Depuis 1998, l’Établissement français du sang Aquitaine-Limousin (EFS AL) a mis en place le dépistage systématique du parvovirus B19 (PV B19) sur chaque plasma servant à la préparation du plasma ...viro-atténué par solvant–détergent (PVA-SD). La parution en 2005 de la monographie européenne du plasma humain (mélange de) traité pour viro-inactivation impose la recherche systématique du génome viral du parvovirus B19 dans les pools de plasmas. L’Agence française de sécurité sanitaire des produits de santé (Afssaps) a mis en place cette recherche dans le cadre du contrôle de qualité externe des produits sanguins labiles (PSL). Cette démarche s’inscrit dans le cadre de l’harmonisation de contrôle de la qualité des produits préparés en Europe même si le PVA-SD est considéré en France comme un produit sanguin labile. La mise en place de ce nouveau contrôle a nécessité une validation de la méthode et une étroite collaboration entre l’Afssaps et l’EFS AL pour l’organisation logistique. La validation a consisté en la mise au point d’une méthode semi-quantitative par
Polymerase Chain Reaction (PCR) en temps réel avec extraction automatisée. Ce travail collaboratif nous a permis de contrôler 1642 pools de plasmas. Tous les résultats sont inférieurs au seuil de 10,0 UI/μL requis par la Pharmacopée européenne. Les résultats de l’Afssaps sont concordants avec ceux de l’EFS AL qui effectue à la fois le dépistage du parvovirus B19 sur les plasmas unitaires avant la constitution du mélange de plasmas et sur le mélange de plasmas avant inactivation virale.
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/μL. 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.
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.
Today, haematopoietic stem cell graft from placental blood concerns more than 15 % of allogeneic grafts. An inter-laboratory study of the quality control of defrosted cord blood units has been ...coordinated by the French society for cell and tissue bioengineering (SFBCT), with the cord blood bank of Bourgogne Franche-Comté and controlled by the French health products safety agency (Afssaps). The aim of this study is to ensure the inter-laboratory reproducibility of the quality controls practised by the banks during defrosting. The cellular outputs were analyzed according to the defrosting techniques, according to the method used in flow cytometry: single-platform (SP) versus double-platform (DP), or the product nature, i.e. in total blood or miniaturized.
Forty-two units of placental blood (USP), which were out of range were provided for defrosting to 14 participating sites. USP were defrosted and controlled according to the procedures of each bank. Once the USP is defrosted, a part of the product was controlled by the site and the other part by Afssaps. Following controls were carried out: numeration of the total nucleated cells (TNC) and of CD34+ cells (made by a SP method in Afssaps) and functional assay.
Concerning TNC, the defrosting sites obtained a cellular output of 94 %+/-28 in day 0 compared with an output of 72 %+/-24 in Afssaps showing a rather good stability of the USP transmitted with an average deviation of 23 %+/-22. The freezing process with or without reduction of volume does not affect this variation. Concerning the numeration of CD34+ cells, the average deviation between the participating sites and Afssaps was 29 %+/-23 compared with 21 %+/-16 for the sites using a SP method against 47 %+/-25 for those using a DP method. The CD34+ outputs are equal to 82 % +/- 60 in day 0 for the participating sites against 52 %+/-20 for Afssaps. For the sites using a DP method, it is stressed that this output is particularly high with a rate of 126 %+/-90 (n=15) whereas it is 62 %+/-20 (n=32) for the sites using a SP method.
These results underline a good stability of viable CD34+ cells and a greater reliability of the SP methods for the CD34+ cell numeration for these defrosted USP. Lastly, the results of the functional assay regarding the average clonogenicities (equal to 15 %) reinforce the conclusions on the quality of the defrosted products.
La greffe de cellules souches hématopoïétiques (CSH) placentaires représente aujourd’hui plus de 15 % des greffes allogéniques. Une étude multicentrique du contrôle de qualité des greffons de sang ...placentaire décongelés a été coordonnée par la Société française de bio-ingénierie cellulaire et tissulaire (SFBCT), en liaison avec la banque de sang placentaire de l’Établissement français du sang (EFS) Bourgogne Franche-Comté et contrôlée par l’Agence française de sécurité sanitaire des produits de santé (Afssaps). Cette étude a pour objectif de s’assurer de la reproductibilité inter-laboratoires des contrôles qualité pratiqués par les banques lors de la décongélation. Les rendements cellulaires ont été analysés selon les techniques de décongélation, selon la méthode utilisée en cytométrie en flux : simple plateforme (SP) versus double plateforme (DP), ainsi que selon la nature du produit congelé (sang total ou miniaturisé).
Quarante-deux unités de sang placentaire (USP) déclassées ont été adressées pour décongélation à 14 banques. Les USP ont été décongelées et contrôlées selon les procédures de chaque banque. Une fraction du produit décongelé a été contrôlée par l’Afssaps. Les contrôles suivants ont été réalisés : numération des cellules nucléées totales (CNT) et des cellules CD34+ (méthode SP à l’Afssaps) et test fonctionnel.
