Cell Response to Surface Stimulation

The injection of anti-D into Rh-negative subjects who have Rh-positive red cells in the circulation results in the inhibition of immunization against the D-antigen¹. On the other hand, subjects who have had a primary anti-D response to Rh-positive red cells frequently give a good secondary response to small doses of red cells despite the presence of anti-D in their plasma. The difference in action between the passively-administered and the actively-produced anti-D might lie in the fact that the injected IgG anti-D is derived from a pool of donors and therefore contains a number of IgG antigens which are foreign to the recipient, compared with the autologous nature of the anti-D present after a primary response.     

抗-D抗体及其应用

Rh血型是仅次于ABO血型系统的人类红细胞抗原系统,至今已发现40多种抗原,但与临床密切相关的是D,C、c、E、e等5种抗原,其中最主要的是D抗原。相应的抗-D抗体无论是在临床输血检测,还是在Rh(D)新生儿溶血病、溶血性输血反应等的防治方面均具有非常重要的意义。传统的抗-D抗体的制备需用人的血清,来源受限。各种抗-D人源性单克隆抗体和基因工程抗体已经成为发展方向。     

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for sup-pressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human trans-formed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.     

Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

Anti-leuko-platelet allo-immunization in blood replacements. Do transfusions of platelets from single donors present an advantage over platelets from multiple donors?

42 patients with acute leukaemia, treated with cytotoxic drugs, have been evaluated retrospectively: --group I: 11 patients received packed red blood cells and platelets from single donors; --group II: 6 patients received packed red blood cells and platelets from multiple donors; --group III: 25 patients received packed red blood cells and platelets from single or multiple donors and granulocytes transfusions. There was no difference in age, sex, time of follow up, number of transfusions, in the three groups. The rate of alloimmunization defined as lymphocytotoxicity against more than 20% of a panel of 24 lymphocytes, was 33% (36% group I--33% group II--32% group III). This study shows that platelets from single donors are of no use in preventing or delaying alloimmunization. On the other hand, their major interest is to provide alloimmunized patients with compatible platelets.     

Occurrence of non-A, non-B post-transfusion hepatitis in heart surgery. Prospective study on the value of the determination of serum alanine aminotransferase and detection of anti-HBc antibodies in blood donors

We studied a group of 64 patients undergoing cardiac surgery for the occurrence of post-transfusion hepatitis during a follow-up period of 5 months. They received blood units (packed red cells in saline-adenine-glucose medium and/or fresh frozen plasma exclusively) from 447 volunteer donors. Post-transfusion hepatitis was identified in 5 patients: 1 patient had cytomegalovirus hepatitis and the remaining 4 cases were defined, by exclusion, as non-A, non-B hepatitis (with prevalence and incidence rates of 80% and 6.25% respectively). We found no statistically significant differences between the numbers of transfused blood product units in patients who developed non-A, non-B hepatitis as compared to those who did not. Our analysis of the predictive effectiveness of alanine aminotransferase and anti-HBc antibodies screening in blood donors to prevent non-A, non-B post-transfusion hepatitis led to the following conclusions: we failed to confirm the association between anti-HBc in blood donors and enhanced risk of non-A, non-B hepatitis in recipients since no case developed among patients receiving blood products from anti-HBc positive donors. So, 20 donors (4.5%) would have been discarded without any reduction of the incidence of non-A, non-B hepatitis. we could not confirm nor exclude the possibility that screening donor blood for elevated alanine aminotransferase levels would have reduced the number of non-A, non-B hepatitis in recipients.     

Antibodies against the common polysaccharide and protein protective antigens of Pseudomonas aeruginosa in normal blood donors.

Screening of normal plasma obtained from 172 blood donors from the Helsinki area and from 46 blood donors from the Moscow area was performed in order to reveal 'natural' antibodies to the common polysaccharide (rhamnan) and protein antigens of P. aeruginosa. Antibodies were detected by ELISA. Among blood donors from the Helsinki area high titres of antibodies to the protein antigens were detected in 42 active blood donors (24.4%) and very high titres in nine (5.3%) highly-active blood donors, whereas in the Moscow area in 15 (34.9%) and in one case (2.3%), respectively. Antibodies to the common polysaccharide antigen were determined in the Helsinki area in 23 active blood donors (13.4%) and in one (0.5%) highly active blood donor, whereas in the Moscow area in four active blood donors (8.6%). The plasma contained both polysaccharide and anti-protein antibodies. The level of antibodies to the polysaccharide antigen was lower than the level of antibodies to the protein antigens. There was no statistically significant difference between the corresponding values of blood donor groups from the Helsinki and Moscow areas.     

