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SUMMARY: Objectives: To study the rate of ABO haemolytic anaemia of fetus and newborn (HDFN) in one institution over 6 years. Background: ABO major incompatibility between mothers and their newborns occurs in about 10% of births. So, mothers with an O blood group may form IgG‐class antibodies against A and B antigens, which could pass across the placenta and lead to a variable degree of HDFN in the newborn. Methods: At our institution, we have reviewed data regarding ABO‐based HDFN in the last 6 years. Results: We found that, in 28 089 deliveries, an ABO major incompatibility between mothers and newborns occurs in 11% of cases, with 72% of O/A and 28% of O/B incompatibility. In turn, 23% of these newborns had an eluate‐confirmed positive direct antiglobulin test [DAT; 74% (511) were due to anti‐A and 26% (179) to anti‐B], with 1·0% requiring invasive treatments (exchange transfusion or intravenous immunoglobulin). Overall, 2·5% of the total newborns had a positive DAT for an anti‐A or anti‐B antibody, and 0·11% required invasive treatment in addition to phototherapy for their HDFN. Conclusions: Serological ABO HDFN is a relatively frequent event when an O‐A/O‐B incompatibility between mothers and their newborn occurs and, in most cases, translates into a self‐limiting disease, with a small number of newborns requiring invasive treatments. The DAT test, although not predictive of disease severity, appears to be a useful tool to monitor babies born from O‐A/O‐B‐incompatible pregnancies and to identify those who may require treatment. [ABSTRACT FROM AUTHOR]

The article reports that cloning of blood group genes and subsequent elucidation of the molecular backgrounds to blood group polymorphisms have made it possible to predict blood group phenotypes from the DNA of a patient or donor, when a suitable red cell sample is not available. It mentions that purpose of Vox Sanguinis International Forum is to find out where fetal blood grouping is being carried out.

Background and Objectives A photochemical treatment process ( PCT) utilizing amotosalen and UVA light ( INTERCEPT™ Blood System) has been developed for inactivation of viruses, bacteria, parasites and leucocytes that can contaminate blood components intended for transfusion. The objective of this study was to further characterize the safety profile of INTERCEPT-treated platelet components ( PCT- PLT) administered across a broad patient population. Materials and Methods This open-label, observational haemovigilance programme of PCT- PLT transfusions was conducted in 21 centres in 11 countries. All transfusions were monitored for adverse events within 24 h post-transfusion and for serious adverse events ( SAEs) up to 7 days post-transfusion. All adverse events were assessed for severity (Grade 0-4), and causal relationship to PCT- PLT transfusion. Results Over the course of 7 years in the study centres, 4067 patients received 19 175 PCT- PLT transfusions. Adverse events were infrequent, and most were of Grade 1 severity. On a per-transfusion basis, 123 (0·6%) were classified an acute transfusion reaction ( ATR) defined as an adverse event related to the transfusion. Among these ATRs, the most common were chills (77, 0·4%) and urticaria (41, 0·2%). Fourteen SAEs were reported, of which 2 were attributed to platelet transfusion (<0·1%). No case of transfusion-related acute lung injury, transfusion-associated graft-versus-host disease, transfusion-transmitted infection or death was attributed to the transfusion of PCT- PLT. Conclusion This longitudinal haemovigilance safety programme to monitor PCT- PLT transfusions demonstrated a low rate of ATRs, and a safety profile consistent with that previously reported for conventional platelet components. [ABSTRACT FROM AUTHOR]

Red blood cell ( RBC) transfusions are a milestone in the treatment for sickle cell anaemia ( SSA) and for thalassaemia. RBC alloimmunization remains a major challenge of chronic transfusion therapy, and it can lead to adverse life-threatening events. The alloimmunization risk could depend on multiple factors such as the number of transfusions and, most of all, the genetic background. Different ethnic groups are predisposed to immunization because of a significant degree of RBC antigenic mismatch between donor and recipient. There is no universal agreement and standards for the most appropriate selection of RBC units in chronically transfused subjects. Current practice only deals with compatibility of ABO, Rh and K antigens. Molecular RBC antigenic matching extended to other blood group systems is an innovative strategy to ensure a better quality and effectiveness of transfusion therapy. [ABSTRACT FROM AUTHOR]

