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Objectives: Reconstitution of hematopoiesis by means of peripheral blood stem cells is a valid alternative to autologous bone marrow transplantation. The aim of this investigation was to increase the efficiency of collection of circulating blood progenitor cells and to obtain a purer product for transplant., Methods: We carried out leukapheresis procedures with the Fresenius AS 104 blood cell separator, using two different protocols, the previously used PBSC-LYM and a new mononuclear cell collection program., Results: Both programs were highly effective in collecting mononuclear cells (MNC) and CD34+ cells. Some differences were found, especially regarding MNC yield and efficiencies. There are remarkable differences in the efficiency of collection of CD34+ cells (62.38% with the new program as opposed to 31.69% with the older one). Linear regression analysis showed a negative correlation between blood volume processed and MNC efficiency only for the PBSC-LYM program. Differences were also observed in the degree of inverse correlation existing in both programs between patients' white blood cell precount and MNC collection efficiency. The inverse correlation was stronger for the PBSC-LYM program. Seven patients with solid tumors and hematologic malignancies received high dose chemotherapy and were subsequently transplanted with peripheral blood stem cells collected using the new protocol. All patients obtained a complete and stable engraftment with the reinfusion product collected with one or two leukapheresis procedures., Conclusions: High efficiencies and yields were observed in the new protocol for MNC and CD34+ cells. These were able to effect rapid and complete bone marrow recovery after myeloablative chemotherapy.

The present report describes the non-haematological toxicity and the influence of growth factor administration on haematological toxicity and haematopoietic recovery observed after high-dose carboplatin (1200 mg m(-2)), etoposide (900 mg m(-2)) and melphalan (100 mg m(-2)) (CEM) followed by peripheral blood progenitor cell transplantation (PBPCT) in 40 patients with high-risk cancer during their first-line treatment. PBPCs were collected during the previous outpatient induction chemotherapy programme by leukaphereses. CEM administration with PBPCT was associated with low non-haematological toxicity and the only significant toxicity consisted of a reversible grade III/IV increase in liver enzymes in 32% of the patients. Haematopoietic recovery was very fast in all patients and the administration of granulocyte colony-stimulating factor (G-CSF) plus erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) plus EPO after PBPCT significantly reduced haematological toxicity, abrogated antibiotic administration during neutropenia and significantly reduced hospital stay and patient's hospital charge compared with patients treated with PBPCT only. None of the patients died early of CEM plus PBPCT-related complications. Low non-haematological toxicity and accelerated haematopoietic recovery renders CEM with PBPC/growth factor support an acceptable therapeutic approach in an adjuvant or neoadjuvant setting.

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were culture with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/ monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/ chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.

In this report we analyzed sixty leukapheresis procedures on 35 patients with a new protocol for the Fresenius AS 104. Yields and efficiencies for MNC, CD 34+ cells, and CFU-GM indicate that the new protocol is able to collect large quantities of hemopoietic progenitors. Procedures were performed processing 8.69 +/- 2.8 liters of whole blood per apheresis and modifying 3 parameters: spillover-volume 7 ml, buffy-coat volume 11.5 ml, centrifuge speed 1,500 rpm; blood flow rate was 50 ml/min and the anticoagulant ratio was 1:12. No side effects were observed during apheresis procedures except for transient paresthesia episodes promptly resolved with the administration of calcium gluconate. Yields show a high capacity of the new program to collect on average MNC 17.28 +/- 10.85 x 10(9), CD 34+ 471 +/- 553.5 x 10(6) and CFU-GM 1278.7 +/- 1346.3 x 10(4) per procedure. Separator collection efficiency on average was 49.91 +/- 23.28% for MNC, 55.1 +/- 35.66% for CFU-GM, and 62.97 +/- 23.09% for CD 34+ cells. Particularly interesting are results for MNC yields and CD 34+ efficiency; these results make the new program advantageous or similar to the most progressive blood cell separators and capable to collect a sufficient number of progenitor cells for a graft with a mean of 1.80 +/- 0.98 procedures per patient.

Circulating CD34+ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34 contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3+ T-lymphocytes (range 2-12%), CD3+/CD16+/CD56+ natural killer cells (range 2-11%) and rare mature CD15+/ CD11b+ granulocytes (range 1-2%). CD34+ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor. stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34+ cells until day 35. These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans: additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)2D3. parathyroid hormone interleukin-11 and of other cytokines on osteoclast development in humans.

