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Focuses on a procedure for red blood cell (RBC) depletion and different cryoprotective solutions with or without rate-controlled freezing in an attempt to develop an inexpensive and simple method for processing and cryopreserving umbilical cord blood (UCB) hemopoietic cells. Cooling rate and concentration of the cryoprotectant; Analysis of RBC depletion by double gravity sedimentation and hyroxyethyl starch; Implications for UCB banks.

BACKGROUND PBPC transplantation has become a widely accepted therapeutic option for patients with chemotherapy-sensitive malignancies. PBPCs facilitate even the transplantation of mismatched allogeneic cells, because of their better engraftment. PBPCs are harvested by leukapheresis after mobilization with G-CSF or chemotherapy and G-CSF. To improve the purity and yield of PBPC grafts, to ensure reproducibility of the procedure and to reduce platelet loss in the patient/donor, different fully automated PBPC collection systems were developed. We report here our experience with the new Amicus blood cell separator. METHODS From July 2000 to March 2001, 46 procedures were performed. Six donors underwent leukaphereses after G-CSF administration (10-12 microgr/Kg/day) beginning on day 4; 14 patients were harvested after chemotherapy and G-CSF or GM-CSF mobilization, when the peripheral CD34+ cell count was >20 cells/micrL. Peripheral venous access was preferred whenever possible, otherwise femoral or subclavian catheters were used. Complete cell counts and CD34+ cell evaluations were done both in patients and donors, before and after each procedure and in all the products of the apheresis. RESULTS A median of 8.959 L of blood (range 3.585-17.021 ) was processed with a duration of 315 minutes (range 215-400) at a blood flow rate of 40 ml/min (range 25-70). ACD was used at a ratio of 1:14. The products of apheresis had a median volume of 143 ml (range 70-306) and a Hct of 12.9% (range 1.6-21). Platelet contamination was low (722 × 10[sup 9]/L, range 55-1,620), according to a low collection efficiency (12%, range 2-34) and a low decrease of platelet level both in donors and patients (21 × 10[sup 9]/L, range 3-83); in contrast, CD34 + cell collection efficiency was high (59.5%, range 15-97). No adverse affects were observed. CONCLUSIONS Amicus blood cell separator has a high collection efficiency for PBPC collections and improves donor safety by reducing platelet loss. [ABSTRACT FROM AUTHOR]

TLR9 activation by unmethylated CpG provides a homeostatic mechanism to maintain B cell memory in the absence of Ag. In this study, we demonstrate that CpG also triggers the generation of somatically mutated memory B cells from immature transitional B cells. In response to CpG, a fraction of transitional B cells proliferates and introduces somatic hypermutations in the H chain V regions. The nonproliferating pool of transitional B cells mostly maintains germline configurations. Mutations are VH specific: VH5 is the least mutated family, whereas VH1 and VH4/6 are the most mutated families. CpG stimulation also results in upregulation of VH5 transcripts in proliferating cells. Therefore, early recognition of bacterial DNA preferentially expands VH5-expressing B cells while inducing somatic hypermutations in other families. The mutation frequency, range, and type of substitutions observed in vitro are comparable to those found in memory B cells from the peripheral blood of Hyper IgM type 1 patients and the spleen of normal infants. The process triggered by TLRs may represent a first step leading to additional diversification of the germline repertoire and to the generation of memory B cells that will further refine their repertoire and specificity in the germinal centers.

The authors compared the efficiency of two different blood cell separators (Amicus and Cobe-Spectra) in collecting peripheral blood progenitor cells for autologous or homologous transplantation. A total number of 129 procedures were performed, 36 with Spectra, 93 with Amicus. There was no difference between Spectra and Amicus efficiencies for CD34+ cell collection (46.685% vs 46.235%; p=n.s) but the platelet efficiencies were 17.31% and 12.54% respectively (p=0.04) and, if autologous and allogeneic collections were considered separately, a marked difference resulted in allogeneic platelet efficiency between 6 Spectra and 23 Amicus procedures (26.83% vs 8.68%, p=0.0004). The authors were able to demonstrate that in 70 Amicus autologous collections there was a different platelet efficiency, if peripheral count was considered: 12 procedures performed with a platelet count > 100 x 10(9)/l had a very low efficiency (6.86%), but this value increased if platelet count lowered (13.02% if between 100 and 50 x 10(9)/l, 22.63% if between 50 and 0 x 10(9)/l, 23 and 35 procedures respectively). The study is preliminary and the number of collections is little, but the overall data suggest that Spectra (AutoPBSC, V 6.0) and Amicus separators have the same efficiency for collecting CD34+ cells while Amicus procedures have a very low platelet contamination, especially with donors.

