Variations of factor VIII:C plasma levels with respect to the blood group ABO.
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Biomedical subjects
Publications and source records attributed to Y Piquet.
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Three major steps are very important to increase transfusion safety: epidemiological step: donor selection; biological step: qualification of each blood donation; technological step: obtention of final product. The final therapeutical blood products must meet the requirements concerning their quality and safety, two factors with which the population is very demanding. These requirements can be ensured only if ethical, clinical epidemiological and biological parameters are clearly defined. The strict application of these criteria to each step of the transfusion process, from the blood donation to the final product, allows us to ensure the security of recipients with regard to known risks but also to anticipate other emergent risks.
Viral inactivation is one of the possibilities to reduce the residual risk of blood products. It is now applied to all plasma derived products (PDP). Application of such techniques to labile blood products (LBP) is difficult for two main reasons: any method should inactivate cell-associated viruses and should avoid any injury of the cells constituting the active ingredient. Physical techniques may reduce the viral content of cellular BPL (leucodepletion, washing, gamma irradiation), but none of them is active enough to comply with the present requirements for efficacy. An important work has been dedicated to the development of virus photoinactivation techniques. They consist of the addition of a photoreagent followed by illumination at an appropriate wavelength which results in a photochemical reaction responsible for the viral inactivation. Treatment of platelet concentrates by psoralen derivatives and UV-A illumination significantly inactivate in vitro enveloped and naked viruses, free and cell-associated viruses and also sequences integrated in the viral genome. Recent progresses have led to these results without detectable functional alteration of platelets and mutagenicity. Viral inactivation of red blood cells yet did not reach the same level because hemoglobin does not allow the use of the photoreagent compounds applicable to platelet concentrates. Viral decontamination of fresh frozen plasma by solvent and detergent, active on enveloped viruses, has been used in France since 1992. Other techniques of comparable efficacy, have received an agreement in other countries. The research on viral inactivation of LBP could prove to be of great importance in the near future in bringing additional safety to patients not only for the residual viral risk but maybe also for the residual bacterial risk of LBP.
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Leukocyte contamination of various blood products is implicated in febrile transfusion reactions, in alloimmunization phenomena and immunosuppression, and in the transmission of viral infections. The biggest difficulty in interpreting published reports lies in the methods used to quantify residual leukocytes. As automated cell counters are not precise and sensitive enough to detect low cell counts, we compared two techniques: i) the classical method using the Nageotte haemocytometer; and ii) fluorescent staining with propidium iodide (PI). We performed these two techniques to evaluate the residual white cell count in 41 platelet units collected by cytapheresis. The difference in results obtained with the two techniques was borderline significant (P = 0.05). To evaluate the accuracy of both techniques, we added a known number of lymphocytes to platelet concentrates that had been filtered three times. There was a good correlation between the values obtained using the two techniques (r > 0.99). However, though the difference between the known value and PI results was not significant, the difference for Nageotte cell counter results was significant (P < 0.0001). From a practical point of view, the PI fluorescent technique is simple, rapid, and easy to carry out. Our results demonstrate the reliability of the PI technique for the evaluation of residual leukocytes.
In order to increase the safety of blood products, we have developed a procedure for the virus inactivation of fresh frozen plasma. Several batches have been prepared and with the first 10 batches, each of them composed of 60 litres of plasma, we have determined a set of biological parameters. Virus inactivation was realised using TnBP (1%) and Octoxynol 9 (1%). After their elimination with castor oil using chromatography on insolubilized C18 resin, glycine was added and the pH of the plasma was adjusted to 7.4. Plastic bags were aseptically filled with a mean volume of 200 ml of plasma. The mean levels of coagulation factors were all over 0.7 U/ml and their recovery from initial plasma was nearly the same as total protein except for factor VIII:C. The net loss in factor VIII:C was 16%, when including the dilution of plasma. In vivo and in vitro tests demonstrated that in the final product there were no activated factors. As in fresh frozen plasma, the protein concentration was over 50 g/l and the potassium level lower than 5 mmol/l. According to these results, virus-inactivated plasma has the same qualities of fresh frozen plasma and could now replace it.