Counterflow centrifugal elutriation (CCE) has been extensively employed in T cell depletion of bone marrow cells for allografting. Nevertheless very little is known about CCE properties of mobilized hematopoietic progenitors. In this study five leukapheresis products collected after chemotherapy and G-CSF from patients with non-Hodgkin's lymphoma were elutriated. Two mononuclear cell fractions were obtained containing smaller and less dense cells (lymphocyte fraction) and larger and denser cells (monocyte fraction), respectively. The presence of immature CD34+ progenitor cells, not co-expressing CD33, CD38 and HLA-DR antigens, was demonstrated in both cell fractions. CD34+ cells were isolated from each fraction and grown in various culture conditions (CFU-GM and BFU-E assay, blast cell colony assay, cytokine supplemented liquid culture). CD34+ cells isolated from the monocyte fraction showed a longer lasting expansion in liquid culture and a higher number of blast cell colonies than CD34+ cells selected from the lymphocyte fraction. Moreover a significant reduction of T cell number was obtained in the monocyte fraction. These data suggest that chemotherapy plus G-CSF-mobilized progenitor cells show a characteristic behavior when subjected to CCE, allowing an efficient T cell depletion without losing more immature progenitors.

In order to investigate the effects of erythropoietin (EPO) plus granulocyte colony-stimulating factor (G-CSF) administration after peripheral blood progenitor cell transplantation (PBPCT) we performed a phase I/II study in patients with high-risk cancer. 15 consecutive patients were treated wit recombinant human G-CSF (rhG-CSF) at the dose of 5 micrograms/kg subcutaneously (s.c.) every 24 h until day + 12 and with recombinant human EPO (rhEPO) at the dose of 150 IU/kg s.c. every 48 h until day + 11 following PBPCT. Their haemopoietic recovery was compared to that obtained in eight historic and control patients who did not receive any cytokines after PBPCT. No side-effects were observed during EPO plus G-CSF treatment and the treatment was not discontinued in any of the patients before completion of the treatment plan. The administration of EPO plus G-CSF after PBPCT produced a significant increase in the rate of white blood cell (WBC) (P = 0.0005), polymorphonuclear leucocyte (PMN) (P = 0.0005) and platelet (PLT) (P = 0.0105) recovery compared to the control group. The acceleration in haemopoietic recovery observed in the EPO plus G-CSF-treated patients produced a significant reduction of the days with WBC < 1 x 10(9)/l (P = 0.0009), PMN < 0.2 x 10(9)/l (P = 0.0030) and PMN < 0.5 x 10(9)/l (P = 0.0006). EPO plus G-CSF-treated patients required a significantly lower number of single donor PLT transfusions (P = 0.0142) and did not experience neutropenic fever, but historic control patients experienced fever > 38 degrees C for a median period of 4 d (0-12) with a medial period of parenteral antibiotic administration of 7.5 d (0-17). The length of the hospital stay was significantly shorter in the study group than in the historic control group (P = 0.0264). In conclusion, we can confirm that EPO plus G-CSF treatment is feasible and potentiates the haemopoietic recovery after PBPCT, thus simplifying the clinical management of cancer patients who undergo high-dose chemotherapy.

20 patients with stage III-IV ovarian cancer were submitted to induction chemotherapy (ICT) (40 mg/m2 cisplatin, days 1-4; 1.5 g/m2 cyclophosphamide, day 4; every 4 weeks for 2 cycles) followed by intensified CT (100 mg/m2 cisplatin, day 1; 650 mg/m2 etoposide, day 2; 1.8 g/m2 carboplatin by 24 h infusion, day 3). Haematological support consisted of autologous peripheral stem cells (APSC) and bone marrow (ABM) transplant (T) in 16 and 4 patients, respectively. All patients were evaluable for toxicity and 19 for pathological response (PR), one patient dying of systemic mycosis after ABMT. Severe (grade 3-4) non-haematological toxic effects were gastrointestinal (100%), neurological (10%) and hepatic (10%). PR was observed in 84% of patients (complete response 37%, partial response with microscopic residual disease 26%, partial response with macroscopic residual disease 21%). Five year overall survival was 60% and progression-free survival was 51% with 9 patients still disease-free (DFS). APSCT significantly reduced the duration of aplasia compared with ABMT, and toxicity was acceptable in those patients undergoing APSCT. The prolonged DFS in patients showing PCR suggests that this new approach may have a therapeutic impact.

Recovery phase infection patterns in 55 patients who had undergone autologous blood progenitor cell transplantation (ABPCT) were evaluated retrospectively. The results were compared to those obtained in a group of 41 patients who received autologous bone marrow transplantation (ABMT). Fever related to documented or suspected infection developed in 38 of 55 patients in the ABPCT group and in 37 of 41 in the ABMT group (p < 0.05). The percentages of patients with positive blood cultures did not differ significantly (ABPCT, 8/55 vs. ABMT, 8/41, p > 0.05). However, fewer acquired systemic fungal infections (1/55 vs. 5/41, p < 0.05) as well as fewer days of antibiotic usage were observed in the ABPCT group.

