommes ; ensuite ils diminuent avec l’âge mais sont plus eleves chez les hommes que chez les femmes. Pour le VHC, alors que chez les femmes la prevalence augmente avec l’âge, chez les hommes elle atteint un pic dans le groupe d’âge 30–39 ans, puis elle diminue jusqu’au groupe d’âge 50–59 ans et se stabilise ensuite. Le risque devenu extremement faible de transmettre une infection virale par transfusion homologue de produits sanguins labiles et l’evolution des techniques de transfusion autologue semblent etre les deux principaux facteurs ayant contribue a la diminution recente du nombre de candidats a une transfusion autologue. La diminution de la prevalence de l’Ag HBs et des Ac anti-VHC entre 1993 et 2000 est multifactorielle mais la chute importante observee pour le VHC est, en partie, le reflet d’une diminution de la prevalence dans la population generale au cours de ces dix dernieres annees.

Dépistage des bactéries dans les concentrés de plaquettes : perspectives

Abstract: Bacterial contamination of blood components represents today the highest infectious risk of blood transfusion, the risk is particularly high when it affects platelet concentrates. In France the prevention methods developed over the past six years (donor selection, phlebotomy site preparation, first 30 ml diversion, systematic leuko-reduction...) aimed at limiting the introduction of bacteria in blood and bacterial proliferation. Several methods have been tested for the detection of bacterial contamination in platelet concentrates but none have been generalised. Difficulties were met, due to the necessity of 1) detecting only the platelet concentrates presenting a real infectious risk, when the presence of bacteria is observed in 2.2% (2-4%) of donated blood and 2) guaranteeing the availability of platelet concentrates. New methods have been developed which seem able to bring responses to these difficulties. Several processes are being (or will be) assessed, including automated blood culture, bacterial genomic detection with or without amplification, flow cytometric methods. In parallel, an indirect method able to detect the presence of bacteria, based on oxygen consumption, will also be evaluated. One (or several) of these processes should allow, in the short-term, to detect platelet concentrates presenting an infectious risk. In the future, the interest of bio-chips for bacterial detection in biological fluids must be investigated.

Section 5: Structural/genetic analysis of mAbs to blood group antigens. Coordinator’s report

Abstract: The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.

Étude régionale de validité, dans les services de soins, du test d’agglutination du contrôle ultime prétransfusionnel

Abstract: Resume Le risque d’accident transfusionnel ABO persiste en France a un niveau juge eleve par l’Agence Francaise de Securite Sanitaire des Produits de Sante. La verification de compatibilite pretransfusionnelle au lit du malade comporte en France, pour les globules rouges, un test d’agglutination obligatoire. Sa validite a ete peu evaluee dans les conditions de terrain. 847 soignants de 9 etablissements de sante publics et prives du Languedoc-Roussillon, representant 60 % de la transfusion regionale, ont participe a une etude de validite du test d'agglutination pretransfusionnel dans les services de soins. La sensibilite mesuree est de 93,9 % ± 3 %. Les seuls facteurs augmentant le risque de non-detection des incompatibilites sont l’anciennete dans le service superieur a 4 ans et, a un degre moindre, l’absence de formation continue a ce test. Il existe quelques possibilites d’ameliorer ce niveau de sensibilite. Ce test est juge suffisamment sensible pour recommander son maintien, mais insuffisamment sensible pour constituer a lui seul une protection contre les erreurs de transfusion. Il est necessaire, en plus des ameliorations du test, de le coupler veritablement a une verification des concordances scripturales normalisees.

Peut-on hiérarchiser les contre-indications au don du sang ?

Abstract: Because it symbolizes henceforth the sanitary risk and fears which are linked to it, the blood transfusion must master the potential or real risks associated to its practice. The analysis of these risks leads to confirm the initial selection phase of candidates for blood donation as an always original stage of the blood transfusion safety towards the identified infectious risks, but also mainly towards the emergent or modelled risks. The evaluation of the current system of prevention leads to consider two potential dangers: the ineffectiveness of the selection because of the lack of meaning given to this stage, and the donors' disaffection caused by badly accepted or badly justified deferrals. The deficit of meaning can be due to an insufficient information of the population as for the policy of collective prevention of transfusion-transmitted infectious diseases. It can be worsened by the construction of the pre-donation interview which can appear as a succession of questions without visible links. This paper suggests risk analysis to blood transfusion, and a reflection to improve this important stage of blood product safety approach represent by the selection of candidates to a blood donation.

