ALBERT

Description: Introduction: At moment PBSC collections can be performed using semiautomated or automated cell separator devices. The collection with semiautomated methods implies an augmented working load for the dedicated personnel and is strongly influenced by the operator. On the contrary, the automated methods offer the advantages of a diminuished working load for the dedicated personnel and an high standardization of the collection procedure. Herein we report our experience on 60 PBSC collections employing the new automated COM.TEC Fresenius autoMNC program that provides the possibility to predict the total number of CD34+ cells collected basing on the CD34+ cell count (x μL) pre-leukapheresis (LKF) collection in peripheral blood. Materials and Methods: 39 patients affected with various onchohematological diseases and10 healty donors were mobilized with chemotherapy + G-CSF or G-CSF alone, respectively, and subsequently underwent LKF collection for auto or allotransplant. According to our internal protocol 60 LKF collections were performed starting with a CD34+ cell count in peripheral blood at least of 20/μL. Net weight of the final LKF product and its CD34+ cell content were evaluated at the end of each PBSC collection procedure and then compared to the expected data calculated by the cell separator device. Moreover a post collection peripheral blood Plt count was evaluated for each patient/donor. Results: The mean starting WBC count was 25.86x103/μL (range: 4–82.3), Plt count was 151.38x103/μL (20–395), CD34+ cells was 96.63/μL (20–332). The mean WBC and CD34+ cells in the LKF collection were 224.78x103/μL (20.71–425.3) and 565.45x106 (59.3–1609.3), respectively. The mean volume of the LKF collection was 237.28 ml (120–503). The mean estimated CD34+ cell content was 498.37x106 while the real mean CD34+ LKF cell content was 623.32x106. The mean CD34+ cell collection efficiency was 91% (66–126). Finally, the mean post procedure Plt count in patient/donor was 77.91x103/μL (12–164). Conclusions: The automatized PBSC collection with the new program COM.TEC Fresenius autoMNC demonstrated a very high CD34+ cell collection efficiency. Moreover the possibility to predict the CD34+ cell yield permits an optimal management of the LKF collection, reducing the number of procedures per patient/donor. The difference observed between the mean estimated CD34+ cells and the real CD34+ cell content may be due to the intra-procedure stem cell mobilization phenomenon. Finally, this new automatized collection system demonstrated to limit the collection related thrombocytopenia either in patient or in donor.

Description: There is large interest in the use of mesenchymal stromal cells (MSCs) in approaches of cell-therapy and tissue engineering. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns in the case of clinical grade cellular preparations because of the theoretical risk of transmission of zoonoses and triggering immune reactions in the host. Therefore, the identification of a serum-free medium appropriate for both the extensive expansion necessary to reach the large numbers of MSCs required for clinical application, and the exclusion of risks connected with the use of animal products, is warranted. Aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL) are endowed with biological properties appropriate for cell-therapy approaches. MSCs were generated from bone-marrow of 8 healthy hematopoietic stem cell donor; 4 different culture conditions were tested: 10 % FCS; 5% PL; 2,5% PL; 1% PL. MSCs were harvested when reaching ≥ 80% confluence and replated for expansion at 4.000 cells/cm² until passage 5. CFU-F frequency, proliferative capacity, morphology, surface phenotype and differentiation capacity were evaluated. In particular, the immune regulatory effect on alloantigen-specific immune response, the kinetics of cytokine production and the resistance to spontaneous transformation into tumor cells of MSC expanded in the presence of either PL or FCS were investigated. Our results demonstrate that MSCs expanded in either FCS or PL display comparable morphology, phenotype and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. Immune-regulatory effect of MSCs was investigated on alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: decrease alloantigen-induced cytotoxic activity; favor differentiation of CD4+ T-cell subsets expressing Treg phenotype; increase early secretion of IL-10 in MLC supernatant, as well as to induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and in reducing early IFNg-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by both molecular karyotyping (array-comparative genomic hybridization) and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the hypothesis of a remarkable immune functional plasticity of human MSCs and suggest that the use of MSCs-PL, which seem to be endowed with a relatively low immune suppressive activity, could be more appropriate in approaches of reparative/regenerative cell-therapy or in strategies aimed at improving hematopoietic/immune recovery after hematopoietic stem cell transplantation (HSCT). On the contrary, as MSCs-FCS seem to display a more pronounced immune suppressive function, they might be more suitable for preventing or treating alloreactive-related immune complications, such as severe Graft-versus-host disease (GvHD) in HSCT and graft rejection in HSCT and solid organ transplantation.

