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BACKGROUND: The freezing phase is a critical step of the freezing process of the hematopoietic stem cells. To standardize the decrease of the temperature, the use of a programmable freezer is recommended. There is no available protocol, neither to describe exactly the validation of a programmable freezer, nor to prove the performance of the freezing/thawing step of the grafts. METHOD: We describe a validation protocol with three phases: first a qualification of installation, then an operational qualification and finally, a qualification of performance. The validation is performed in tandem between the freezer which is routinely used (Nicool Plus) and a new one (Freezal). RESULTS: With this protocol, we demonstrate the efficacy of the freezing program and its ability to assure the quality of the grafts reinjected to the patients, particularly in terms of cellular efficiency on CD34+ hematopoietic stem cells. On these cells, we measured a significant increase of cellular efficiency (+10%) after freezing with the Freezal. CONCLUSION: Here, we propose a validation protocol which is able to qualify a programmable freezer. This protocol can optimize the capability of the freezer and is able to prove its performance.

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PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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Splenic lymphoma with villous lymphocytes (SLVL) is a low grade lymphoproliferation characterized by a massive splenomegaly, an absence of lymphadenopathy and the presence in the peripheral blood of atypical B-lymphocytes with hairy-cell appearance. We have studied the morphological, immunological and molecular characteristics of 3 cases of SLVL. SLVL presented on blood smears characteristic irregularities of the plasma membrane consisting in thin and short villi unevenly distributed. The main phenotype was CD5-, CD11c+, and CD25-, but individual SLVL cases can not be identified by using immunohistochemical criteria alone. Clonal rearrangements of the immunoglobulin heavy chain gene were found in all 3 cases and in one case presented a bcl2-JH rearrangement. SLVL are clonal B-cell lymphoproliferations and can be associated with t(14; 18) translocation.

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Human recombinant erythropoietin (rHu-Epo) is now extensively used in chronic renal failures; this treatment, resulting in a correction of the severe anemias seen in hemodialysed patients, may in turn lead to a resistance to rHu-Epo therapy by reason of the shortage of erythropoiesis factors, such as iron, vitamin B12 and folates. The utility of the red cell indices (MCV, MCH, RDW) for detection of early iron, folate and B12 deficiencies was studied in eighteen hemodialysed patients with end-stage renal failure treated with rHu-Epo; Microcytosis (MCV < 80 fl) was found ineffective in detecting iron deficiencies as well as macrocytosis (MCV > 100 fl) in folate and B12 deficiencies, partly due to the high incidence of associated iron and folate deficiencies. Lowered MCH (< 27 pg) was not more efficient than microcytosis in detecting early iron deficiencies. Increased RDW was the most sensitive feature for folate, iron and B12 deficiencies with respective sensitivities of 62.5%, 72% and 75%. The global specificity for detecting all deficiencies was 74%. However, high RDW values were not indicative of any type of deficiency; it may thus be concluded that RDW is a non expensive, non invasive and sensitive test, which allows a selection of hemodialysed patients treated with rHu-Epo for a complete investigation program, in order to detect early iron, B12 and folate deficiencies.

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