Collection, processing and testing of bone, corneas, umbilical cord blood and haematopoietic stem cells by European Blood Alliance members.

BACKGROUND AND OBJECTIVES: A questionnaire study was carried out in collaboration with the European Blood Alliance (EBA) Tissues and Cells (T&C) working group. The aim was to assess the level of involvement and commonality of processes on the procurement, testing and storage of bone, corneas, umbilical cord blood (UCB) and haematopoietic stem cells (HSC) in order to identify different practices and to explore whether recommendations can be made for harmonization. MATERIALS AND METHODS: An online questionnaire was used for data collection in 2011, and 43 replies were received covering 71 product answers from 13 countries. RESULTS AND CONCLUSIONS: Estimated percentages of tissue and cell banking covered by EBA member blood banks as a proportion of all collections of each individual country varied markedly. There were also major differences in the amounts of products collected and discarded and in proportions tissues provided for grafting. However, discarding of certain collections also reflects the practice of increasing the likelihood of the very best units being used for transplantation. Harmonization of possible practices should focus on matching supply with demand and on identifying the most efficient operators. This could allow for the development of practices for minimizing unnecessary collections.

[Assessment of the usefulness of autologous serum skin testing in chronic urticaria: a retrospective single-centre study of 74 patients]. / Evaluation de l'utilité du test au sérum autologue dans l'urticaire chronique idiopathique: étude rétrospective chez 74 patients.

BACKGROUND: Chronic urticaria (CU) is a debilitating disease, and patients and their physicians often seek an aetiological explanation. Studies have suggested that idiopathic CU is associated with the presence of serum auto-antibodies that may be detected by autologous serum skin test (ASST). AIM: To confirm the frequency of positivity of ASST and to evaluate its usefulness and possible correlation with the severity of urticaria (greater resistance to AH1, greater activity score or longer duration). PATIENTS AND METHODS: Patients referred for CU between 1 October 2001 and 31 March 2005 were submitted to standardized explorations including clinical examination, physical tests, CBC, ESR, CRP and anti-thyroperoxidase antibodies, and an ASST was ordered. Inclusion criteria included no discernible cause of CU, acceptance of the protocol, including blood sampling and injection of ASST 3 weeks later, serological tests for HBV, HCV and HIV, and discontinuation of anti-H1 agents and corticosteroids. Exclusion criteria comprised the presence of dermographism, physical urticaria, urticarial vasculitis, and failure to discontinue anti-H1 drugs or corticosteroids. In April 2006, we contacted patients by mail to assess their current treatment, their CU activity score or its resolution. RESULTS: Seventy-four patients (67 women, seven men) of mean age 43 years were included. ASST was positive in 43 patients (58 %) and negative in 31 (42 %). The only noticeable difference, although not statistically significant (p=0.23), was a positive anti-thyroperoxidase antibody result in 12 % of patients with negative ASST versus 24 % of patients with positive ASST. The occurrence of angioedema, the duration of CU, the severity score, the relative inefficiency of AH1 and the use of corticosteroids or cyclosporine were similar between the two groups. DISCUSSION: ASST was positive in more than half of the patients with idiopathic CU both in our study and in the literature, with sensitivity of around 70 % and specificity approaching 80 %. However, while this test can help patients understand their disease better and avoid both a fruitless search for other causes and unnecessary proscription of food allergens, our study shows that positive or negative ASST results have no bearing on treatment and are not associated with greater severity of urticaria or greater resistance to treatment. Consequently, we do not recommend routine use of ASST in patients presenting idiopathic CU.

The role of autologous bone marrow transplantation in 46 adult patients with non-Hodgkin's lymphomas.

Forty-six patients with non-Hodgkin's lymphoma (NHL) were treated with autologous bone marrow transplantation (ABMT) in two different institutions. All patients were pretreated with conventional chemotherapy. Three different conditioning regimens were used, and 20 patients underwent bone marrow purging. Twelve patients were treated in first complete remission (CR); eight are in unmaintained CR 8 to 104 months after ABMT. Five patients were grafted in first partial remission (PR) after conventional therapy; all achieved CR, and all remain in prolonged CR (first CR for four patients, second CR for one patient). Of 21 patients with chemosensitive relapses, 13 patients are in prolonged unmaintained CR 8 to 94 months after ABMT. Eight patients with resistant disease remained uncured by ABMT; all eight died, six from progressive illness and two from toxicity. The current 3-year disease-free probability is 60% for all patients, 0% for refractory disease; 82% for first PR or CR, and 60% for sensitive relapses (SRs). These results confirm the efficacy of ABMT in the treatment of chemosensitive NHL with bad prognosis.