Sero-prevalence of RBC alloantibodies among Han and minority patients in Xinjiang

The aim of the study was to examine the sero-prevalence and frequency of red blood cell (RBC) alloantibodies (RAAbs) and to investigate the risk factors for producing RAAbs among the Han and Uyghurs in Xinjiang. The RBC antibody screening test and identification were conducted for 45,163 Han and 70,633 Uyghurs admitted to the First Affiliated Hospital of Xinjiang Medical University from October 2010 to December 2014. RBC alloantibodies against the Rh antigens were the most frequent, including anti-E(21.7% in the Han, 21.6% in Uyghurs) and anti-D(18.5% in the Han, 18.2% in Uyghurs). Notably, the sero-prevalence of anti-K and anti-Fy^a was also high in Xinjiang. Transfusion and pregnancy were risk factors among both the Han and Uyghurs; furthermore, Uyghurs had a higher sero-prevalence of RBC antibodies compared to that of Han because of a higher incidence of these risk factors. We concluded that RBC alloantibodies against the Rh factor showed the highest frequency, and antibodies against Asian-related high-frequency antigens, including Fy^a and low-frequency antigens, such as K were notably high in Xinjiang.     

Quantitative comparison of Rh1 (Rho, D) antigen on D, Du and d red cells by radioimmunoassay using 125I-protein A

Relative Rh1 (Rho, D) antigen contents of the red Rh: 1 (Rh positive, D), Rh: wl (Rh variant, Du) and Rh: -1 (Rh negative, d) cells were estimated from the quantity of 125I-protein A bound to the sensitized red cells. The isotope binding activity to both D and Du cells decreased in parallel with the dilution of anti-D serum. The relative amount of the 125I-protein A bound to Du cells was about one-sixth that of D cells without papain treatment, while no isotope binding was observed in d cells. The Du red cells were quantitatively deficient in Rh1 (Rho, D) antigen activity compared with the D cells. A radioimmunoassay using 125I-protein A was a very useful method for studies regarding measuring the relative amounts of various blood group antigens.     

Study of equivalents of rhesus antigens in non-human primates]

Human alloantibodies specific of some Rh antigens cross-react with non human primates red blood cells. These crossreactions demonstrated that only African apes express equivalents of Rho (D) and hr' (c). The antigenic resemblance between these two human antigens and their primate homologues is confirmed by the reactivities of human anti-D and anti-c monoclonal antibodies. The use of a human Rh cDNA probe allowed to confirm by Southern blot hybridization that nonhuman primates possess Rh-like genes. The number of Rh-like genes per haploid genome was deduced from the results obtained with exon-specific probes.     

Relevance of blood groups in transfusion of sickle cell disease patients

Blood groups are clinically significant in sickle cell disease (SCD) as transfusion remains a key treatment in this pathology. The occurrence of a delayed haemolytic transfusion reaction (DHTR) is not rare and is a life-threatening event. The main cause of DHTR is the production of alloantibodies against red blood cell antigens. The high rate of alloimmunization in SCD patients is mainly due to the differences of red blood groups between patients of African descent, and the frequently Caucasian donors. From an immuno-haematological point of view, DHTR in SCD patients has specific features: classical antibodies known to be haemolytic can be encountered, but otherwise non significant antibodies, autoantibodies and antibodies related to partial and rare blood groups are also frequently found in individuals of African descent. In some cases, there are no detectable antibodies. As alloimmunization remains the main cause of DHTR, it is extremely important to promote blood donation by individuals of African ancestry to make appropriate blood available.     

Collection of anti-D immune plasma after labor. Apropos of 4370 plasmaphereses

4 370 plasmaphereses have been realized on women in the post delivery period for collection of immune anti-D plasma. 1.550 liters of such plasma have been collected and submitted to Cohn fractionation, leading to the preparation of about 100 000 vials of 100 micrograms. Thus, most of the Paris area needs for anti-D IgG have been covered for the past 13 years. Medico-legal aspects of blood donation at this particular period of a woman's life and proper motivations of these donors are underlined. Prospects for this type of blood collection are analyzed as a conclusion.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data.

BACKGROUND: Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification. METHODS: By eight-colour cytofluorimetric analysis, we investigated the T cell compartment in donors of different age (young, middle-aged, and centenarians). T cell subsets were identified by combining positive and negative expression of antigens. The resulting data set was organized into a matrix and subjected to CA and PCA. RESULTS: CA clustered people of different ages on the basis of cytofluorimetric profile. PCA of the cellular subsets identified centenarians within a different cluster from young donors, while middle-aged donors were scattered between these groups. These approaches identified T cell phenotypes that changed with increasing age. In young donors, memory T cell subsets tended to be CD127+ and CD95- whereas CD127-, CD95+ phenotypes were found at higher frequencies in people with advanced age. CONCLUSIONS: Our data suggest the use of bioinformatic approaches to analyze large data-sets generated by PFC and to obtain the rapid identification of key populations that best characterize a group of subjects.     