The bisdioxopiperazine ICRF 187 is a potent intracellular chelating agent which effectively diminishes Adriamycin cardiotoxicity without compromising its antitumor activity. Our study aimed at verifying whether ICRF 187 can modulate the cytotoxic action of cisplatin (CDDP) on ovarian cancer cells. We used the A2780 ovarian cancer cell line and a subline resistant to CDDP (A2780-CDDP) obtained in our laboratory by continuous exposure of the parenatal cells to progressively increasing CDDP doses. In both cell lines ICRF 187 (0.1-0.5 microgram/ml) used in combination with CDDP (0.01-1 microgram/ml) produced a dose-dependent reduction of CDDP IC50 (the concentration inhibiting 50% of cell growth). Moreover, when ICRF 187 was used in combination with CDDP, analysis of the data by the isobole method showed that the combination of the two drugs produced a synergistic antiproliferative activity in both cell lines, with a CDDP potentiation up to fivefold. Our in vitro data show that ICRF 187 can synergize with CDDP. Prospective clinical trials are now needed to verify whether the addition of ICRF 187 to CDDP-containing regimens will result in an improved clinical response in ovarian cancer.

Twenty patients with advanced (stage III-IV), previously untreated ovarian carcinoma were treated by: (a) induction chemotherapy (40 mg/m2 cisplatin, days 1-4; 1.5 g/m2 cyclophosphamide, day 4; every 4 weeks for two cycles) followed by (b) intensification chemotherapy (100 mg/m2 cisplatin, day 1; 650 mg/m2 etoposide, day 2; 1.8 g/m2 carboplatin, day 3). Eligibility criteria further included: age less than 55 years, moderately good to poor tumour grade, macroscopic (> 0.5 cm) residual tumour. Autologous peripheral stem cells were recruited after the induction cycles and, to ensure haematological support, autologous bone marrow harvesting was routinely performed in the first 14 cases. Haematological support consisted of autologous peripheral stem cells and autologous bone marrow transplant in 16 and four patients, respectively. All patients are evaluable for toxicity and 19 for pathological response, one being dead of systemic mycosis 35 days after the autologous bone marrow transplant. Severe extra-haematological toxicities were the following: gastrointestinal (100%), neurological (10%), hepatic (10%). Pathological response was detected in 84% of cases (CR 37%, microscopic PR 26%, macroscopic PR 21%). Median follow-up times of 48 and 41 months have been reached respectively from enrolment and second-look. Four-year 62% overall and 57% progression-free survivals have been reached. Ten patients are still alive with NED (six of seven with CR, three of five with microscopic PR, and one of four with macroscopic PR). Autologous peripheral stem cell transplant significantly reduced the duration of aplasia compared with autologous bone marrow transplant, and toxicity was proved to be manageable in those patients undergoing autologous peripheral stem cell transplant. The prolonged disease-free survival in patients showing CR and microscopic PR suggests that further investigation on this new approach is worthwhile.

Six patients with advanced ovarian carcinoma (OvCa), and six patients with stage II or III resectable breast cancer (BrCa) were treated with low-dose CY (LD-CY, 1500 mg/m2) and cisplatin (CDDP) 100 mg/m2 (OvCa) or epirubicin (EPR) 120 mg/m2 (BrCa) plus recombinant human G-CSF (rhG-CSF). Twelve days after chemotherapy, all patients underwent PBSC collection on an outpatient basis. Following the completion of the induction programme, all patients underwent high-dose chemotherapy (HDC) with carboplatin 1200 mg/m2, etoposide 900 mg/m2 and melphalan 100 mg/m2 with the reinfusion of PBSC. LD-CY plus rhG-CSF in combination with CDDP or EPR mobilised a very large number of PBSC. After a median of 13 days from chemotherapy, the concentration of PBSC in the peripheral blood was 40-fold higher than the same patient's baseline value. Each collection yielded a median of 10.8 x 10(4)/kg colony-forming unit granulocyte-macrophage. Severe myelosuppression occurred in all patients following HDC, but the infusion of PBSC produced a rapid and sustained haemopoietic recovery. After a median of 11 days from reinfusion, haemopoietic engraftment was complete and 80% of the patients had platelets > 100 x 10(9)/l and PMN > 1 x 10(9)/l within 14 days after reinfusion. We can conclude that the present therapeutic approach is an excellent option for mobilisation, collection and transplantation of PBSC during intensive dose adjuvant polychemotherapy of high-risk cancer.

