[Hepatitis E virus: Blood transfusion implications]. / Virus de l'hépatite E, implications en transfusion sanguine.

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementation's impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by French's health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.

[Assessment of a transfusion emergent risk: the case of HEV]. / Estimation d'un risque transfusionnel émergent: l'exemple du VHE.

BACKGROUND: The risk assessment for blood transfusion is an essential step that must precede any screening strategy of a pathogen transmitted by transfusion. After several cases of HEV transmission by transfusion in France, a risk assessment for this virus was performed. METHODS: We used a method based on the prevalence of HEV-RNA in plasmas collected for the preparation of SD-plasma. To estimate the rate of HEV-RNA positive among all blood donations, data on SD-plasma were adjusted on the following HEV risk factors: gender, age group and region of residence. We assumed that HEV risk factors were the same in plasma donors and whole blood donors. RESULTS: Among 57,101 plasma donations tested for HEV-RNA in 2013, 24 were positive (crude rate of 4.2 per 10,000 donations). After adjustment, the total number of HEV-RNA positive blood donations was estimated at 788, accounting for a rate of 2.65 per 10,000 donations (95% CI: 1.6-3.7) or 1 in 3800 donations (1 in 6,200-1 in 2,700). This rate was 12 times higher in men than in women, increased with age, and varied according to region of residence. CONCLUSION: The risk of blood donation contamination by HEV has been estimated to be 1 in 3800 donations in 2013. An essential input is still missing to assess now the risk in recipients: the minimum infectious dose. Furthermore, the risk in recipients has to be analyzed according to characteristics of transfused patients: presence of anti-HEV immunity, existence of chronic liver disease or immunodeficiency.

[Detection of B19 parvovirus in plasma pools before solvent-detergent treatment of plasma: AFSSAPS and EFS Aquitaine-Limousin's experience]. / Détection du génome viral du parvovirus B19 dans les pools de plasmas servant à la préparation du plasma frais congelé traité pour viro-atténuation par solvant-détergent : expérience de l'Afssaps et de l'EFS Aquitaine-Limousin.

Since 1998, the Aquitaine-Limousin branch of the French Blood Institute has set up a parvovirus B19 (PV B19) systematic screening on each unit of plasma to be treated by solvent-detergent procedure for virus inactivation. Parvovirus B19 nucleic acid systematic testing in plasma pools became mandatory since 2005 (European monograph "Human plasma" - pooled and treated for virus inactivation). The French competent state authority (AFSSAPS) has decided to introduce this test as a part of the external quality control of labile blood products. This process is related to the harmonization of quality control practice realised on blood products in Europe even if the human plasma pooled and treated for virus inactivation by solvent-detergent is considered in France as a blood labile component. Implementation of this test required a validation step and a close cooperation between AFSSAPS and Aquitaine-Limousin blood transfusion centre. Validation consisted in perfecting a semi-quantitative, real-time nucleic acid testing method with automated extraction. This collaborative study leads us to control 1642 plasma pools. All the results were under the threshold of 10,0 IU/microL. AFSSAPS's results were in agreement with those of Aquitaine-Limousin's blood transfusion center who carry out the parvovirus B19 screening both on fresh frozen plasma units composing the pool and on plasma pools.

[Nucleic acid testing: Biomerieux Roche technology. Setting up of limits concerning temperature, volume and incubation time during the nucleic extraction step]. / Dépistage génomique viral : technique combinée biomérieux/roche. Etude des limites de tolérance des parametres températures, temps d'incubation et volumes au cours de l'extraction des acides nucléiques par le nuclisens extractor.

Nucleic acid testing is carried out in several steps: plasma pooling, nucleic acid extraction, amplification and detection. The target values of temperature, volume and incubation time are mentioned in the initial protocol without giving their limit values. The objective of this work was to determine the range of values in which the test remains positives. So we have tested: 1) the temperature and incubation time of plasma in lysis buffer (37 degrees C +/- 3 et 30 min +/- 10); 2) the influence of volume and incubation time of silica (50 mul +/- 10 et 10 min +/- 5); 3) the variation of the eluate volume after nucleic extraction. We have also studied the influence of the internal control volume variation. For each parameter the assays were performed at sensitivity limit of the technique and repeated several times. Our results showed that: 1) for all parameters evaluated the tests remain positive within a wide range of values; 2) It is not necessary to set up a sophisticated measurement process; 3) the technique is robust.

[Clinical and biological qualifications for blood donations]. / Qualification clinique et biologique des dons sanguins.

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 attenuation of labile blood products]. / Atténuation virale des produits sanguins labiles.

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.

Measurement of residual leukocytes in platelet concentrate units.

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.

Virus inactivation of fresh frozen plasma by a solvent detergent procedure: biological results.

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.

Characterization of a murine monoclonal antibody specific for the human P1 blood group antigen.

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.

Platelet kinetics in stable atopic asthmatic patients.

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.

Soluble fibrinogen derivatives generated by thrombin: affinity for elastin.

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.

[Production and characterization of monoclonal antibodies with P1 specificity]. / Obtention et caractérisation d'anticorps monoclonaux de spécificité anti-P1.

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.

Technical aspects of different donor plasmapheresis systems and biological results obtained in collected plasma.

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.