La biovigilance, une vigilance exercée sur l'utilisation des produits issus du corps humain

Abstract: Organ transplantations and tissue/cells grafts are efficacious in many diseases. Nevertheless, beside the risk due to the technology which permits to carry out transplantations and grafts (surgery, tissue and cells collection, preservation, storage, cell expansion technics, immunosuppressive regimen,...), the microbiology risk must be controlled throughout the process leading to the transplantation or the graft. The structures, the organizations, the procedures, the information network and the controls assure the control of the risk. It is the main objective of human product vigilance. Future regulations will define the objectives of this sanitary vigilance more precisely.

Évaluation des publications scientifiques et médicales

Abstract: Quality assessment of publications article is now teached during medical studies and will soon be included into the program of the internship examination. The impact factor of medical journals is frequently used to evaluate the scientific quality of an article and consequently as an index of scientific productivity. Impact factor is an indicator of citation but not of the quality of an article and should not be used as a unique criterion for evaluating the quality of medical or scientific research works.

La cytométrie en flux en immunohématologie

Abstract: Flow cytometry is an objective, sensitive and quantitative technique which allows rapid and simultaneous analysis of several parameters on a great number of cells. Hence, flow cytometry is particularly suitable for the analysis of complex cell populations, rare events and quantitative studies. In immunohematology, flow cytometry is a very powerful approach to the study of mixed red cell populations (hematopoietic chimerism, transfusion or bone marrow transplantation), the detection of low frequency cell populations (reticulocytes, fetomaternal hemorrhage) and the quantitative analysis of red blood cell antigens.

Spécificité des techniques sérologiques de dépistage des anticorps anti-paludéens en immunofluorescence : étude comparative dans la pratique de qualification du don de sang

Abstract: Resume Grâce aux criteres de selection des dons de sang, aucun cas de paludisme transfusionnel n’a ete declare en France depuis 1994. Cependant, une technique serologique peu specifique conduit a une perte injustifiee en produits sanguins labiles. Materiels et Methodes : Une etude retrospective sur 55 serums disqualifies pour presence d’anticorps antipalustres a permis de comparer les resultats de differentes techniques serologiques : Paludix™, Falciparum-spot IF™ sans et avec elimination des antiglobulines naturelles selon deux methodes. Resultats : Avec le test Paludix™, 87 % des serums s’averent negatifs (p < 0,001) ; Falciparum–spot IF™, apres elimination des antiglobulines naturelles par neutralisation ou absorption, donne des resultats negatifs dans des proportions respectives de 87 % et 94,5 % (p < 0,001). Conclusion : Le nombre de faux positifs diminue considerablement avec les methodes evitant les fluorescences non specifiques provoquees par les antiglobulines naturelles. Le nombre de donneurs ayant sejourne en zone d’endemie palustre etant en constante augmentation, ce gain de specificite devrait permettre une economie des dons collectes.

Immunothérapie génique du cancer

Abstract: Resume Les concepts de l'immunotherapie anti-tumorale ainsi que les ameliorations technologiques qui en decoulent, ont considerablement evolue durant les dix dernieres annees. Cependant, l'utilisation de cytokines comme l'IFN-α et l'IL-2 represente encore a ce jour les principales armes therapeutiques dont l'efficacite clinique a ete demontree, notamment dans le cancer du rein, le melanome et certains lymphomes. Les approches d'immunotherapie, comme les approches vaccinales, la therapie genique et cellulaire, n'ont toujours pas demontre une efficacite clinique suffisante dans le traitement de tumeur solide. L'objet de cette revue est de faire le point sur les differentes approches actuellement developpees en immunotherapie des cancers. Nous envisagerons successivement les approches specifiques comme la vaccination antigenique et non-specifiques comme le transfert de genes « immuno-stimulants ».

Création et organisation d'une collecte mobile

Abstract: In France, nearly eighty per cent homologous blood donations are given in mobile settings. The collection site requirements, and particularly premises, are conditioning the security of persons, blood products quality, and blood collection efficiency. They also take a decisive part in the public image of the transfusion network. This paper describes an easy method for evaluating and validating premises used for mobile setting of blood collection.

Dossier transfusionnel et techniques de groupage sanguin : éléments essentiels de la sécurité transfusionnelle

Abstract: The clinical and biological control of the whole transfusion process is a major preoccupation for everyone dealing with blood transfusion. Specially when the patient is a female recipient or belongs to a group with a high prevalence of alloimmunisation. This case report points out the outstanding importance of the immune compatibility, which must be strongly maintained to prevent any harmful consequences. The transfusional record transmission and a simple and sensitive blood grouping test are essential to increase transfusion safety.