Description: Introduction: Platelet concentrates (PC) collected by apheresis are effective in supporting deeply thrombocytopenic patients. The reduced risk of multiple allogeneic exposure and transmissible infectious diseases together with the high WBC depletion and diminished transfusion reactions are the main advantages offered by PC transfusion. At moment, the availability of several synthetic solutions for platelets storage permits to prepare hyperconcentrate(dry) apheresis platelets with the advantage of reducing febrile non-hemolytic transfusion reactions and, in low body weight patients the citrate toxicity, without the necessity of further manipulations. The aim of this study was to test the quality of 20 dry platelets (DP) in comparison to 20 standard plateletpheresis (SP) concentrates. Materials and methods: A total of 40 apheresis procedures were performed by the single-needle Cobe Trima separation device (Gambro BCT, Lakewood, CO, USA) collecting either DP or SP concentrates. Within 1h after collection, the bag containing DP was added with the appropriate amount (70% of DP final volume) of synthetic solution for platelets storage (SSP, MacoPharma). Both DP and SP concentrates were stored at room temperature with gentle agitation for 4 days. For both concentrates, platelet yield was calculated and in vitro studies of membrane glycoproteins expression and aggregation at day +1 and day +4 were carried out. Results: The comparison between 20 DP and 20 SP concentrates in terms of ability to aggregate in vitro and membrane glycoproteins expression at day +1 and day +4 of storage is reported in table A and B respectively. Conclusions: The in vitro tests documented a major activation of dry platelets. In particular, the ability to aggregate was reduced in the 20 DP concentrates analised and this phenomenon was more evident at day +4 of storage. The alteration of membrane glycoproteins expression (markers of storage lesion) confirms the lower in vitro quality of DP concentrates. The effectiveness of this new blood component in vivo should be evaluated in a controlled clinical trial. Table A. At collection Day +1 Day +4 SP DP SP DP SP DP Collagen μg/ml 4 93 88 97 82 80 53 ADP 10 μM 32 11 24 16 16 5 Ristocetin 1.5 mg/ml 91 97 77 81 71 52 Collagen 10 μg/ml + Adrenaline 10 μM 98 95 93 94 93 80 ADP 10 μM + Adrenaline 10 μM 87 72 76 61 57 30 Table B. At collection Day +1 Day +4 SP DP SP DP SP DP GPIb alfa (MFI) 5.06 5.75 6.31 6.13 5.26 4.54 GPIIb-IIIa (MFI) 35.47 36.71 34.61 37.7 31.76 40.9 GP IV (MFI) 11.49 11.4 11.67 10.28 11.65 11.38 GP 53 24.23 27.11 21.74 25.91 21.46 31.84 GMP 140 21.79 29.29 22.65 30.38 20.58 34.89

Description: Extracorporeal photochemotherapy (ECP) is an effective, relatively new technique, FDA approved for cutaneous T-cell lymphoma, employed as second-third line treatment in patients affected with GVHD not or poorely responsive to standard immunosuppressive drugs. Both aGVHD and cGVHD are the major complications after stem cell transplantation (till to 50% and 80% of the patients, respectively). Corticosteroids, cyclosporine, micofenolate, tacrolimus and various experimental monoclonal antibodies (anti CD40 ligand, anti TNFα etc) are the drugs employed to control GVHD and are burdened with important short and long term side effects. Recently we revised our data about 102 pts (children and adults) treated by ECP in our Institution. The overall response was 75%, permitting to taper or suspend the immunosuppressive therapy (IST) in 67.6% of the pts. The economical and social impact (cost analysis and quality of life) of ECP vs standard IST was calculated basing on the National Price List and on Short Form Quality of Life (QoL) Scoring System. The cost of ECP comprehensive of the leukapheresis procedure, waste matherials and dedicated personnel was estimated as 684.51 Euro/procedure. On the other side, when the solely costs of the most common IST drugs (corticosteroids, cyclosporine) were considered for 5 months of treatment, an