Summary Introduction: Accurate white blood cell counting (WBC) and differential count by blood analyzers could allow a more informative characterization of granulocyte colony-stimulating factor (G-CSF) mobilized blood (MB), leukapheresis products (LP), and cord blood (CB). However, reliable counting by a blood cell analyzer in this setting is a major challenge owing to quali-quantitative abnormalities of blood cells. Methods: We evaluated the performances of the analyzer Pentra DX 120 by Horiba ABX working with dedicated cell-gating profiles, which generate three-part differential counts in samples obtained from donors' MB, LP, and CB. The results of the analyzer were compared to counts obtained by flow cytometry and manual counts, the latter performed for reference validation and in the case of discrepant results between study and reference counts. Results: Pentra DX 120 generated highly correlated counts ( R > 0.91 in all cases) to those obtained by flow cytometry in all samples (MB, LP, and CB) with high degree of count accuracy in most cases and referred to WBC absolute count and differential count including lymphocytes (LYM) %, monocytes (MON) %, and polymorphonuclear leukocytes (PMN) %. Accuracy, judged by the difference between study and reference counts and expressed as percentage of reference count, ranged from 0.8% to 8.6%, and sporadic loss of accuracy occurred for MON % only in no more than 10% of CB samples. Conclusion: The ABX Pentra DX 120 provided accurate WBC count and differential count during MB, LP, and CB analyses and allowed a better characterization of donors' hematologic status and graft composition. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate a set of reference counts applied to multiparameter pre-counts in blood donors. Aim: Analyse the impact of pre-donation counts and specific reference intervals on donors' management. Background: Multiparameter blood counts allow an improved enrolment process of blood donors due to a prompt identification of abnormalities involving haemoglobin (Hb), white blood cells (WBC) and platelets (PLT). Methods/Materials: Multiple pre-donation capillary counts were applied in the enrolment process of 13 347 consecutive donors. The rate of specific alterations of permanent exclusion and donor readmittance to donations for temporary exclusion had been evaluated, applying a set of multiparameter reference intervals. Results: Alterations involved Hb in 72·55% of cases, mean corpuscular volume (MCV) in 20·99%, total WBC in 9·39%, lymphocytes in 7·55% and PLT in 6·07%. Among donors with initial alterations (543; 4·06%), 12·70% were readmitted to donations within 15 days, 14·36% had permanent exclusion, 36·83% underwent prompt supplementation treatment and 36·09% were lost at follow-up or refused treatments. Discussion: The systematic use of blood count reference intervals and pre-donation multiparameter blood counts allowed prompt identification of WBC, PLT and MCV alterations, readmittance within 15 days of 12·70% of initially excluded donors and contributed to prompt management of supplement deficiency. [ABSTRACT FROM AUTHOR]

Background and Objectives Blood donor enrolment process is frequently based on the sole capillary haemoglobin (Hb) evaluation while platelet donors by apheresis also requires platelet (Plt) count. The ‘sole Hb’ approach prevents a complete donor evaluation and does not allow Plt donor enrolment. To extend blood counts before donations, we evaluated the performances of a multiparametric counter using capillary blood. Materials and Methods The ABX Micros 60 (Micros 60) blood analyzer was employed on capillary blood and compared with venous counts by a reference counter (Coulter AcT 5diff) in a first series of 416 donors and in a second series of 136, after a 3-month period of routine use of this study counter. An average of 50 μl of capillary blood was collected whose 10 μl had been aspirated by Micros 60. Results High correlations were found between capillary counts using Micros 60 and venous counts using the reference counter. Mean Plt counts differed of 37 × 109/l less for capillary approach in the first series of comparisons, but decreased to 10 × 109/l less in the second series due to a greater expertise of operators in capillary sampling. All other parameters were accurate and never reached clinical relevance albeit they showed statistically significant differences. Conclusion Data on Micros 60 demonstrated that capillary predonation counts may represent a feasible and effective approach to realize an accurate enrolment process of blood and Plt donors. [ABSTRACT FROM AUTHOR]