We have monitored the serum concentrations of hematopoietic growth factors (HGFs; ie, stem cell factor [SCF], leukemia inhibitory factor [LIF], interleukin-3 [IL-3], IL-6, IL-8, and granulocyte colony-stimulating factor [G-CSF]) in 15 lymphoma/leukemia and 6 ovarian cancer patients undergoing autologous bone marrow (BM) or peripheral blood (PB) stem cell transplantation (SCT). Thus, the analysis was performed during and after high-dose chemotherapy (from day -6 to day -1), at the time of SCT (day 0), and thereafter (through day +17). Despite the heterogeneity of these patients and their conditioning regimens, a consistent kinetic pattern was observed for all analyzed cytokines. Particularly, (1) SCF serum concentration did not significantly fluctuate. (2) High levels of LIF (approximately 250 to 450 pg/mL) before chemotherapy rapidly declined to markedly lower concentrations (approximately 10 ng/mL) starting from day -1 through day +17; (3) conversely, IL-3 level was low before treatment, sharply increased during chemotherapy, and rapidly returned to base-line level after SCT. Hypothetically, the sharp LIF decrease and IL-3 increase during chemotherapy may underlie the induction of stem cell cycling and differentiation caused by hematopoietic ablation. Furthermore, (4) IL-6 concentration was low before and immediately after chemotherapy, but increased starting from day +5, peaked at day +6 through 9 and then declined to baseline level from day +10 onward; (5) a strictly similar pattern was consistently observed for both G-CSF and IL-8 levels, in agreement with our previous studies. It is relevant that peak IL-6, G-CSF, and IL-8 concentrations were directly correlated to peak neutrophil numbers in the recovery phase, thus suggesting an important role for these cytokines in granulocyte rescue; in line with this interpretation, hematologic patients undergoing PBSCT (10 of 15) exhibited higher peaks of IL-6, G-CSF, and IL-8 and a more pronounced increase of neutrophil/platelet number than did hematologic cases undergoing BMSCT (5 of 15). Altogether, these studies indicate a coordinate pattern of cytokine release during hematopoietic ablation/recovery after chemotherapy and autologous SCT, the fluctuations of LIF and IL-3 levels during chemotherapy are seemingly related to stem cell recruitment, whereas the post-SCT increase of IL-6, G-CSF, and IL-8 may underlie the neutrophil recovery.

Peripheral blood (PB) mononuclear cells mobilized with chemotherapy and granulocyte colony-stimulating factor (G-CSF) were enriched in CD34+ cells; aliquots were seeded in long-term cultures (LTC) on bone marrow (BM)-derived stromal layers and in liquid cultures containing various growth factors. The final recovery of PB CD34+ cells was similar to normal BM controls, and no difference was found in the expression of CD33 and CD13 antigens; a lower number of CD34+/HLA-DR- cells was found in PB with respect to BM samples (p < 0.001). PB cells sustained hematopoiesis in LTC at least as long as BM cells. At week 3 and 4, PB total mononuclear cell (MC) and CD34(+)-selected cell cultures showed a higher nonadherent cell recovery compared to the respective BM controls (p = 0.05 and p < 0.01). The liquid culture of PB CD34+ cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and stem cell factor (SCF) resulted in a marked and long-lasting increase of colony-forming units-granulocyte/macrophage (CFU-GM). Taken together, our data suggest that chemotherapy and G-CSF-primed cells contain a considerable number of both committed and early precursors, accounting for the rapid hematopoietic recovery observed after their reinfusion following myeloablative chemotherapy.

We tested in vitro the effect of recombinant human erythropoietin (rhEPO) plus recombinant human G-CSF (rhG-CSF) on purified human CD34+ haemopoietic progenitors (HP) and in vivo in patients who had undergone anti-cancer chemotherapy for advanced ovarian cancer. In this preliminary experience we found that, in vitro, rhEPO potentiates the effect of rhG-CSF on HP growth and differentiation toward the granulocyte-macrophage lineage. rhEPO plus rhG-CSF produced in vitro a proliferative stimulus of HP which represents 26% of the maximum stimulation obtained using IL-1, IL-3, IL-6, G-CSF, GM-CSF and stem cell factor in combination. In the patients treated with rhEPO plus rhG-CSF after chemotherapy, we observed a favourable trend for platelet and neutrophil recoveries compared with a control group treated with rhG-CSF alone and a significantly higher haematocrit nadir was observed in the rhEPO plus rhG-CSF series. In the patients treated with rhEPO plus rhG-CSF we observed a significant increase of circulating colony-forming unit granulocyte-macrophage (CFU-GM) and burst forming unit-erythroid (BFU-e) compared with the rhG-CSF series. Our results, in vitro and in vivo, encourage the in vivo use of rhEPO plus rhG-CSF to improve blood cell recoveries of patients who have undergone conventional or high-dose chemotherapy. Moreover, rhEPO plus rhG-CSF was demonstrated to be a good HP mobilising treatment for blood stem cell collection after chemotherapy.

We enrolled 18 patients affected by refractory or relapsed lymphoma (HD, NHL) in a two-step protocol that included salvage chemotherapy with mitoxantrone, carboplatinum, methylprednisolone, and cytosine arabinoside (MiCMA) plus G-CSF (5 micrograms/kg/day), peripheral blood progenitor cell (PBPC) collection, and subsequent transplantation after BUCY2 regimen. After MiCMA chemotherapy, four patients (22%) achieved complete response, eight patients (44%) obtained a partial response, and six showed progression of disease (PD). Fourteen out of 18 patients (78%) were considered eligible for PBPC transplantation. Three patients with complete response refused PBPCT; they are currently in continuous complete remission (CCR) at 15, 13, and 15 months, respectively. One patient has been recently transplanted but is too early to be evaluated. Ten patients so far completed the study, eight of whom are currently alive in CR, with a median follow-up of 7.5 months (range 2-13). Hematologic reconstitution was very rapid with a median time to achieve WBC > 1 x 10(9)/L, PMN > 0.5 x 10(9)/L, platelets > 50 x 10(9)/L and > 100 x 10(9)/L of 13 (range 9-15), 12 (range 9-14), 10 (range 0-22), and 14 (range 5-49) days, respectively. Our protocol is highly effective as a salvage treatment, while permitting PBPC collection after G-CSF administration. Hemopoietic reconstitution after transplantation of PBPCs collected with this procedure is complete, rapid, and sustained.