evident and obvious economical advantage emerged (120,3 E). On the contrary, when the costs of hospitalisation and day hospital regimen derived from the most common side effects related to the solely use of IST were included in our cost analysis studies, an economical advantage for long term ECP treatment (calculated on 16 procedures) was demonstrated (14952.16 E vs 17553 E). Moreover, when the “real life implication” calculated on the QoL parameters were considered, the advantages were more evident. In conclusion, the tie of respecting strictly an imposed program of budget calculated on the short period may exert an inhibitory effect in introducing new diagnostic or therapeutic procedures ignoring that the improvement or the cure of the patient has always a positive economical counterpart, expecially when the impact of a new technology is considered in a long term view.

Description: Background. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective treatment option for patients with malignant and non-malignant hematologic disorders lacking an HLA-compatible donor. Strategies for T-cell depletion (TCD) of the graft, such as positive selection of CD34+ cells, offer the potential to prevent acute and chronic graft-versus-host disease (GVHD). The risk of graft rejection associated with the extensive depletion of both T lymphocytes and accessory cells can be overcome by infusing a very high number (megadose) of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) (exceeding 10x106/kg recipient body weight) to overcome the HLA barrier (Aversa F. et al. Blood 1994). Moreover, the infusion of a megadose of CD34+ cells (higher than 20x106/kg and 12.4x106/kg, respectively) has been shown to result in faster immunological recovery and improved leukemia-free survival probability in children (Handgretinger R. et al. Bone Marrow Transplant 2001; Klingebiel T. et al. Blood 2010). Nevertheless, in the case of donors considered “poor mobilizers” (10-30% of cases), the threshold dose of CD34+ cells needed to ensure the inoculum of a megadose of stem cells might not be achieved. In the setting of cord blood (CB) transplantation, one of the strategies aimed at overcoming the problem of low cellularity is represented by the intrabone injection of CB stem cells, with good engraftment rates even in adult patients. We explored the same strategy in the context of T-cell depleted haplo-HSCT and low graft cellularity due to poor donor mobilization, ensuing in inadequate dose of CD34+cells available after positive selection TCD. Patients and methods. From September 2009 to April 2013, 11 pediatric patients affected by malignant or non-malignant hematological disorders (5 acute lymphoblastic leukemias, 1 acute myeloid leukemia, 1 myelodysplastic syndrome, 2 dyskeratosis congenita, 1 Fanconi anemia) received a T-cell depleted CD34+positively selected PBSC allograft from an HLA-haploidentical related donor. Due to the failure to achieve a target cell dose higher than 12x106 purified CD34+ cells/kg, part of the stem cell inoculum was infused as intrabone injection. The procedure was carried out at the patient bedside by multiple intrabone injections in the superior-posterior iliac crests under sedoanalgesia, as previously described (Frassoni F. et al. Lancet Oncol 2008). The median dose of CD34+ cells infused was 9x106/kg (range, 5-12) while the median number of CD3+ lymphocytes was 0.7x104/kg recipient body weight (range, 0.3-11). About one third of the stem cell inoculum, corresponding to a total volume of 20-40 ml, was given intrabone, while the remaining stem cell portion was infused intravenously. Results.No complication occurred during, or immediately after, the intrabone injection. Nine out of the 11 patients achieved a complete donor engraftment, while graft rejection occurred in 2 patients. The median time for neutrophil engraftment was 13.5 days (range, 12-20), while the median time for platelet recovery was 14 days (range, 13-24). One patient developed