High-dose chemotherapy and radiation used as a preparative regimen for allogeneic stem cell transplantation (SCT) produces a considerable morbidity and mortality. An alternative strategy, developed to reduce transplant-related toxicity and to induce graft versus malignancy effect in the presence of full hematopoietic engraftment, includes the application of RIC that provide sufficient immunosuppression. We studied the efficacy and toxicity of RIC followed by allogeneic SCT in elderly patients or with relative contraindications to conventional SCT. In all, 22 patients with hematologic malignancies and HLA-identical sibling donors were included in this study. All patients were either refractory to therapy or beyond first complete remission (CR). The majority of patients received fludarabine 120 mg/m(2)+thiotepa 10 mg/kg as conditioning regimen and cyclosporine as graft versus host disease (GVHD) prophylaxis. Organ toxicity was acceptable and all evaluable patients achieved engraftment. Three patients (15%) showed grade >II aGVHD; extensive chronic GVHD occurred in three out of 15 evaluable patients (20%). With median follow-up of 63.5 months, survival was 31%, disease-free survival (DFS) was 23%. All the durable responses occurred in patients who developed GVHD. Transplant related mortality (TRM) at 100 days was 27.3%, 67% of that caused by infections, while 6-year cumulative incidence of TRM was 38%. Our data show that RIC: (1). allows the engraftment of HLA-matched hematopoietic stem cells (2). provide durable responses in patients not eligible for conventional SCT exploiting the graft-versus-malignancy effect and (3). is loaded by an high rate of fatal infections.

In this study, we investigated whether hematopoietic stem cells (HSC) and progenitors present in human cord blood can express nerve growth factor (NGF)-specific receptors, TrkA and p75. Our results showed a marked expression of TrkA and NGF in cord blood CD34(+) cells. A gradient of TrkA and NGF expression exists and is highest in cord blood CD34(+) cells, reduced in cord blood mononuclear cells (MNC) and minimal in mononuclear cells isolated from adult peripheral blood. Our findings suggest that NGF may play a role in the differentiation of hematopoietic progenitors and indicate a different requirement for NGF by immune cells, depending on their state of maturity.

There is a progressive increase in the use of selected hematopoietic progenitor cells after myeloablative therapy in patients affected by malignancies. Our goal was to determine which blood parameters, in the starting cell population, influence the concentration of CD34+ progenitors and the removal of unwanted cells in the final product. Also, we evaluated the hematopoietic recovery and toxicity associated with peripheral blood stem cell infusion. We retrospectively reviewed 53 procedures of positive selection of CD34+ cells, performed with the Ceprate SC immunoadsorption system, in 47 paticnts affected by various hematologic malignancies and solid tumors. An increased percentage of CD34+ cells in the starting fraction was associated both with the final purity and enrichment of CD34+ cells and with a decreased percentage of CD3+ and CD19+ cells in the final product. A low platelet count before selection had a borderlinc influence on the recovery of CD34+ cells. Forty patients received a median of 5 x 10(6) CD34+ cells per kg; the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l in a median of 10 days whereas a PLT count above 20 x 10(9)/l was observed in 14 days. The reinfusion of selected CD34+ cells, containing a very low amount of dymethylsulfoxide. was well tolerated and no adverse reactions were observed. Autologous transplantation with selected CD34+ cells is a safe and well-tolerated procedure in patients affected by hematologic malignancies and solid tumors. Positive selection of CD34+ cells seems to be related to the quality of the apheresis products, particularly to the initial CD34+ cell and PLT content.

Central venous access is necessary in patients candidate for peripheral blood stem cell (PBSC) collection. We report our experience with a dual lumen femoral catheter (Gamcath, 11 french), initially designed for hemodialysis. We studied 147 patients and performed 488 collections after mobilization with either G-CSF alone or chemotherapy + G-CSF, when the white blood cell count exceeded 1 x 10(9)/L, or when a measurable population of CD34+ cells (20/microL) was detected in peripheral blood. All patients received systemic anticoagulation with a low weight heparin and ultrasound examination was performed after the removal of the catheter. Seven patients developed thrombosis (4.7%), ten experienced hematomas at the site of catheter placement (6.8%) despite prophylactic platelet transfusions, while only one patient (0.6%) had a catheter-related infection. In conclusion, the short-term use of large bore femoral catheters in setting up PBSC collection seems to be associated with minimal risk of infection and low thrombotic incidence.

Determination of CD34+ cells was performed in bone marrow and G-CSF mobilised peripheral blood samples. We adopted three different protocols of analysis: the Milan/Mulhouse protocol, the ISHAGE guidelines for CD34+ cell determination and our own protocol based upon the use of PAINT-A-GATEPRO software analysis program. An excellent correlation was demonstrated between the three methods (r2 0.98); however the analysis of variance showed a statistically significant difference between the results generated with the three methods (P=0.001). The differences between the three procedures are discussed with a special focus on the value of CD34+dim cells and the role of CD45 in the setting of a double staining. We have in fact identified a minor subset (CD34+CD38+CD45-) which would go unrecognised based upon its CD45 negativity.