Impedance-Based Monitoring of Mesenchymal Stromal Cell Three-Dimensional Proliferation Using Aerosol Jet Printed Sensors: A Tissue Engineering Application.

One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and -9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.

Human leukocyte antigens class II in CIDP spectrum neuropathies.

CIDP spectrum encompasses several clinical variants and the reasons of the heterogeneous clinical expression and the variable response to therapy are scarcely known. HLA associations are common in dysimmune conditions. In CIDP, few studies reported no associations or HLA-DR13/DQ6 association in some populations but, to date, a clear confirmed association is lacking. We analyzed expression of HLA-DR and DQ haplotypes in 24 CIDP patients and 216 healthy subject. HLA-DR3 and DR3/DQ2 were significantly more frequent in CIDP patients than in the control group. The DR3 and DR3/DQ2 positive patients present with more frequent relapsing course, worse response to IVIg, higher inflammatory neuropathy sensory sumscore (ISS) and Rotterdam Inflammatory Neuropathy Cause and Treatment Scale (INCAT) than negative patients.

Circulating microRNAs and Their Role in Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by bone marrow infiltration of clonal plasma cells. The recent literature has clearly demonstrated clonal heterogeneity in terms of both the genomic and transcriptomic signature of the tumor. Of note, novel studies have also highlighted the importance of the functional cross-talk between the tumor clone and the surrounding bone marrow milieu, as a relevant player of MM pathogenesis. These findings have certainly enhanced our understanding of the underlying mechanisms supporting MM pathogenesis and disease progression. Within the specific field of small non-coding RNA-research, recent studies have provided evidence for considering microRNAs as a crucial regulator of MM biology and, in this context, circulating microRNAs have been shown to potentially contribute to prognostic stratification of MM patients. The present review will summarize the most recent studies within the specific topic of microRNAs and circulating microRNAs in MM.

Counting circulating endothelial cells in allo-HSCT: an ad hoc designed polychromatic flowcytometry-based panel versus the CellSearch System.

Physio-pathologic interrelationships between endothelial layer and graft-versus-host disease (GVHD) have been described leading to assess the entity "endothelial GVHD" as the early step for clinical manifestations of acute GVHD. The availability of the CellSearch system has allowed us to monitor Circulating Endothelial Cells (CEC) changes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as useful tool to help clinicians in GVHD diagnostic definition. We have compared CEC counts generated by an ad hoc designed polychromatic-flowcytometry (PFC) Lyotube with those of the CellSearch system. CEC were counted in parallel at 5 timepoints in 50 patients with malignant hematologic disorders undergoing allo-HSCT (ClinicalTrials.gov, NCT02064972). Spearman rank correlation showed significant association between CEC values at all time points (p = 0.0001). The limits of agreement was demonstrated by Bland Altman plot analysis, showing bias not significant at T1, T3, T4, while at T2 and T5 resulted not estimable. Moreover, Passing Bablok regression analysis showed not significant differences between BD Lyotube and CellSearch system. We show that CEC counts, generated with either the CellSearch system or the PFC-based panel, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as a suitable tool to assist clinicians in GVHD diagnosis.

A real-life evaluation of two platelet cross-matching programmes for the treatment of patients refractory to platelet transfusions.

BACKGROUND AND OBJECTIVES: Strategies for overcoming alloimmune refractoriness to random donor platelets are based on the use of compatible platelets selected from large panels of HLA-typed donors or cross-matching (XM). The aim of this study was to review the effectiveness of a platelet XM programme for treating refractory haematological patients at Milan's Policlinico Hospital (PHM) 2002-2014 and Spedali Civili in Brescia (SCB) 2013-2016. MATERIALS AND METHODS: A commercially available solid-phase antibody detection system was used for platelet antibody detection and XM. Forty-nine alloimmune refractory patients at PHM and 13 at SCB, respectively, received a median [IQR] of 12 [6-13] and 18 [13-15] XM compatible platelet transfusions after the detection of refractoriness. The absolute increases in post-transfusion platelet counts obtained using random, and XM platelets were retrieved from the patients' hospital records. RESULTS: The critical review at SCB showed that the median [IQR] 1 h post-transfusion increase in platelet counts was 3 × 109 /L [1-5] after 47/47 random platelet transfusions, and 10 × 109 /L [2-25] after 325/326 XM compatible platelet transfusions. The documentation concerning the outcomes of XM platelet transfusions at PHM was incomplete, and so the findings of the review were inconclusive. CONCLUSION: This retrospective analysis confirmed the effectiveness of the XM programme at SCB, but revealed defective data collection and retrieval methods at PHM, thus underlining the importance of such methods. The literature review accompanying this retrospective analysis identified a recently described algorithm for ensuring platelet support in refractory patients that optimally integrates the combined use of XM and HLA typing.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Mesenchymal stromal cells (MSCs) induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells (CB-CD34+ cells).

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

Extracorporeal Photopheresis for Treatment of Acute and Chronic Graft Versus Host Disease: An Italian Multicentric Retrospective Analysis on 94 Patients on Behalf of the Gruppo Italiano Trapianto di Midollo Osseo.