grade II acute GVHD and only 1 case of limited chronic GVHD was observed. No transplant-related deaths were observed. Conclusions. Our data suggest that, in the haplo-HSCT setting, the intrabone injection of positively selected CD34+ cells, can be safely used in cases of low graft cellularity due to poor donor mobilization, with the aim of minimizing the risk of graft rejection or poor engraftment. Our preliminary data need to be confirmed in larger series of patients and compared with those obtained with conventional intravenous administration of comparable dose of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Primitive hematopoietic progenitor cells may be characterized by the detection of the intracellular enzymatic activity of aldehyde dehydrogenase (ALDH ). The use of a fluorescent substrate for ALDH (Aldefluor) permits the cytofluorimetric study of different stem cell populations in the functional way rather then as markers of cell surface. Aim of this study was to evaluate the ALDH activity of the stem cells in pre-freezing (fresh) leukapheresis (LKF) colllections and in cord blood (CB) units, to verify the feasibility of detecting the ALDH activity in the post-thawing units and finally to compare the results between the fresh and post thawed stem cells to be transplanted. Materials and methods: Samples containing 2 x106 total nucleated cells obtained from 5 LKF and 5 CB units immediately after collection and from the same units after thawing, were incubated with Aldeflour (StemCo Biomedical, Durham, NC, USA) at 37°C for 45 min. and successively for 15 min. with anti CD34 PE, (Beckman Coulter, Fullerton, CA, USA) and anti CD45 PerCP (BD Biosciences, San Jose, CA, USA). Viability of CD34+ cells was evaluated using 7AAD, (Beckman Coulter, Fullerton, CA, USA). The setting of thawed samples was carried out immediately after thawing, taking care to make the sample dilution very quickly, in order to avoid the detrimental action on the cells exerted by DMSO at 37°C and at room temperature. All samples were analized using a BD Biosciences FACSCalibur flow cytometer device (San Jose, CA, USA). Results: The results of the ALDH and CD34+ cell analysis and stem cell viability on fresh and thawed samples are detailed in table 1. Conclusions: Our results demonstrate the feasibility of ALDH determination even in post thawed samples on condition that the test is conducted with accuracy with particular attention at the dilution step that must be completed in a very short time. The ALDH activity tends to decline in thawed stem cells demonstrating the detrimental action on stem cell functionality of the entire cryopreservation and thawing processes. In particular the contact of the stem cell with cryoprotectant mixture (DMSO) at room temperature has a negative, time dependent, impact on stem cell quality, confirming that the reinfusion phase of the graft must be carried out as soon as possible. The introduction of a functional test to evaluate the graft quality may be helpful in transplant clinical practice. Table 1: Results of ALDH assayes on pre-freezing and post-thawing leukapheresis and cord blood collections. CD34+ (%) ALDH+ (%) CD34+/ALDH+ (%) ALDH+/CD34+ (%) Viability (%) Fresh LKF n=5 0.56 0.12 64.6 97.8 99.2 Fresh CB n=5 0.28 0.23 76.7 99.8 98.1 Thawed LKF n=5 0.48 0.09 65.5 94.4 81.2 Thawed CB n=5 0.24 0.19 71.5 96.8 77.3

Description: Introduction: Nowadays UCB represents an established source of hematopoietic stem cells for unrelated transplants in children and the employ in adults is quickly growing up. Nucleated cells (NCs) content is one of the main predictors to evaluate UCB for clinical use; however, other indicators of the hematopoietic potential, such as CD34+ cell and colony-forming cells (CFUs), have recently showed similar correlations. According to Netcord-FACT standards, it is recommended to test all the above mentioned parameters before releasing UCB to the transplant center; a segmented tubing of the UCB bag should be used but