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Five blood banks of Regione Lazio implemented a multicomponent collection program using apheresis technology. The automated collection of blood components included: red blood cell concentrate and fresh plasma (RBCP), plasma and platelet concentrate (P-PLT), red blood cell and platelet concentrates (RBC-PLT). 334 voluntary blood donors and 30 patients - as autologous donors- were involved. Apheresis collection of RBCP, P - PU, RBC - PLT yielded a standardized product (adequate volume, low residual leucocyte counts, adeguate hematocrit, low platelet contamination) was well tolerated by donors, was performed without technical problems. We conclude that multicomponent collection is a new feasible alternative to conventional whole blood collection.

Peripheral blood stem cells (PBSCs) collected following stimulation with cytokines are commonly used for autologous haematopoietic transplants. Currently, PBSCs are being used for syngeneic or allogeneic transplants from matched or haploidentical donors. However, many issues are still unanswered regarding the early or late side-effects cytokines have on recipients and on healthy donors. The aims of this paper were to evaluate the experience acquired worldwide in this field, to define the acceptability of stem cell donation by G-CSF-stimulated apheresis from unrelated donors after the failure of a first donation, and to assess side-effects of G-CSF on unrelated donors. The use of PBSCs has increased tremendously over the last few years and in the near future PBSCs will probably become the most relevant source of stem cells. Studies conducted so far have definitely concluded that G-CSF is safe and well tolerated. Results observed in transplants utilizing marrow stem cells compared with results obtained in transplants utilizing PBSCs have shown that patients undergoing this latter procedure recover earlier, require a lower number of transfusions and spend fewer days in hospital with a consequent decrease in costs. We concluded that a second transplant by G-CSF-stimulated apheresis from an unrelated donor is definitely acceptable and we designed a prospective study to better define all controversial aspects. Donors will be given 10 microg/kg/day of G-CSF subcutaneously for 5 days. One or two PBSC collection procedures will be performed: the first on day 5 and the second, if necessary, on day 6. Donors will be surveyed and blood counts monitored in a standardized manner during the process.

Circulating CD34+ cell populations characterized by a low rate (up to five) or high rate (more than five) of cell divisions were isolated from 8 d cultures in the presence of stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), Flt3 ligand and Peg-rHu megakaryocyte growth and development factor (Peg-rHuMGDF) using the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometric cell sorting. Phenotypic characterization of cells which had experienced up to five divisions (CFDA-SEbright) showed a similar surface antigen expression to starting, freshly isolated CD34+ cells. Conversely, cells which experienced more than five divisions (CFDA-SEdim) showed a differentiating behaviour, down-regulating CD34 antigen and acquiring differentiation markers. CFDA-SEbright cells were significantly enriched in CD105 (endoglin) positive precursors as compared to both freshly isolated CD34+ and CFDA-SEdim cells. Functional analysis indicated that CFDA-SEbright had a 3-fold and 10-fold greater cumulative cloning efficiency as compared to freshly isolated CD34+ cells and CFDA-SEdim cells, respectively. CFDA-SEbright cells retained the vast majority of LTC-IC and showed a LTC-IC frequency 2.8-fold higher than that found in freshly isolated CD34+ cells. RT-PCR and Western blot analyses showed significantly higher bcl-2 RNA and protein levels in CFDA-SEbright cells as compared to freshly isolated CD34+ and CFDA-SEdim cells. This study indicates that cytokine low-responding circulating CD34+ cells (CFDA-SEbright cells) represent a functionally, phenotypically and molecularly distinct multipotent progenitor population with biological properties associated with primitive precursors.

A retrospective study was undertaken to assess the factors affecting the yield of peripheral blood stem cell (PBSC) collections after chemotherapy. Fifty-five patients with malignancies, observed in 4 Italian Institutions from January 1987 to June 1991 were eligible for evaluation. This series included 19 non-Hodgkin lymphoma, 11 multiple myeloma, 9 ovarian cancer, 7 Hodgkin disease, 7 acute non-lymphocytic leukemia, 1 acute lymphoblastic leukemia, 1 neuroblastoma. Five hundred and twenty two PBSC collections were performed on 55 patients after a median of 18 days after the start of chemotherapy. The yields of PBSC collections were related to the dose of cytoreductive chemotherapy exploited for PBSC mobilization and to the number of circulating white blood cells, colony forming unit granulocyte/macrophage (CFU-GM) and the percentage of monocytes at the time of collection. Forty-eight patients out of 55 transplanted (87%) had rapid, complete and sustained engraftment. Three patients (5%) died of transplant related complications.