Long-term cultures to evaluate engraftment potential of CD34+ cells from peripheral blood after mobilization by chemotherapy with and without GM-CSF.

In this study we used a long-term culture system to evaluate engraftment potential of human peripheral blood (PB) cells mobilized by chemotherapy (CT) associated or not with granulocyte-macrophage colony-stimulating factor (GM-CSF). In six patients who underwent blood cell transplantation, PB CD34+ cells were cultured after mobilization and were compared to CD34+ cells in steady state from PB and bone marrow (BM). Qualitative differences were shown between PBC samples obtained after CT with and without GM-CSF. Despite similar CFU-GM counts at culture initiation, GM-CSF-mobilized CD34+ cells might contain a lower proportion of primitive stem cells, as suggested by the significant decrease in CFU-GM numbers produced beyond week 5 compared to CT-mobilized CD34+ cells (p = 0.033). Likewise, the percentage of CFU-GM produced beyond week 5 in relation to initial input was significantly lower than steady-state PB (p = 0.039) and than CT-mobilized CD34+ cells (p = 0.033). However, this CFU-GM production with GM-CSF-mobilized PB CD34+ cells was not different from cultures with BMC CD34+ cells. These results suggest that GM-CSF can mobilize CFU-GM in the blood mainly by differentiation at the expense of the primitive stem cell compartment. It appears valuable to define clearly for each mobilizing procedure a particular threshold of CFU-GM which reflects sufficient numbers of primitive stem cells to ensure long-term engraftment.

Granulocyte-macrophage colony-stimulating factor accelerates hematopoietic recovery after autologous bone marrow or peripheral blood progenitor cell transplantation and high-dose chemotherapy for lymphoma.

The use of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) as an adjunct to autologous bone marrow transplantation (ABMT) or peripheral blood progenitor cell (PBPC) transplantation was evaluated in 59 lymphoma patients. Patients were divided into three groups. In group I (n = 21) patients received rhGM-CSF (5 micrograms/kg daily) at the time of PBPC collection and during the recovery phase post-infusion. In group II (n = 12) patients received rhGM-CSF as an adjunct to ABMT. In group III (n = 26) they were grafted with bone marrow without rhGM-CSF. Administration of rhGM-CSF (groups I and II) significantly reduced the time to myeloid engraftment, the number of febrile days and the median duration of antibiotics administration and of hospital stay when compared with the group in which patients did not receive rhGM-CSF. The only difference between ABMT and PBPC, given with rhGM-CSF support, was observed in the duration of hospitalization (group I > group II, P < 0.05). These data show that rhGM-CSF is highly effective in reducing the duration of aplasia following BMT and PBPC transfusion, and there appears to be little difference in efficacy between these techniques, provided that patients also receive rhGM-CSF.

Sensitivity of human CFU-GM to mafosfamide: analysis of the factors affecting individual variations.

A study of CFU-GM sensitivity to mafosfamide was carried out in 67 candidates for ABMT. A lethal dose 95 (LD95) was calculated from the dose-response curve. Despite standardized treatment conditions of marrow buffy-coat cells, LD95 values that were normally distributed (median 100 micrograms/ml; mean +/- SEM 95.6 +/- 4.0 micrograms/ml) varied considerably from patient to patient (range 30-160 micrograms/ml). Three independent factors appeared to confer greater sensitivity of CFU-GM: low patient age, low CFU-GM rate in treated cells and prolonged delay of CFU-GM sensitivity test from last chemotherapy course. In contrast, sex, pathology, disease status, number of previous chemotherapies and use of CY before the test did not influence CFU-GM sensitivity. Forty-six patients were autografted with mafosfamide-treated marrows according to their LD95. The mean percentage of CFU-GM effectively recovered after graft purging was 3.96 +/- 2.09%. All patients engrafted well and their peripheral blood cell recoveries were correlated with graft CFU-GM content evaluated before purging but not after purging or after freezing. In multivariate analysis, this parameter remained the only factor predicting hematopoietic recovery among other patient variables such as sex, age, pathology, disease status, previous chemotherapies or TBI in conditioning regimens. In a subgroup of 39 patients with lymphoid malignancies compared with 25 patients autografted for non-Hodgkin's lymphomas with unpurged marrows, the delay in days (medians) was similar for leukocytes > 1 x 10(9)/l (19 vs 17 days) and for neutrophils > 0.5 x 10(9)/l (18 vs 17 days) while it was longer for platelets > 50 x 10(9)/l (34 vs 128 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent decrease in proliferative potential of marrow CD34(+)cells exposed to early-acting growth factors after autologous bone marrow transplantation.