a satellite cryotube is more often available. We preliminarily report the results of quality controls performed on thawed cryovials corresponding to each of 15 units delivered by our UCB Bank. Material and methods: in our policy, all UCBs are stored accompanied by 3 satellite cryovials, treated under the same conditions of the unit. For each of the 15 UCBs released for transplantation, one cryotube was thawed in a 37°C water bath with a gentle agitation, without washing out DMSO. NCs and mononucleated cells (MNCs) were estimated with an automated cell counter. Viability and CD34+ cell count were evaluated by flow cytometry, with a no-wash, single-platform technique and 7-aminoactinomycin D. CFU assay was performed using commercial reagents (Methocult GF H4434, StemCell Technologies) and colonies were classified after a 14 days incubation. The same parameters referring to fresh UCBs (before cryopreservation) were always available. Results: the UCB characteristics measured after thawing a cryovial were compared with those of the fresh cord and are detailed in the table below. fresh UCB before cryopreservation UCB cryovial after thawing Yield (%) NC (x106) 1491.3 (148–2262) 1354.2 (167.4–2119.9) 92 (83–113) MNC (x106) 662.7 (96.5–900.3) 638.8 (295–1238.7) 95 (63–159) CD 34+ cells (x106) 3.47 (0.38–10.87) 3.19 (0.4–9.27) 85 (37–118) Viability (%) 96 (88–100) 62 (39–77) Viability of CD34+ cells (%) 92 (71–99) CFU-GM (x104) 838.5 (57.2–3581.2) 424.11 (65.29–917.64) 69 (16–184) BFU-E (x104) 1709.61 (159.84–5116) 473.34 (39.69–1204.14) 41 (5–139) total CFU (x104) 2565.17 (217.04–8953) 911.68 (164.43–2075.22) 57 (9–160) Excellent yields were found for NCs, MNCs and CD34+ cells. Despite of the decrease in the overall viability, the viable CD34+ cells as percentage was always highly satisfactory. The colonies growth was found lower in the thawed sample in comparison with fresh UCB before cryopreservation. Conclusion: in our experience, highly satisfactory evaluations of UCB content could be obtained using thawed cryotubes with regard to NC, MNC and also CD34+ cell. However, concerning the latter, the different methods employed on fresh UCBs, such as CD34+ cells detection without beads, may be advocated to explain some discrepancies in the yield range. The results of CFU assay confirmed to be poorly useful, essentially because affected by a subjective interpretation even if the reduced cell growth may be also related to the presence of DMSO as inhibiting factor.

Description: INTRODUCTION: Extracorporeal photoapheresis (ECP) is an immunomodulatory therapy for patients with acute or chronic graft-versus-host disease (aGVHD/cGVHD). So far, few studies have explored the molecular regulation of GVHD, and to date no studies have addressed how specific is miRNA expression change during ECP, and whether selected miRNA profiles might be predictive of clinical responses to ECP. OBJECTIVE: The aim of the study was to analyze the expression profile of miRNAs in plasma of patients with GVHD candidates for ECP, and their changes in responding and non-responding patients to this therapy. PATIENTS AND METHODS: Patients with GVHD underwent ECP therapy by off-line methods according to internal protocols. Peripheral blood samples were drawn pre-ECP and after 6 months of treatment. Data on patient characteristics, medical therapies and responses were obtained from medical records. We included the following study cohorts: 1) Initial cohort of 10 GVHD patients (7 cGVHD, 3 aGVHD) and 3 controls; 2) Internal validation cohort with 21 GVHD patients (14 cGVHD, 7 aGVHD) and 10 controls; and 3) External validation cohort (Policlinico S. Matteo, Pavia) composed of 24 GVHD patients (17 cGVHD, 7 aGVHD) and 12 controls. Additionally, samples from 12 patients undergoing ECP due to lung transplantation were also included. Plasma miRNAs were purified with NucleoSpin miRNA Plasma (Macherey-Nagel). In the initial cohort, we analyzed 178 miRNAs, using the Plasma focus miRNAs PCR array (Exiqon). In the validation cohorts we quantified, by qRT-PCR, candidate miRNAs using miRcury LNA RT miRNA PCR (Exiqon) and specific Exiqon primers. RESULTS: In the initial cohort, 4 miRNAs (miR-22-5p, miR-34a-5p, miR-148a-3p, and miR-505-3p) showed higher expression in patients with GVHD compared to controls (p