Due to immunological immaturity, the fetus is the ideal recipient as well as donor of haemopoietic stem cells (HSCs); thus intrauterine therapy may prove to be effective in all major haemopoietic disorders when early prenatal diagnosis is available. In man, "fetus-to-fetus" transplantation has demonstrated the possibility of grafting donor HSCs and reconstituting immunodeficient fetuses. The limitations of fetal tissue use for transplantation derive from the origin of tissues from elective abortions. Early and late live spontaneous abortions may constitute an alternative to elective abortions, but are widely considered as unsuitable for fetal tissue collection because of rapid loss of viability and/or infections. The aim of this retrospective study was to assess the number of live abortions in a population of women who underwent spontaneous abortion in a single centre. In a 19-month period, 9 spontaneous abortions alive at the moment of delivery and 8 with a heart beat at the last ultrasound scan before abortion were recorded. In 1 case, fetal liver (FL) harvesting was easily performed and the tissue was cryopreserved, subsequently thawed and injected into a monkey fetus. This case shows the feasibility of this approach. The majority of cases reviewed consisted of late abortions. These findings clearly show that fetal tissue collection from spontaneous abortions is feasible for research purposes, for postnatal FL transplantation and for intrauterine transplantation, postnatal FL transplantation and for intrauterine transplantation, provided that depletion of more mature cells is performed when FL of later gestational age are used.(ABSTRACT TRUNCATED AT 250 WORDS)

A retrospective study was undertaken to evaluate the efficacy of autologous blood stem cell transplantation (ABSCT) in terms of haemopoietic reconstitution after ablative chemotherapy or chemo-radiotherapy. 55 patients with malignancies, observed in four Italian institutions from January 1987 to June 1991, were eligible for evaluation. This series included 19 non-Hodgkin's lymphoma, 11 multiple myeloma, nine ovarian cancer, seven Hodgkin's disease, seven non-lymphocytic leukaemia, one acute lymphoblastic leukaemia, one neuroblastoma. 522 PBSC collections were performed on 55 patients. Following ABSCT, the rate of engraftment was positively related to the dose of CFU-GM infused and negatively to the presence of bone marrow involvement at conditioning. 48 patients out of 55 transplanted (87%) had rapid, complete and sustained engraftment. Three patients (5%) died of transplant-related complications. Considering that 60% of the patients in this series were in partial remission or in progressive disease at the time of ABSCT, we conclude that ABSCT is a safe approach for the use of ablative conditioning therapy in patients with a wide scope of malignancies, provided that a large number of CFU-GM have been collected after mobilizing treatment.

We have analyzed the results of a multicenter study on peripheral blood stem cell transplantation (PBSCT) performed on 55 patients suffering from various neoplastic diseases. After myeloablative therapy, they received a median of 6.8 x 10(8)/kg MNC and 11.4 x 10(4)/kg CFU-GM harvested by a median of 9 apheresis after mobilization with chemotherapy alone. As of date, 34 of the 55 patients are alive and 28 of them are in continuous complete remission after a follow-up of 30 months. The probability of survival was related to the disease status at transplant, CR/PR vs. PD (p = 0.0001) and the bone marrow involvement, BM-vs. BM+ (P = 0.009). Furthermore, a comparative study on speed of engraftment and clinical management was conducted on the 55 PBSCT patients as well as on 41 autoBMT and 52 alloBMT patients. Days to reach WBC > 1.0 x 10(9)/L, PMN > 0.5 x 10(9)/L and PLT > 50 x 10(9)/L was 12/14/33 for PBSCT, 17/20/23 for ABMT and 15/16.5/18 for BMT, respectively. Days with fever > 38 degrees C, systemic antibiotic therapy and length of hospitalization was 3/12/36 for PBSCT, 5/18.5/42 for ABMT and 9/25/46 for BMT respectively.

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.

Several authors have reported a faster immunological and hemopoietic post-transplant reconstitution using autologous peripheral blood stem cell (PBSC) than using autologous bone marrow stem cells. A large number of PBSC can be collected by leukapheresis during the hematological recovery after induction or salvage chemotherapy. In our experience we demonstrated that several separators, even if they have different results in mononuclear cell (MNC) yields, red blood cell and platelet contaminations, are able to collect PBSC for autotransplantation in patients with several malignant diseases and different status of disease. Eighty three patients were submitted to 590 leukapheresis procedures using 4 different blood cell separators: the results showed that all employed protocols are efficient in the collection of peripheral MNC even if after the widespread use of granulocytecolony stimulating factor (G-CSF) in the harvesting phase, the blood cell separator efficiency in terms of MNC is reduced. The use of GCSF in combination with other growth factors, during chemotherapy mobilization could simplify, in the future, this therapeutical program even if improvements in the efficiency of PBSC collection protocols are required.