Post-graft hematopoiesis is characterized by long-term quantitative deficiency in marrow progenitor cells in both autologous and allogenic settings. In order to evaluate the function of post-graft progenitor cells, the proliferative capacity of marrow CD34(+) cells was evaluated in 10 patients 6 months after autologous bone marrow transplantation (ABMT) for non-Hodgkin's lymphoma and compared to that of 10 patients before ABMT and 10 normal controls. Immuno-selected CD34(+) cells were cultured for 7 days in liquid serum-free medium with a combination of early-acting GF consisting of stem cell factor, IL-3 and IL-1beta. Clonogenic efficiency of unselected cells for CFU-GM and BFU-E was decreased in post-graft patients compared to pre-graft and control patients. However, clonogenic efficiency of selected CD34(+) cells for CFU-GM was not different in post-graft, pre-graft and control patients but BFU-E values of post-graft patients remained lower than those of control patients. Decreased percentages of CD34(+) CD38(-) cells were observed in both post-graft and pre-graft patients while those of CD34(+) c-kit(+) cells were similar in all three patient groups. After 7-day liquid culture, expansion yields of total progenitor cells were significantly lower in post-graft patients (147 +/- 28%) than in pre-graft (255 +/- 27%) and control patients (246 +/- 23%). Post-graft deficiency in progenitor cell expansion was particularly marked for BFU-E (61 +/- 24%) compared to pre-graft patients (220 +/- 82%) and to controls (349 +/- 82%). These results indicate impaired proliferative potential of marrow CD34(+) cells several months after ABMT involving erythroid progenitor cells and/or commitment towards erythroid lineage from a more immature stage (pre-CFU).

High dose chemotherapy with autologous marrow transplantation in follicular lymphomas.

Twenty six adult patients with low grade nodular non Hodgkin's lymphoma (NHL) were treated with autologous bone marrow transplantation. Conditioning regimen was BEAM-BEAC in 15 patients and TBI + Cyclophosphamide in 11 patients. Twenty one patients were grafted with haematopoietic stem cells, 12 after bone marrow purging and five with peripheral blood stem cells (PBSC). Two patients were treated in CR1 of leukemic phase, six in PR1 and eighteen in sensitive relapse. With a median follow-up of 30 months, the actuarial survival is 91% and actuarial event free survival 67%. These data confirm some interest of ABMT in the treatment of low grade follicular NHL.

Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation.

Marrow stromal cells were evaluated several months after autologous BMT for their capacity to support both normal hemopoiesis and secrete the main growth factors involved in its control, G-CSF, GM-CSF, IL-3 and SCF. Stromal layers (SL) were obtained by long-term marrow cultures (LTMC) established from 15 patients (9 with hematologic malignancies and 6 with solid tumors) 3 months after autologous BMT and were compared to pre-graft patients. After irradiation, both post-graft and pre-graft SL were recharged with the same inoculum of normal marrow cells. As compared to pre-graft values, CFU-GM production on post-graft SL was significantly increased during the first 2 weeks of culture whereas it was decreased from week 3 to week 8. These findings were only observed in patients with hematologic malignancies and not in patients with solid tumors. Growth factor secretion was evaluated by ELISA in the supernatants of unstimulated and IL-1-stimulated SL from 10 post-graft patients, 13 pre-graft patients and 5 normal controls. In any group of patients, IL-3 was undetectable either spontaneously or after IL-1-stimulation. As compared to controls, secretion by IL-1-stimulated SL was not different for GM-CSF in pre- and post-graft patients but tended to be decreased for G-CSF in post-graft patients. SCF secretion, which was not induced by IL-1, appeared dramatically decreased in both pre- and post-graft patients. The capacity of post-graft SL to support CFU-GM growth in LTMC was correlated at week 1 with G-CSF secretion and from week 3 to week 8 with SCF secretion. These results suggest that microenvironment remains qualitatively damaged several months after BMT involving a decreased capacity both to support early hemopoiesis and to secrete SCF, particularly in patients grafted for hemopoietic malignancies.