We investigated the serum concentrations of a variety of cytokines [granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interleukin (IL) 1 alpha, IL-3, IL-6, IL-8, erythropoietin, tumor necrosis factor alpha, gamma-interferon in 10 patients with advanced ovarian cancer undergoing autologous peripheral blood stem cell (PBSC) harvesting followed by treatment with high-dose cisplatin, etoposide, and carboplatin and PBSC transplantation (chemotherapy was administered on days 1 through 3, PBSCT on day 6). Preliminary observations on cytokine serum levels were performed for 4 patients; on this basis, the kinetics of cytokines was then investigated in greater detail at closely sequential times in 6 further patients. We observed a consistent pattern of sequential GM-CSF, G-CSF, and IL-8 release after chemotherapy/PBSCT in all 10 cases, including the 6 patients monitored in detail: (a) at days 5-10 a GM-CSF peak; (b) at days 12-14 a pronounced release of both G-CSF and IL-8, which always preceded granulocyte recovery by approximately 7 days. At days 17-23, a second GM-CSF peak was monitored in 5 of the 6 patients analyzed in detail, as well as in the other 4 cases. Particularly relevant are the observations that: (a) the peak of G-CSF serum concentration and neutrophil number in the recovery phase are strikingly and directly correlated, thus indicating a key role for G-CSF in granulocyte rescue; (b) the time courses of G-CSF and IL-8 levels are strictly parallel, thereby suggesting a coordinate stimulus for production of granulocytes, mediated by G-CSF, and their activation/migration capacity, mediated by IL-8. Results were essentially negative for IL-3, tumor necrosis factor alpha, and gamma-interferon concentrations (except in one case for each cytokine). An early peak of IL-1 alpha was observed in all 3 analyzed patients, while an IL-6 peak was monitored at days 13-15 in all 4 patients analyzed in detail. The present results indicate a sequential coordinate pattern of cytokine release after ablative therapy and PBSCT and shed light on the mechanisms mediating the recovery of granulocytes, and more generally of hematopoiesis, after stem cell transplantation. Furthermore, these studies may contribute to the design of improved protocols for cytokine administration following myelosuppressive anticancer therapy, as well as to the prediction of granulocytic response.

We used an in vitro clonogenic assay in semi-solid medium to test the sensitivity of normal bone marrow and acute myeloid and lymphoid leukaemia progenitors to the flavonol quercetin. We have studied 14 acute myeloid (AML) and four acute lymphoid (ALL) leukaemias. All ALL and the vast majority of AML (12/14) had a high sensitivity to quercetin with more than 50% growth inhibition at 2 x 10(-6) M quercetin. One M3-AML was partially quercetin-sensitive displaying 60% surviving AML-colony forming units (CFU-AML) at a quercetin concentration of 10(-5) M. One M1-AML was resistant to the growth inhibitory effect of quercetin at a concentration of 2 x 10(-5) M. The clonogenic efficiency of both AML and ALL positively correlated with leukaemic colony-forming unit (CFU-L) sensitivity to quercetin suggesting that this parameter can be useful in predicting quercetin responsiveness of leukaemic cells. We have also studied the effect of various quercetin concentrations on colony formation by normal bone marrow cells. At a quercetin concentration of 10(-5) M, we observed (in five different experiments) a mean recovery of 53% and 65% of erythroid blast-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM), respectively. Thus, normal bone marrow appeared partially resistant to quercetin, being inhibited less than 50% by quercetin concentration higher than 2 x 10(-5). When normal bone marrow were deprived in CD34+ haematopoietic progenitors the resultant population became highly sensitive to quercetin, with a mean recovery of BFU-E and CFU-GM of 5% and 12% of controls respectively in the presence of 2 x 10(-5) M quercetin. Furthermore, CD34 progenitors, positively selected, appeared fully resistant to quercetin concentrations as high as 2 x 10(-5) M. Thus, CD34+ progenitors are a quercetin-resistant component in normal bone marrow. In conclusion, our results further provide a biological basis for the therapeutic use of quercetin, considering that this compound could inhibit leukaemic cell growth without suppressing normal haematopoiesis.

Many years have passed since the first attempt in marrow grafting was performed (1939). During this period several techniques have been developed in marrow processing and manipulation to overcome bone marrow transplant complications: the ABO barrier in case of major incompatibility between donor and recipient, the graft-versus-host disease due to the presence of allogeneic mature T-lymphocytes in cellular suspension and the neoplastic cell residue in autografts. At the end, the final volume of autologous mononuclear cell suspension must be frozen and an optimized cryopreservation allows a cell viability and subsequently an adequate medullar repopulating capacity.