Background Despite improvements in hepatitis B surface antigen (HBsAg) test sensitivity, post-transfusion hepatitis B virus (HBV) infection still occurs because HBsAg is undetectable during the early window phase (WP) of the infection, in the convalescence core window phase of the infection, or in serologically silent chronic hepatitis or in mutant forms of HBV. HBV-DNA screening using high sensitivity nucleic amplification technology (NAT) assays has recently been introduced to reduce the residual risk of transmission of HBV by transfusion of blood components. Materials Over 1 year 75 063 donations were individually screened for HBV-DNA by the Ultrio Procleix assay on the Tigris platform. The donations were collected in the Latium region, an area of the central Italy, and they accounted for the 40% of the total blood units collected in this area per year. The initial reactive samples were re-tested and confirmed by the discriminatory HBV assay. Additional HBV serological markers were also performed. Suspected WP infections were followed-up to monitor the development of the immune response. All HBV-DNA-positive donors were called back to check up their infectious status. Results The results of testing the 75 063 donations are: 33 donations HBsAg positive, 31 out of them HBV-DNA-positive and two HBV-DNA negative; 22 donations HBsAg-negative but HBV-DNA positive with low viral load. Six of the 22 were found to be consistently HBV-DNA reactive whereas the remaining 16 donations showed inconsistent results on multiple NAT retesting. One WP infection was confirmed by the follow-up of the donor for 3 months following the index blood donation. Conclusions In the donor population of the Latium region, NAT screening has revealed a higher than expected number of donors who were HBsAg non-reactive but HBV-DNA-positive with three donors showing HBV-DNA as the only marker of infection. The adoption of genome screening has increased the safety of the blood supply and has also contributed to the protection of donor health by identifying either WP or clinically silent infections. [ABSTRACT FROM AUTHOR]

Background and Objectives Stem cell collection is a standard procedure for the procurement of autologous grafts to rescue myelosuppression induced by high-dose treatments. Accurate prediction of collection yields may contribute to optimize planning and quality control of collection. Materials and Methods Data of 313 autologous haematopoietic stem cell (AHSC) evaluable collections performed in 208 patients with haematologic and non-haematologic neoplasms from seven centres were prospectively analysed to test the accuracy of yield predictions generated by a formula that required the input of peripheral blood (PB) CD34+ cell precount and desired PB volume to be processed. Data were matched in a standard linear regression, in a zero-point regression analysis and tested for prediction accuracy. Further 165 AHSC collections were analysed on a single-centre basis, using yield predictions as reference standards. Results Analysis showed high levels of correlation between measured collection yields (my) and predictions (py) ( R = 0·85; P = 0·000000) as well as high degree of prediction accuracy (my vs. py at paired t-test: P = 0·114781; median my/py ratio = 1·23). Analysis of additional 165 AHSC collections on a single-centre basis showed that the analysed centres had 70% or more measured yields comprising the 0·6–1·8 interval of the my/py ratio. The observance of the ‘efficiency’ my/py interval assured collection quality control in these centres confirming the reliability of the method. Conclusions This prediction method generates accurate and immediate yield predictions allowing collection planning and rapid efficiency control. As a consequence of our study, four centres out of seven use the described method to plan both leukapheresis number and single-procedure blood processing volume while the remaining three centres plan leukapheresis number on the basis of our predictions, maintaining a fixed single-procedure 200 ml/kg blood volume processing, according to their centre AHSC collection policy. [ABSTRACT FROM AUTHOR]

Background: Peripheral blood progenitor cell (PBPC) transplantation (PBPCT) combined with post-PBPCT administration of myelopoietic growth factors is a valid therapeutic intervention to rapidly restore hematopoiesis after the delivery of intensive, myeloablative cancer chemotherapy. On the other hand, the best growth factor regimen to potentiate PBPC-mediated immunohematopoietic recovery has yet to be determined.Study Design and Methods: In a randomized evaluation, the effects produced by post-PBPCT G-CSF and GM-CSF on myeloid/lymphoid recovery and transplant outcome in women with chemosensitive cancer were compared. Thirty-seven ovarian cancer patients and 34 breast cancer patients ranging in age from 24 to 60 years were treated with carboplatin, etoposide, and melphalan (CEM) high-dose chemotherapy and then randomly assigned to receive G-CSF (5 microg/kg subcutaneously) or GM-CSF (5 microg/kg subcutaneously) until Day 13 after PBPCT. Patients were compared in regard to hematopoietic recovery, posttransplant clinical management, and immune recovery. Finally, clinical outcome was estimated as time to progression and overall survival.Results: Hematopoietic recovery and posttransplant clinical management were comparable in both the G-CSF and GM-CSF series. Conversely, significantly higher T-cell counts were observed in G-CSF-treated patients during the early and late posttransplant follow-up. Patients who received G-CSF showed a significantly longer median time to progression. A parallel analysis revealed that patients in whom a higher CD3+ count was recovered had a significantly longer overall survival and time to progression.Conclusion: The enhancement of post-PBPCT T-cell recovery observed in G-CSF-treated patients encourages the use of G-CSF to ameliorate immune recovery, which seems to play a role in post-PBPCT control of disease in cancer patients. GM-CSF might be administered to prolong immunosuppression after autologous PBPCT for autoimmune diseases or allogeneic PBPCT. [ABSTRACT FROM AUTHOR]