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Four monoclonal antibodies (MAbs) directed against the P1 blood group antigen were produced by hybridomas obtained from mouse immunized with turtle-dove avomucoid. One of the MAb (154 IX B6) selected as a blood typing reagent agglutinated native P1 and Pk1 red cells with a high titer but was inactive against native P2, Pk2 and p erythrocytes. After papain treatment the reactivity towards P1 and Pk1 erythrocytes was enhanced whereas p erythrocytes remained unreactive. A weak cross-reactivity of the MAb with the Pk antigen was suspected since enzyme-treated Pk2 erythrocytes became significantly agglutinated. Further analysis of the antibody specificity was established by binding studies using neutral glycolipids prepared from P1 and P2 erythrocytes, affinity immunoabsorbents carrying known oligosaccharide structures and hapten inhibition with synthetic oligosaccharides. The MAb bound weakly to the Gal alpha 1-4Gal structure common to P1 and Pk antigens but had a marked preference for the P1 determinant (Gal alpha 1-4 Gal beta 1-4 GlcNAc) and the binding was abolished by prior treatment of oligosaccharide antigens by alpha(not beta)-galactosidase, which supports evidence that a terminal alpha-galactose residue is involved in the blood group P1 and Pk specificities. The MAb has a slightly broader specificity than the human anti-P1 counterpart but can be used safely for routine blood typing.
When human citrated plasma is dialysed against a phosphate buffer containing Ca++, citrate anions are removed, thrombin is generated and soluble fibrinogen derivatives (fibrin monomers and/or soluble fibrin polymers) are formed. These derivatives are able to combine with human or bovine elastin to form a very stable addition product or adduct. The formation of the adduct is dependent on time, Ca++ and thrombin concentrations.
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Four plasmapheresis systems were comparatively studied: a centrifugation system (Haemonetics Model 50) and three filtration ones (Dideco BT 810, Organon Teknika PLASMAPUR and HemaScience Autopheresis Plasmacell). For each separator we studied the technical conditions for plasma separation, the biological characteristics of the collected plasma and the in vitro recovery of factor VIIc after preparation of cryoprecipitates. Concerning the plasma extraction rate, the HemaScience apparatus was the most efficient but values were quite similar to those obtained with the Organon Teknika machine. Contaminating cells were only found in plasma separated with the Haemonetics. Total protein and immunoglobulin levels were higher in plasma collected with the Haemonetics and HaemaScience systems. Factor VIIIc activity was comparable in plasma separated by filtration or by centrifugation while fibrinopeptide A levels were higher in plasma collected by Haemonetics. Whatever the machine, no statistical difference was observed when in vitro recovery of factor VIII was studied.
The kinetics of platelets labeled with indium-111 were investigated in 13 healthy subjects as well as in 9 patients in the asymptomatic interattack stage of asthma. The survival times of platelets in healthy subjects was 8.9 +/- 1 days; in asthmatic subjects it was 4.7 +/- 1.3 days (p less than 0.001). The survival curve is of a biexponential form in asthmatics, thus suggesting the presence of 2 populations: one with a short life span (23 +/- 7 h), representing a third of the total population (33 +/- 9%), and the other with a normal life span. No single preferred site of platelet sequestration was found. These results suggest the presence of functional or anatomic lesions of platelets in asthmatic patients, which can be explained only hypothetically at the present time.
Four monoclonal antibodies, with P1 specificity were obtained after fusion of myeloma cells and spleen cells from mice immunized with turtle dove ovomucoid. Immediately after the fusion, the culture supernatants were studied for specificity with panels of erythrocytes and red blood cells sharing rare phenotypes (P1K, P2K, p) in the P system. After cloning, four monoclonal antibodies were produced, these antibodies strongly agglutinate P1 red blood cells, specially when they are used with 3% of dextran or with a 350 mmol/l concentration of sodium.
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A non specific immunosupressive factor able to block an in vitro cytotoxicity reaction is demonstrated in the serum of pregnant women. A solution containing this blocking factor is obtained by gel filtration and precipitation of plasma by polyethylene glycol 4 000. Then after immunisation of rabbits the immune serum can be used for affinity chromatography. An alpha 2 glycoprotein has been separated which inhibits the in vitro cytotoxicity reaction and whose molecular weight determined by gel filtration and polyacrylamide gel is about 200 000 daltons.
A blocking factor present in the serum of pregnant women is able to inhibit non-specifically a cellular cytotoxicity reaction. It is a glycoprotein, migrating in the alpha-globulin region with a molecular weight of about 210,000 daltons. Its absence from the serum of women with recurrent abortions could predict the outcome of pregnancy.
A comparative study was performed on platelets frozen with dimethyl-sulfoxide (D.M.S.O.), glycérol and dimethyl-acetamide (D.M.A.C.). The results of tests realized in vitro (platelet yields after thawing platelet aggregation, hypotonic stress, release of enzymes and ultrastructure), and in vivo (life span and recoveries after transfusion) have clearly shown that D.M.S.O. is the best cryoprotective agent. The results obtained with glycerol were less interesting and more clinical data are needed in order to study the hemostatic effectiveness of platelets preserved with this agent. Dimethyl-acetamide gave the worse results and in our opinion, must not be used for the cryopreservation of platelets.
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