Transfusion-induced malaria: risks, prevention and cost (1 year's experience)

Three cases of transfusion induced paludism have been seen in Rouen since 1976. Two of them were due to Plasmodium falciparum and the third to Plasmodium malariae. In order to improve the prevention of this disease, a survey was carried out in 1980 which concerned 51,000 blood samples, 1,020 blood donors were selected because they had lived in a malarial endemic zone. Among them, 92 subjects were detected by an indirect immunofluorescence reaction as having a titer exceeding 1: 20 and their blood was kept aside any transfusional use. These results were studied according to the geographical area concerned, the ethnical origin, the time elapsed since the return and whether or not a chemoprophylaxis had been used. The present study gives further evidence of the risk linked to a stay in Africa. But neither the time since the return, nor the use or not of a chemoprophylaxis seemed to influence the serological results. The five years exclusion rule, used up until now, came to be at the same time too much and insufficiently limitative, since it would have eliminated 1.4% blood donors while the serological controls would eliminate only 0.18%. On the other hand, 14 blood donors, who were back in France for more than 5 years and presented with a positive serology, would have been considered as normal. In the light of these results, a practical line is given based on the association of a simple inquiry to a malarial serology.     

Molecular and cellular biology of malaria

Thanks to the extensive use of recombinant DNA technology and immunological methods, much insight into cellular functions of the human malaria parasite Plasmodium falciparum has been gained since it was learnt ten years ago how to grow this organism in culture. The amino acid sequence of over a dozen surface proteins of the parasite and of several proteins the parasite excretes into its most important host cell, the erythrocyte, have been determined. Interestingly many of these proteins show blocks of repeated amino acids. Several proteins have been shown to be involved in specific aspects of the complex hostparasite interaction, such as penetration of host cells or increased stickiness of infected red blood cells in the blood vessels. Some of the proteins described here may be protective antigens and may become important in vaccine development.     

Specific anti-Yersinia G- and M-antibodies in healthy blood donors and in patients with alimentary intoxication

The work deals with the results of determination of specific antibodies in blood donors of Moscow and Tula and in patients with alimentary toxicoinfection, made with the use of enzyme immunoassay on the basis of Yersinia enterocolitica lipopolysaccharides (LPS), serovars O3 and O9. The sera of patients with alimentary toxicoinfection were found to yield positive reactions with Y. enterocolitica LPS in 35.9% of cases (the number of such reactions obtained with blood donor sera was 3 times less). The presence of cross reactions between Y. enterocolitica LPS and the microsomal antigens of the thyroid gland was established. A high detection rate of antibodies to the microsomal antigens of the thyroid gland among blood donors of Tula was registered.     

A procedure for decreasing the level of 2,3-bisphosphoglycerate in red cells in vitro

The physiological adaptation to anemia and other hypoxic states includes an increase in the level of 2,3-bisphosphoglycerate (2,3-DPG) in the red cell. We suggest that the high level of 2,3-DPG may have adverse effects in vivo. It has been found that red cells incubated with glycolate lose 2,3-DPG at a rapid rate relative to controls. ATP is stable. Net 2,3-DPG synthesis is observed after the glycolate is removed from the cells suggesting that they are not harmed. The effect appears to be specific for glycolate since lactate, glyoxylate, glycerate, acetate, and citrate were without effect. This procedure could be used to assess the effects of decreasing the 2,3-DPG level to normal in the erythrocytes of sickle cell and other anemias.     

Cytoskeleton alterations of erythrocytes from patients with Fanconi's anemia

Fanconi's anemia (FA) is a very rare genetically heterogeneous disease which has been hypothesized to be defective in the detoxification of reactive oxygen species. In this work we report the results obtained by morphometric and biochemical analyses on the red blood cells (RBCs) from FA patients. With respect to RBCs from healthy donors the following changes have been detected: (i) a variety of ultrastructural alterations, mainly surface blebbing typical of acanthocytes and stomatocytes; (ii) a significant quantitative increase of these altered forms; (iii) modifications of spectrin cytoskeleton network; (iv) an altered redox balance, e.g. a decreased catalase activity and significant variations in the GSSG/GSH ratio. We hypothesize that remodeling of the redox state occurring in FA patients results in cytoskeleton-associated alterations of red blood cell integrity and function.     

In vitro immunization of human lymphocytes with Epstein-Barr virus capsid antigen]

Conditions for in vitro immunization of human lymphocytes from adult peripheral blood, tonsils and cord blood with Epstein-Barr Virus (EBV) capsid antigens have been studied. Pokeweed mitogen and B cell growth factor from Namalva cell line were shown to induce a significant production of specific antibodies by human lymphocytes stimulated with EBV. This effect made it possible to generate primary immune response in vitro using lymphocytes from EBV seronegative donors.