After high-dose chemotherapy, autologous cryopreserved bone marrow infusion is employed to restore rapidly the compromised hematopoietic function. An efficient bone marrow processing reduces the infusion toxicity produced by hemolized red cells, granulocytes and platelets clumping and DMSO amount; moreover it increases freezing efficacy, a critical step in autologous bone marrow grafting techniques. Gravity sedimentation technique with 6% hydroxyethyl-starch (HES) or a semiautomated procedure using a blood cell processor were used in our center to manipulate ex-vivo the collected bone marrow. In our experience we compared these two different procedure and we evaluated their efficiency.

The side effects of a series of 2418 hemapheresis procedures performed in a total of 570 subjects (patients and donors) are described. Patients with various diseases were subjected to plasmapheresis (926 procedures in 181 patients) or cytapheresis (305 procedures in 89 patients). One hundred twelve plasmapheresis procedures and 1075 of cytaphereses were also performed in 300 blood donors. A total of 225 complications involving 107 patients (39.6%) occurred during 196 (15.9%) therapeutic procedures. Among the blood donors only 45 complications, involving 35 patients (11.6%) occurred during 45 procedures (4.2%). The complications seen with therapeutic plasmaphereses were circulatory disturbances (38% of all those observed), citrate reactions (27%), technical problems (20%), allergic reactions (9%) and miscellaneous complications (6%). Therapeutic cytaphereses were complicated by citrate reactions (44%), technical problems (25%), circulatory disturbances (14%), allergic reactions (11%) and miscellaneous complications (6%). Complications in the blood donor group included circulatory disturbances (51%), technical problems (36%) and various other problems (13%). No infectious complications or deaths were observed. The probability that adverse reactions will occur depends on the condition of the patient, the frequency of the sessions and the volume of fluid exchanged. Evaluation of the main risk factors, use of less intensive protocols and interruption of the session at the first sign of disturbances will help improve patient tolerance of these procedures.

Six patients suffering from refractory malignancies (3 NHL, 1 MM, 1 AML, 1 neuroblastoma) received high dose of chemotherapy and autologous peripheral blood stem cell transplantation (APBSCT). The recruitment of PBSC was performed using conventional salvatage schedules of therapy. The patients received a median of 8.69 MNC/kg bw and 20.87 CFU-GM x 10(4)/kg bw. Prompt engraftment occurred in all patients and the median number of days to achieve WBC greater than 1 x 10(9)/l was 16.5 (range 7-26), PMN greater than 0.5 x 10(9)/l was 21.5 (range 6-37) and PLTs greater than 50 x 10(9)/l was 17.5 (range 4-31). Four patients achieved a complete remission. One patient (neuroblastoma) died of progressive disease after a partial response. One patient died in relapse because of drug related toxicity.

This paper describes the development of a semiautomated procedure for autologous bone marrow processing, prior to ex vivo manipulation and/or cryopreservation. This procedure was employed with a pediatric bowl (125 ml Latham bowl) and the automated DuPont Stericell processor. We have obtained a mononuclear cell recovery of 85% and a hemopoietic progenitor cell recovery of 81% (CFU-GM; BFU-E), with a red cell removal of 84%. We believe that a reliable and standardized bone marrow processing procedure is the basic necessity for a bone marrow transplantation program.

In HIV infection, numerous alterations of the hematopoietic system, with frequent cytopenias in peripheral blood and dysplasia of the bone marrow, have been observed. In order to assess the incidence of the myelodysplastic anomalies, 69 bone marrow aspirate smears from 47 patients with group IV HIV infection, as classified by CDC, have been studied. Various degrees of myelodysplastic alterations were found in all cases; however, dysgranulopoiesis was more frequent and more accentuated than other kinds of dyshematopoiesis. Intense vacuolization, especially in the granuloblastic series, was very frequent. It is felt that a bone marrow configuration that is highly indicative of overt AIDS can be sketched. The human immune deficiency virus may be either directly or indirectly responsible for myelodysplastic alterations.

The following report describes the initial stage of the activity of the Italian Cooperative Study group on cellular manipulation in hematology consisting of a retrospective evaluation of data regarding bone marrow (BM) harvesting processing, and proceedings from 20 Italian Centers. Two thousand, three hundred and eighty-four BM have considered: 1073 were performed for autografts and 1311 for allografts. A cohort of adverse effects in marrow harvesting were reported by 7 Centers out of 20, including one death caused by tracheomalacia during the anesthesia. Twelve Centers used blood separators as a mean for marrow processing. Eight Centers used the RBC removal technique in major donor/recipient AB0 incompatibility. Ficol-Hipaque gradient was employed in 7 Centers. T-depletion were accomplished with monoclonal antibodies in 7 Centers and elutriation in two Centers. Fifteen Centers provided to purge the residual tumour cells with chemicals (11), immunological (4) and chemo-physical means (1).