Hemopoietic CD34+ progenitors were isolated by immunomagnetic method from normal bone marrow (BM) or from peripheral blood (PB) of patients with non-Hodgkin's lymphoma treated with chemotherapy and granulocyte colony-stimulating factor (GCSF). Aliquots were seeded in long-term cultures (LTC) on bone marrow-derived stromal layers; non-adherent and adherent clonogenic content of the cultures was assayed weekly. The final recovery and the clonogenic efficiency of the CD34+ cells were slightly higher in PB samples than in BM controls. In long term cultures PB cells sustained hemopoiesis as much as BM cells; at week 3 and 4 PB total mononuclear cells and CD34+ cells showed a non-adherent cell recovery higher than the respective BM controls. Furthermore, PB CD34+ cells were expanded in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF alone or combined with interleukin 3 (IL3), stem cell factor (SCF), interleukin 1 (IL1), interleukin 6 (IL6). The combination of GM-CSF, IL3, SCF, IL1 and IL6 produced the maximum increase of both mononuclear cells (30-fold) and granulocyte-macrophage colony forming units (CFU-GM) (4.6-fold) after 7 days of cultures; yet after 14 days a strong decrease of the CFU-GM occurred. These data suggest that G-CSF following chemotherapy mobilizes both early and committed hemopoietic progenitors.

Background and Methods: Bone marrow transplantation is increasingly used to overcome the bone marrow toxicity from myeloblative therapy. Peripheral blood progenitor cell transplantation (PBPCT) has been recognized as an alternative source of bone marrow for hemopoietic recovery after myeloblative therapy. In the steady state condition hemopoietic progenitors are scarce in peripheral blood; chemotherapy and growth factors are both able to increase PBPCs. Twenty-five patients affected by resistant lymphoproliferative diseases [4 multiple myeloma (MM), 19 non Hodgkin's lymphoma (NHL) and 2 Hodgkin's disease (HD)] were submitted to autologous peripheral blood progenitor cell transplantation (PBPCT). PBPCs were collected after chemotherapy (CT) alone (8 patients) or plus filgrastim (17 patients). Filgrastim was not administered after PBPCT in any case., Results and Conclusions: A statistically significant difference between the two groups was found for the following parameters: number of leukaphereses administered, amount of CFU-GM infused, time to hemopoietic recovery, amount of supportive care, number of days of antibiotic therapy, length of hospitalization. PBPCs primed with filgrastim CT appeared to be markedly superior to CT-recruited PBPCs in reducing the period of neutropenia and, surprisingly, of thrombocytopenia. Reduction in hematologic toxicity resulted in a decrease of transplantation-related toxicity and mortality, even in elderly and/or heavily pretreated patients.

Forty-seven peripheral blood stem cell (PBSC) collections were carried out on patients mobilized with chemotherapy and 63 on patients mobilized with chemotherapy plus G-CSF (Filgrastim), using the Fresenius AS104 cell separator and a novel automated PBSC collection protocol. As expected, cell yields were significantly higher in the series mobilized using chemotherapy plus G-CSF. The low platelet and red blood cell contamination permitted freezing of the apheresis product without further manipulation, other than plasma removal in both series. In patients mobilized with chemotherapy we obtained a MNC and a hemopoietic progenitor (CFU-GM, BFU-e, and CD34+ cells) collection efficiency comparable or superior to those reported by Bender (1992) with the Baxter CS3000 Plus after mobilization with cyclophosphamide. A significant decrease in MNC, BFU-e, and CD34+ cell collection efficiency was found in patients mobilized with chemotherapy plus G-CSF compared to those obtained in patients mobilized with chemotherapy alone. Ten patients achieved a prompt and stable engraftment after high dose chemotherapy and the infusion of cryopreserved PBSC collected using this protocol. Studies are in progress in order to improve MNC and hemopoietic progenitor collection efficiency in patients mobilized with G-CSF to obtain a graft in no more than one or two procedures.

We evaluated the HLA-DR, CD33 and CD13 antigen expression on CD34+ haematopoietic progenitor cells (HPC) isolated from the bone marrow (BM) and peripheral blood (PB) of normal donors. The majority of both BM and PB CD34+ HPC expressed CD13 and HLA-DR. The coexpression of CD34 and CD33 was found in a minor CD34+ subset. After 7 d of culture in the presence of interleukin-3 and granulocyte-macrophage colony-stimulating factor, CD33 expression was detected in about 50% of HPC. At this point CD34 antigen expression was lost and CD13 and HLA-DR expression was partially lost. After 14 d of culture, the majority of HPC were CD33+. HPC maintained the capacity to generate colony forming unit granulocyte-macrophage but they lost the capacity to generate burst forming unit-erythroid. A correlation was found between the percentage of CD34+/HLA-DR+ cells and the total number of colony forming cells in unfractionated samples from BM and PB of patients with malignancies. These studies demonstrate that, in normal conditions, only a minor subset of CD34+ cells coexpress CD33 antigen either in BM or in PB and CD33 antigen is a lineage marker which is coexpressed with HLA-DR and CD13 on a progenitor committed to the granulocytic-macrophagic lineage.