High dose chemotherapy including vepeside, cyclophosphamide and carboplatin with autologous bone marrow transplantation in one case of chemoresistant testicular cancer.

Non seminomatous germ cell testicular tumors (NSGCTT) is a very chemosensitive cancer; vepeside, cyclophosphamide or ifosfamide and cisplatin are the most effective drugs. Carboplatin is also active with a lack of nephrotoxicity, but there is probably a cross-resistance to cisplatin. High dose chemotherapy with autologous bone marrow transplantation is able to cure poor prognosis testicular cancers in first partial remission or in sensitive relapse. We report one case of non seminomatous testicular cancer which was progressive after chemotherapy with cisplatin and vepeside or teniposide, and which is in prolonged complete remission after conventional chemotherapy (vepeside, ifosfamide, carboplatin) and high dose chemotherapy (carboplatin, vepeside, cyclophosphamide) with autologous bone marrow transplantation.

[Normovolemic hemodilution: application to the treatment of refractory cutaneous ulcers. Transfusion Techniques and Therapeutics Group]. / L'Hémodilution Normovolémique: application au traitement des ulcères cutanés rebelles. Groupe Techniques Transfusionnelles et Thérapeutiques.

Resistant cutaneous leg ulcers have a long evolution and are not easily cured by traditional therapeutics. Normovolemic haemodilution was proposed in 1984 by Guenneguez and Ouvry, who studied a small number of patients with good results, by using a local protocol, and a manual procedure. Recent data about physiopathology of cutaneous leg ulcers seem to confirm the benefit that Normovolemic Haemodilution should bring to the treatment of this pathology. Therefore a multidisciplinary group was created to lead a multicentric study about the treatment of resistant cutaneous leg ulcers by long term Normovolemic Haemodilution.

[Peripheral stem cell collection, search for predictive factors: a multicenter study. Working Group on Transfusion and Therapeutic Techniques of the French Blood Transfusion Society ]. / Collection de cellules souches périphériques, recherche de facteurs prédictifs: une étude multicentrique. Groupe de travail Technique transfusionnelle et thérapeutique de la SFTS.

AIMS: A multicentric study involving 12 centers was made to investigate the results of peripheral stem cell collection carried out between 1996 and 1997 from 655 patients with hemopathic syndromes or malignant tumors, The aim of this investigation was to determine the predictive factors for transplant quality, and to thereby optimize collection procedures. PATIENTS AND METHODS: Information sheets were completed for 1,346 cytapheretic sessions, i.e., 655 grafts. The samples were taken after induction chemotherapy and exposure to hematopoeitic colony-stimulating growth factors (except the LMCs). Each graft was defined as being of good or bad quality depending on the number of CD34+ cells that it contained. Based on the data available in the literature, a workgroup consensus was reached that a level of CD34+ cells +/- 2.10(6)/kg recipient body weight constituted a good transplant criterion. The 2 subgroups (good graft versus lower quality graft) were compared by univariate analysis followed by discriminant multivariate analysis. RESULTS: It was established that a number of parameters were significantly linked to the criterion of collection quality; however, 3 predictive factors emerged from the multivariate analysis--the level of circulating CD34+ cells; the number of cytaphereses; the number of blood volumes treated. CONCLUSION: It was concluded that the level of circulating CD34+ cells seems to be an essential aspect in predicting the quality of the transplant and the number of cytaphereses required to obtain a sufficiently rich collection. Moreover, it also appears that at least 2 blood volumes should be treated to optimize the results.

Quality, ISO 9002 and haemapheresis service.