Background: The peripheral blood progenitor cell (PBPC) mobilization capacity of EPO in association with either G-CSF or sequential GM-CSF/G-CSF was compared in a randomized fashion after epirubicin, paclitaxel, and cisplatin (ETP) chemotherapy.Study Design and Methods: Forty patients with stage IIIB, IIIC, or IV ovarian carcinoma were enrolled in this randomized comparison of mobilizing capacity and myelopoietic effects of G-CSF + EPO and GM-/G-CSF + EPO following the first ETP chemotherapy treatment. After ETP chemotherapy (Day 1), 20 patients received G-CSF 5 microg per kg per day from Day 2 to Day 13 and 20 patients received GM-CSF 5 microg per kg per day from Day 2 to Day 6 followed by G-CSF 5 microg per kg per day from Day 7 to Day 13. EPO (150 IU per kg) was given every other day from Day 2 to Day 13 to all patients in both arms of the study. Apheresis (two blood volumes) was performed during hematologic recovery.Results: The magnitude of CD34+ cell mobilization and the abrogation of patients' myelosuppression were comparable in both study arms; however, GM-/G-CSF + EPO patients had significantly higher CD34+ yields because of a higher CD34+ cell collection efficiency (57.5% for GM-/G-CSF + EPO and 46.3% for G-CSF + EPO patients; p = 0.0009). Identical doses of PBPCs mobilized by GM-/G-CSF + EPO and G-CSF + EPO drove comparable hematopoietic recovery after reinfusion in patients treated with identical high-dose chemotherapy.Conclusion: The sequential administration of GM-CSF and G-CSF in combination with EPO is feasible and improves the PBPC collection efficiency after platinum-based intensive polychemotherapy, associating high PBPC mobilization to high collection efficiency during apheresis. [ABSTRACT FROM AUTHOR]

MUC1 is a large surface glycoprotein expressed by epithelial cells, which is overexpressed and aberrantly glycosylated in carcinomas. MUC1 is involved in epithelial cell interactions and appears to function as a signal-transducing molecule. The finding that MUC1 can also be expressed in the haematopoietic lineages prompted us to further investigate the possible function(s) of this molecule in haematopoietic cells. In bone marrow differentiating cells, MUC1 was strongly and selectively expressed during erythropoiesis; it was also weakly expressed during megakaryocytopoiesis and granulomonocytopoiesis; however, no correlation between MUC1 and differentiation marker expression was observed in these lineages. In vitro CD34+ cells, induced towards erythroid differentiation, acquired MUC1 transiently, while expressing increasing levels of the lineage marker glycophorin A. MUC1 was absent in the circulating erythrocytes. During erythropoiesis, MUC1 expression was transcriptionally regulated and the molecule underwent phosphorylation. To investigate the possible role of MUC1 during erythropoiesis, we studied the ability of MUC1 to act as ligand for cell-cell interaction. The sialylated MUC1 glycoforms selectively expressed on erythroid cells were able to bind the macrophage-restricted molecule sialoadhesin. These results suggest that MUC1 can function as a cross-talk molecule between the erythroblasts and the surrounding cells during erythropoiesis.

Stem/progenitor cells endowed with in vitro and in vivo haematopoietic activity express the surface protein CD34. Transforming growth factor beta1 (TGF-beta1) is one of the soluble molecules that regulate cell cycle and differentiation of haematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has previously been shown that TGF-beta1 maintains human CD34+ haematopoietic progenitors in an undifferentiated state, independently of any cell cycle effect. Here, we have shown that TGF-beta1 upregulates the human CD34, an effect that was evident in primary stem/progenitor cells (CD34+lin-) both at the transcriptional and protein levels, and was not associated with any relevant effect on cell growth. The presence of TGF-beta1 influenced differentiation, maintaining primary CD34+/Lin- in an undifferentiated state. This effect was associated with Smad activation and with a dramatic decrease in p38 phosphorylation. Moreover, blocking p38 phosphorylation by the SB202190 inhibitor increased CD34 RNA levels but did not enhance CD34 protein expression in CD34+/Lin- cells, suggesting that modulation of multiple signalling pathways is necessary to reproduce TGF-beta1 effects. These data establish the role that TGF-beta1 has in the modulation of the CD34 stem/progenitor protein and stem/progenitor functions, providing important clues for understanding haematopoietic development and a potential tool for the modulation of human haematopoiesis.