Le rendement en CNT obtenu par les banques est de 94 %
±
28 à j0 pour un rendement de 72 %
±
24 à l’Afssaps, montrant une assez bonne stabilité des USP transmises. Le procédé de congélation avec ou sans réduction de volume n’a pas d’incidence sur cet écart. Concernant la numération des cellules CD34+, l’écart moyen entre les sites participants et l’Afssaps est de 29 % ± 23. Il est de 21 %
±
16 avec les sites ayant utilisé une méthode SP contre 47 %
±
25 avec ceux ayant utilisé une méthode DP. Les rendements en CD34+ sont de 82 %
±
60 à j0 pour les sites participants et de 52 %
±
20 pour l’Afssaps. Pour les sites utilisant une méthode DP, ce rendement atteint un taux de 126 %
±
90 (
n
=
15) alors qu’il est de 62 %
±
20 (
n
=
32) pour les sites utilisant une méthode SP.
Ces résultats mettent en évidence une bonne stabilité des cellules CD34+ viables et une meilleure fiabilité des méthodes SP pour la numération des cellules CD34+ dans les USP décongelées. Enfin, les résultats du test fonctionnel au vu des clonogénicités moyennes observées (égales à 15 %) renforcent les conclusions sur la qualité des produits résultant de la décongélation.
Today, haematopoietic stem cell graft from placental blood concerns more than 15 % of allogeneic grafts. An inter-laboratory study of the quality control of defrosted cord blood units has been coordinated by the French society for cell and tissue bioengineering (SFBCT), with the cord blood bank of Bourgogne Franche-Comté and controlled by the French health products safety agency (Afssaps). The aim of this study is to ensure the inter-laboratory reproducibility of the quality controls practised by the banks during defrosting. The cellular outputs were analyzed according to the defrosting techniques, according to the method used in flow cytometry: single-platform (SP) versus double-platform (DP), or the product nature, i.e. in total blood or miniaturized.
Forty-two units of placental blood (USP), which were out of range were provided for defrosting to 14 participating sites. USP were defrosted and controlled according to the procedures of each bank. Once the USP is defrosted, a part of the product was controlled by the site and the other part by Afssaps. Following controls were carried out: numeration of the total nucleated cells (TNC) and of CD34+ cells (made by a SP method in Afssaps) and functional assay.
Concerning TNC, the defrosting sites obtained a cellular output of 94 %
±
28 in day 0 compared with an output of 72 %
±
24 in Afssaps showing a rather good stability of the USP transmitted with an average deviation of 23 %
±
22. The freezing process with or without reduction of volume does not affect this variation. Concerning the numeration of CD34+ cells, the average deviation between the participating sites and Afssaps was 29 %
±
23 compared with 21 %
±
16 for the sites using a SP method against 47 %
±
25 for those using a DP method. The CD34+ outputs are equal to 82 % ± 60 in day 0 for the participating sites against 52 %
±
20 for Afssaps. For the sites using a DP method, it is stressed that this output is particularly high with a rate of 126 %
±
90 (
n
=
15) whereas it is 62 %
±
20 (
n
=
32) for the sites using a SP method.
These results underline a good stability of viable CD34+ cells and a greater reliability of the SP methods for the CD34+ cell numeration for these defrosted USP. Lastly, the results of the functional assay regarding the average clonogenicities (equal to 15 %) reinforce the conclusions on the quality of the defrosted products.
Purpose Considering the importance of having a precise, robust and especially reproducible ECD counting method, Afssaps organized from April 2008 to June 2009 a second assessment of the reliability ...of the routine cell count within the 18 french Eye banks.
Methods The study design was similar to the first assessment driven by the laboratory ‘Biology, engineering and imaging of Corneal Graft’ in 2003 (Transplantation 2004; 78: 1299‐1302).5 test corneas (1 mm2 of flat mounted, fixed and alizarin stained human corneal endothelium) were selected and sent to the 18 Eye banks. All the usual technicians of each bank had to count the test corneas using the routine method(s) employed to assess grafts.
Results 430 counts were carried out by 70 eye banks technicians, by manual and/or image analysis system. 42% (180/430) deviated by more than 10% from the expected ECD. Among them, 128 were over‐estimated (max +88%) and 52 were under‐estimated (max ‐31%). 2 banks constantly over‐estimated (in the mean +31,7% and +42,7%, no calibration and/or material problem) but the 16 other banks were in average within ±13% from expected ECDs. For manual methods, a statistically significant difference between banks was observed for the 5 test corneas, whereas no difference was observed with image analyzers. ECD obtained with the analysers were closer to expected values than with the manual methods. Compared to the 2003 study, reliability of ECD determination globally improved.
Conclusion Image analysis systems prove more reliable (precise and with a lower intra and inter observer variability) than manual counting methods. This ‘second look’ of Eye banks will allow editing recommendations to improve ECD determination.
PDF