Hemotherapy in developed countries is safer than ever but the present perception of society seems different than in the past. As Isbister wrote, during recent years, as blood safety has been progressively improved, the legal risks have been increasing. The first task is to study and define the risks for each activity. For me the management of risks may be achieved through the management of quality. The confidence of society in blood transfusion can probably best be obtained through a kind of recognized external quality assurance. I intend to describe our targets, our difficulties and the factors that have made our certification a success. I shall focus on several points: what quality represents; what our haemapheresis service is; what ISO 9002 standards are and why we choose them; our difficulties and the factors of success.

[In vitro study of platelet preservation during 5 days in reduced-thickness bags]. / Etude in vitro de la conservation de plaquettes pendant 5 jours dans des poches d'épaisseur réduite.

An in vitro study of platelet concentrates storage for 5 days was performed in PVC bags. Modification of the original three-day containers were introduced by thickness reduction. The results at fifth day were comparable to those at third day in standard plastic bags. During storage, variations of platelet counts were very slight with a low LDH release. PH was stable with a good maintenance of phase microscope platelet morphology. PCO2 and PO2 measurements showed a satisfactory gas permeability which could explain a limited lactate production. If in vivo studies of transfusion recovery confirm these data, platelet concentrates storage could be extended up to 5 days by such modifications of standard three-day PVC bags.

In vitro effect of stem cell factor on human clonogenic marrow progenitors after myeloablative treatments.

Abnormal hematopoiesis, including a deficiency of marrow progenitors and particularly of erythroid progenitors, has been described after autologous stem cell transplantation (ASCT), persisting for several years. In order to explain this deficiency, a resistance of marrow progenitors to stem cell factor (SCF) after ASCT was investigated. Marrow samples were harvested from pregraft patients at graft collection prior to ASCT, transplanted patients 6-24 months after high-dose therapy and control patients. CD34+ cells were cultured in a serum-free clonogenic assay with increasing doses of SCF. The clonogenic efficiency without SCF was lower for BFU-E in treated groups than in controls, whereas it was not different for CFU-GM. With increasing doses of SCF a dose-dependent effect was found on the numbers of both CFU-GM and BFU-E in all groups, although the maximal number of BFU-E remained lower in treated groups. However, the SCF dose that induced 50% of maximal BFU-E growth (D50) was similar in all groups. Furthermore, a dose-dependent effect on the size of BFU-E was found in all groups, with no difference in the proportion of large colonies. Thus, clonogenic erythroid progenitors from patients who have received myelotoxic treatments remain sensitive to SCF, with no evidence for a chemotherapy-related resistance.

Cutaneous toxicity of autologous bone marrow transplantation in nonseminomatous germ cell tumors.

High doses of carboplatin or cisplatin combined with cyclophosphamide and etoposide followed by autologous bone marrow transplantation (ABMT) rescue have been used in the treatment of testicular tumors that have had a bad prognosis. Unusual cutaneous complications, evoking radiation-induced dermatitis, have been seen in two of eight patients with the same regimen. This new type of toxicity seems to be related to high-dose combination chemotherapies. Good results in the treatment of patients with testicular tumors on relapse who continue to respond to chemotherapy lead to the extension of this type of schedule and cutaneous toxicity will probably develop.

Improved pharmacodynamics of L-asparaginase-loaded in human red blood cells.

To evaluate the modification of pharmacodynamic parameters induced by the administration of L-asparaginase loaded into red blood cells, 13 patients received a single dose of L-asparaginase internalised into the carrier. The enzyme was loaded using a reversible lysis-resealing process. The dose per patient ranged from 30 to 200 i.u. kg-1. Considerable heterogeneity occurred between patients. the level of L-asparaginase circulating after 24 h represented 47% of the total injected dose as compared to 74.8% for red blood cells (RBCs). However, the half-life of the enzyme remaining in the circulation was very similar to that of the RBC carrier, i.e. 29 days and 27 days, respectively, compared with 8-24 h for the free enzyme. Sustained elimination of plasma L-asparagine occurred, the duration of which was dependent on the injected dose. A single injection of 30.i.u.kg-1 was sufficient to eliminate plasma L-asparagine over 10 days. With 150-200 IU.kg-1 the elimination period was extended to 50 days. These data show that the use of RBCs as carriers of L-asparaginase greatly improves the pharmacodynamic parameters of the drug.