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte-macrophage colony-forming units (GM-CFU) and burst-forming units - erythroid (BFU-E) were retained in the CD34+/CD105- fraction, whereas rare GM-CFU/BFU-E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti-TGF-beta1 antibody demonstrated CD34+/CD105+ cells capable of colony-forming activity without any significant effect on CD34+/CD105- cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105- cells. CD34+/CD105+ cells had a significantly higher long-term culture-initiating cell (LTC-IC) frequency than CD34+/CD105- cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki-67- cells whereas 82% of CD34+/CD105- were DNA 2n G1Ki-67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105- contained a small CD25+ subset. Three-colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/- primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

The recovery of lymphocyte count, CD4+ and CD8+ T-cell subsets, natural killer (NK) cells and CD19+ B-cells was evaluated in a cohort of 15 patients receiving autologous CD34+ peripheral blood progenitor cells (PBPCs; group A) for haematological malignancies and in 20 patients transplanted with autologous unselected PBPCs (group B). Lymphocyte count recovered in both patient cohorts, being significantly lower in group A than in group B 1 (P = 0.008) and 2 months (P = 0.0035) after progenitor cell infusion. The repopulation of CD3+ T-cells occurred more rapidly in group B than in group A (P = 0.034 on week 4); CD19+ B-lymphocytes did not return to reference ranges in either group of patients. The count of CD4+ T-lymphocytes remained < 200/microl during the study period in patients transplanted with CD34+ PBPCs, significantly lower than group B levels (P = 0.034 and P = 0.021 on weeks 4 and 8 respectively). CD8+ T-cells increased rapidly in both groups; thus, the CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T-cells displayed an activated phenotype in both groups of patients, co-expressing the HLA-DR antigen throughout the study period. NK cells followed a similar repopulation kinetics in both study groups, although their expansion was greater in group B than in group A (P = 0.014 on week 4). In the CD34+ group, post-transplant administration of granulocyte colony-stimulating factor predicted a faster lymphocyte recovery in multivariate analysis (P = 0.025); interestingly, the amount of passively transferred lymphocytes correlated inversely with time to achieve a lymphocyte count > 0.5 x 10(9)/l (r = -0.63, P = 0.01). Further investigations are necessary to characterize T-cell competence after transplantation of CD34+ PBPCs.

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.

The plasma concentrations of erythropoietin (Ep), soluble transferrin receptors (sTfRs), iron, total iron binding capacity (TIBC) and ferritin were monitored in five leukaemia patients undergoing autologous bone marrow stem cell transplantation (BMSCT) and in 10 lymphoma and 21 ovarian cancer patients undergoing autologous peripheral blood SCT (PBSCT); 9/21 ovarian cancer patients received recombinant human G-CSF and Ep and six recombinant human GM-CSF and Ep following SCT. All parameters were evaluated in relation to the kinetics of erythroid reconstitution as evaluated by haemoglobin (Hb) and reticulocyte levels [including the fraction of immature reticulocytes, also called highly fluorescent reticulocytes (HFR)]. Leukaemia patients undergoing BMSCT showed only a delayed (occurring at days 35-50 after SCT) and partial RBC, neutrophil and platelet recovery, whereas all patients undergoing PBSCT exhibited a rapid (occurring at days 10-15 after SCT) and sustained haemopoietic recovery. The various levels of erythroid rescue observed among these patients markedly influenced the kinetics of the different parameters investigated: (i) in leukaemia BMSCT patients sTfRs declined following SCT and remained at low levels thereafter, whereas Ep, iron. TIBC and ferritin showed a progressive and significant increase; (ii) in the different groups of patients undergoing PBSCT: (a) sTfR levels first declined following SCT and then returned to pre-therapy values at days 12-16, this response preceded erythropoietic recovery; (b) Ep, total iron, TIBC and ferritin showed an initial increase in the first days following SCT and then returned to pre-therapy values. Altogether, these observations indicate that: (i) both sTfR levels and reticulocyte counts are predictive parameters of erythropoietic recovery; (ii) coordinated changes of biochemical parameters underlying iron metabolism (iron, TIBC and ferritin) accompany erythroid rescue following SCT.

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.

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.

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.

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.

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 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.