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 evaluated the immunological reconstitution of patients who underwent high-dose chemotherapy and autologous blood stem cell transplantation (ABSCT) for advanced ovarian cancer. Sixty days after transplantation a complete reconstitution of lymphocytes and of the CD3, CD4, CD8, CD19, and CD16/56 subsets was observed in this series. A significant increase in the count of interleukin-2 receptor expressing lymphocyte (CD25) was found on day +60 after transplantation compared to that obtained at diagnosis and before transplantation. A significantly higher lymphokine-activated killer (LAK) precursor activity was seen on day +60 compared to the values obtained at diagnosis and before transplantation while natural killer activity did not show any significant variation. We conclude that ABSCT gives prompt and complete immunohaematopoietic reconstitution after high-dose treatment. Moreover, our data support the feasibility of interleukin-2/LAK therapy as consolidative therapy after ABSCT.

Twenty-three patients with malignant lymphoma, (7 Hodgkin's, and 16 non-Hodgkin's) in different phases of disease were autografted in 4 Italian Haematology institutions using only chemotherapy-mobilized blood stem cells (BSC) collected by apheresis. Clinical and laboratory data were analysed centrally and showed mean collection yields of 8.1 x 10(8) kg mononuclear cells (MNC) (SE 0.5; range 2.6-13.8) and 24.1 x 10(4) kg CFU-GM (SE 7.4; range 1.4-162.9). The mean times required to attain 0.5 x 10(9)/l neutrophils and 50 x 10(9)/l platelets after marrow-ablative high-dose chemo+radiotherapy and BSC reinfusion were 14.9 days (SE 1.5; range 7-38) and 18.6 days (SE 2.6; range 6-49) respectively. The incidence of early deaths was < 5% and the requirement for support with blood product transfusion was moderate. The progression free survival (PFS) is > 50% at 3 years with a median follow-up of 17.3 months. Results were significantly better for patients autografted in remission. These results suggest that autologous blood stem cell transplantation (ABSCT) may be proposed for the primary treatment of poor prognosis malignant lymphomas. However, ABSCT needs to be compared with autologous bone marrow transplantation (ABMT) followed by infusion of growth factors to accelerate recovery.

It has been demonstrated that quercetin (3,3',4',5,7-pentahydroxyflavone) inhibits the growth of several cancer cell lines and that the antiproliferative activity of this substance is probably mediated through a binding interaction with type II estrogen binding sites (type II EBS). The effect of quercetin and cytosine arabinoside (Ara-C) alone or in combination, was tested on HL-60 cell growth. Quercetin significantly synergized the inhibitory activity of Ara-C on HL-60 cell growth while rutin, the 3-rhamnosylglucoside of quercetin, neither competed with [3H]estradiol for type II EBS nor was effective alone or in combination with Ara-C. Based on these results, we studied by a clonogenic assay the effect of quercetin and Ara-C alone and in combination on colony formation by human leukemic cells (CFU-L). In all cases both drugs exhibited a dose-related inhibition of CFU-L in a range of concentrations between 10 nM and 10 microM and 0.01 nM and 10 microM for quercetin and Ara-C, respectively. The combination of the two drugs resulted in a synergistic inhibitory activity on CFU-L. Considering that plasma concentrations of quercetin effective in vitro were obtained in vivo without any apparent side effects, we conclude that this report represents further experimental evidence that quercetin could be used in the treatment of acute leukemias.

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.

Twenty bone marrow aspirates harvested for autotransplantation from 20 patients suffering from several oncohematological diseases were processed using the automated Du Pont SteriCell processor. In 15 bone marrow harvests, the interface buffy coat cells were collected using the SteriCell processor in manual mode with a semiautomated procedure. The procedure yielded an average red cell removal of 84% and an average mononuclear cell (MNC) recovery of 86%. Cloning efficiencies of hematopoietic progenitor cells (CFU-GM and BFU-e) did not differ between processed and recovered MNCs. Four cryopreserved bone marrow buffy coats were thawed and reinfused into four patients who had undergone high dose chemotherapy. Stable engraftment was observed in all cases. In five bone marrow harvests, the SteriCell automated density gradient MNC isolation procedure was performed after buffy coat collection. The whole two-step procedure allowed an average MNC recovery of 69%. CFU-GM and BFU-e assays did not show a significant difference in cloning efficiency between processed and recovered bone marrow MNCs. We conclude that the SteriCell processor offers rapid, safe and feasible procedures for the semiautomated processing of human bone marrow for transplantation. The clinical efficacy of density gradient separated bone marrow employing the automated step and the opportunity to use fully automated processing must be investigated.

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.

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.