Description: Recent evidence suggests that leukemia is not solely a cancer autonomous process, but rather a disease in which the bone marrow microenvironment, the niche, plays a crucial role too (Raaijmakers, 2011). MSCs are key component of the niche. Thus, several studies have tested whether these cells from haematological patients contain chromosomal defects identical or different from those present in leukemic cells. Based on these findings the principal aim of the present study was to evaluate whether leukemic and MSC from six AML patients shared the same cytogenetic defects after examination with three different technologies, conventional cytogenetics (CC), FISH and aCGH/SNPa. At the onset of the disease and after informed consent all the six patients were submitted to bone marrow (BM) aspiration. BM cells were submitted to CC and FISH analyses. In addition, MSC were isolated from BM cell suspension (10-15 ml) as previously described. Briefly, mononucleated cells were isolated from BM by density gradient centrifugation using Lympholyte®-H and seeded in 75 cm2 cell culture flasks at a cell density of 106 cells/cm2. Cells were cultured at 37°C, 5% CO2 in MEM-alpha medium containing 1% Penicillin/Streptomycin, 1% L-Glutamine and 10% fetal bovine serum. After 48-h adhesion, non-adherent cells were removed and culture medium replaced (Achille et al, 2011). Growth medium was changed every three days. MSCs were examined after the first passage and their phenotype was evaluated by flow cytometry. Cells were detached from culture using Tripsin-EDTA, washed twice with PBS and stained for ten minutes with the following fluorochrome-conjugated antibodies: anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD73-PE, anti-CD34-FITC, anti-CD80-PE, anti-CD133-APC, anti-CD31-PE and anti-CD45-APC-Alexa750. Stained cells were acquired with a Beckman Coulter Navios instrument and data analyzed with Kalooza software. The commercial FISH probes used were LSI D7S486/CEP7, LSI AMLETO from Abbot Molecular Inc. (Chicago, Il, USA) and ON c-Myc/SE8, SE10(D10Z1) from Kreatech (Amsterdam, NL). These probes were applied according to manufactures guidelines and cut-off values determined by applying a one-sided 95% confidence interval using a binomial distribution. aCGH/SNPa was carried out with the SureScan Microarray Scanner G4900DA (Agilent Technologies Inc. Santa Clara, CA). CC revealed a monosomy 7 in two patients, a del(7)(q31) in one, a trisomy 8 and a trisomy 10 in one patient each, a t(8;21)(q24,q22) translocation in the last patient. All these defects were confirmed by FISH. In order to establish whether leukemic cells and MSCs shared these same abnormalities, MSCs cultures were tested with FISH. MSC purity assessed by flow-cytometry was 50-87%. FISH revealed a normal pattern in all the cultures examined. In contrast, aCGH/SNPa revealed neither gains/losses nor LOH in four patients, a trisomy 5 in one and the LOH of a 3.8 Mb sized region located on 13q31.1 in one patient. This study, the first one that applied aCGH/SNPa to investigate the MSC chromosomal pattern, suggests that i) MSCs from chromosomally abnormal AML patients may show a normal FISH pattern, but may be either normal or contain chromosomal aberrations different from those present in leukemic cells on aCGH/SNPa analysis; ii) these defects are uncommonly seen in AML; iii) MSCs defects may flag that the leukemogenic event targets not only the hematopoietic tissue but also the stromal cell compartment, i.e. the niche; iii) aCGH/SNPa provides an in-depth view of MSC chromosomal pattern allowing the identification of potential clonal markers. Disclosures: No relevant conflicts of interest to declare.