Authors treated 4 patients suffering from advanced ovarian cancer with high-dose chemotherapy and autologous peripheral blood stem cell (APBSC) or autologous bone marrow stem cell (ABM) as hematopoietic support. In three patients were collected peripheral blood stem cells using a fully automated blood cell separator during hematopoietic recovery following aplasia induced by non-intensive chemotherapy (cisplatin 200 mg/m2 and cyclophosphamide 1,500 mg/m2). Hemopoietic reconstitution of the four patients submitted to APBSC or ABM support after high-dose chemotherapy (cisplatin 100 mg/m2, VP16 650 mg/m2 and carboplatin 1,800 mg/m2) showed the low hematological toxicity of our treatment with APBSC support. Moreover, the drug combination of cisplatin and cyclophosphamide has clinical activity in ovarian cancer and it is an optimal association to mobilize and harvest large number of PBSC.

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.

It has been demonstrated that the flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone; Q) inhibits the growth of several cancer cell lines. There is evidence suggesting that the antiproliferative activity of this substance is mediated by the so-called type II estrogen-binding site (type II EBS). We looked for the presence of type II EBS and the effect of Q on the proliferation of an Adriamycin-resistant estrogen-receptor-negative human breast-cancer cell line (MCF-7 ADRr). By whole-cell assay using estradiol labelled with 6,7-tritium [( 3H]-E2) as a tracer, we demonstrated that MCF-7 ADRr cells contain type II EBSs. Competition analysis revealed that diethylstilbestrol (DES) and Q competed with similar potency for [3H]-Es binding to type II EBSs. The antiestrogen tamoxifen (TAM) competed for type II EBSs, albeit to a lesser extent than either DES or Q. Growth experiments demonstrated that Q and DES exerted a dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 microM, whereas TAM was less effective. Q could also inhibit colony formation in a clonogenic assay. Our results indicate that multidrug-resistant estrogen-receptor-negative MCF-7 cells express type II EBSs and are sensitive to the inhibitory effect of Q. This substance could be the parent compound of a novel class of anticancer agents.

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.

Three patients (2 with high malignant non Hodgkin's lymphomas in partial remission and 1 with acute myeloblastic leukemia in 2nd complete remission) underwent autologous peripheral blood stem cell transplantation (APBSCT). The minimal number of mononuclear cells and CFU-GM collected was 6.18 x 10e8 and 19.77 x 10e4/kg b.w., respectively. Conditioning chemotherapy consisted in BEAM and CVB protocols in non Hodgkin's lymphoma (NHL) patients and busulphan and cyclophosphamide in acute myeloblastic leukemia (AML) patients. All patients achieved complete remission. Bone marrow biopsy performed on day 14 showed complete engraftment. The time to reach 1 x 10e9/l WBC, 0.5 x 10e9/l neutrophil granulocytes and 50 x 10e9/l platelets was no longer than 11, 15 and 8 days, respectively. No major infectious episodes were evident during aplastic phase; fever greater than 38 degrees C was observed in two patients not lasting longer than 2 days. All patients are still in complete remission and continue to have normal hematological values (follow-up lasting 8+ and 3+ months for NHL patients and 4+ months for AML patient).

Twenty collections of peripheral blood stem cells were performed in 3 patients (2 NHL, 1 AML) using the Haemonetics V50S discontinuous flow blood cell separator. A modified lymphocyte collection protocol (Nebraska Surge) was used in all instances. Leukapheresis were performed after 1 or 2 courses of chemotherapy and started when peripheral blood leukocytes count reached 1 x 10e9/l and platelets count 80 x 10e9/l. A mean blood volume of 5.9 +/- 0.6 litres was processed per procedure and the mean yields for mononuclear cells, nucleated cells and CFU-GM were respectively 5.4 +/- 1.4 x 10e9, 4.9 +/- 1.6 x 10e9 and 128.5 +/- 182.3 x 10e4 per procedure. Haemonetics V50S had showed a mean collection efficiency for mononuclear cells of 67.5 +/- 5.0% per procedure. Results obtained are not significantly different from the ones obtained with an automated continuous flow separator even if extracorporeal circulation is consistently high in patients with a low hematocrit when the 250 ml Latham Bowl is used.

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).

Using monoclonal antibodies against CD2, CD4, CD8 and CD19 antigens and an automated biotin-avidin immunoperoxidase technique on whole blood samples, we evaluated the technical performance and clinical usefulness of lymphocyte subset counting by the routine hematology analyzer Technicon H*1. Statistical evaluation demonstrated excellent precision and very good correlation with the immunofluorimetric flow cytometer Ortho Spectrum III. Correlation between manual immunofluorescence at the microscope and the H*1 method was much poorer, owing to the high intrinsic imprecision of the manual method. Reference ranges obtained with the H*1 immunoperoxidase method in 44 healthy subjects closely matched those obtained with the Spectrum III. In 46 subjects with or at risk for HIV infection, we found with the H*1 method a significant decrease in CD4+ cells and in the CD4+/CD8+ cell ratio, which was progressively more marked in HIV- negative patients with lymphadenopathic syndrome, AIDS-related complex, and in patients with full-blown AIDS.