BACKGROUND: Extracorporeal photopheresis (ECP) is considered a valid second-line treatment for acute and chronic graft versus host disease (GVHD). METHODS: Ninety-four patients with acute GVHD (aGVHD) (n = 45) and chronic GVHD (cGVHD) (n = 49), retrospectively recruited in 6 Italian centers, were submitted to ECP for second-line treatment. At the time of ECP, 22 (49%) and 23 (51%) of 45 patients with aGHVD were nonresponsive and in partial remission (PR) after steroids, respectively, and all the 49 patients with cGVHD were steroid refractory. RESULTS: Forty-one (91%) of 45 patients with aGVHD achieved complete remission (CR) after ECP. Fifteen (33%) of 45 patients developed cGVHD. The CR rate in patients who started ECP being nonresponsive and in PR after steroid was 86% and 96%, respectively. After a median follow-up of 20 months (range, 2-72), 15 (33%) of 45 patients developed cGHVD and 16 (35%) of 45 patients died, in 3 cases for aGVHD. A trend for a better survival was seen among patients who started ECP in PR after steroid (80% vs 50% at 2 years; P = 0.07). Overall, 22 (45%) of 49 patients and 17 (35%) of 49 patients with steroid refractory cGHVD achieved CR and PR after ECP, respectively. After a median follow-up of 27 months, 44 (90%) of 49 patients are alive, 21 of whom (48%) are on steroid. CONCLUSIONS: Extracorporeal photopheresis is confirmed as an effective second-line treatment in both aGVHD and cGVHD, because it can induce a response in more than 80% of the patients and a long-term survival in at least 50% of the cases.

Analysis of Circulating Tumor Cells in Prostate Cancer Patients at PSA Recurrence and Review of the Literature.

BACKGROUND: Circulating tumor cells have been described in prostate cancer patients at diagnosis and in the metastatic phase but little is known on their role at biochemical PSA recurrence. PATIENTS AND METHODS: Patients radically cured with either prostatectomy or radiotherapy were sequentially included at PSA recurrence. The presence of CTCs was evaluated by the CellSearch system. RESULTS: Twenty-nine patients were accrued at PSA recurrence. Median PSA at recurrence was 7.2 ng/ml (range=3.86-51.0 ng/ml). The median time to PSA progression was 4.66 years (range=0.1-16 years). CTCs were detected in one patient (3%) with low numbers (1 CTC/7.5 ml). CONCLUSION: In patients radically cured for prostate cancer at biochemical recurrence, CTCs are detected at very low levels in a minority of patients. Further studies are required to investigate alternative methods of CTC detection and the possible role of the bone marrow pre-metastatic niche at biochemical recurrence.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Positive effect of erythrocyte-delivered dexamethasone in ataxia-telangiectasia.

OBJECTIVE: Ataxia-telangiectasia (AT) is a rare, devastating neurodegenerative disease presenting with early-onset ataxia, oculocutaneous telangiectasia, immunodeficiency, radiosensitivity, and proneness to cancer. In a previous phase 2 study, we showed that 6 monthly infusions of autologous erythrocytes loaded with dexamethasone (EryDex; EryDel, Urbino, Italy) were effective in improving neurologic impairment in young patients with AT. The present article reports the results of the extension of this study for an additional 24-month period. METHODS: After the end of the first trial, 4 patients continued to be treated with monthly EryDex infusions for an additional 24 months, and their clinical outcome was compared with that of 7 age-matched patients who stopped the treatment after the first 6 infusions. The protocol included serial assessment of ataxia (by International Cooperative Ataxia Rating Scale) and adaptive behavior (by Vineland Adaptive Behavior Scales) and clinical and laboratory tests revealing treatment- and steroid-dependent adverse effects, if present. RESULTS: Patients in the extended study experienced a continuous neurologic improvement with respect to their pretreatment status, whereas controls showed a progressive neurologic deterioration (according to the natural history of the disease) after the discontinuation of the treatment. The delivery system we adopted proved to be safe and well-tolerated, and none of the side effects usually associated with the chronic administration of corticosteroids were observed during the entire trial. CONCLUSIONS: These promising preliminary results call for a large-scale controlled study on protracted treatment of patients with AT with dexamethasone-loaded erythrocytes.

Circulating tumor cells in patients with recurrent or metastatic head and neck carcinoma: prognostic and predictive significance.

INTRODUCTION: We investigated the frequency of detection and the prognostic and predictive significance of circulating tumor cells (CTCs) in patients with recurrent/metastatic (R/M) head and neck carcinoma (HNC) before starting systemic therapy. PATIENTS AND METHODS: Using the CellSearch technology, CTCs were assessed prospectively in peripheral blood of 53 R/M-HNC patients. We performed spiking experiments to test the diagnostic performance of the CellSearch platform in identifying squamous carcinoma cells. RESULTS: CTCs were identified in 14 (26%) and 22 (41%) patients at baseline and at any time point, respectively. In univariate analysis ≥2 CTCs had a poorer prognostic role than 0-1 CTC. In multivariate analysis, the presence of one CTC or more was associated with a poor prognosis both in terms of progression-free survival (PFS) [Hazard Ratio (HR): 3.068, 95% confidence interval (CI): 1.53-6.13, p 0.002] and overall survival (OS) [HR: 3.0, 95% CI: 1.48-6.0, p 0.002]. A disease control after systemic therapy was obtained in 8% of CTC-positive patients as opposed to 45% in CTC-negative ones (p 0.03). The epidermal growth factor receptor (EGFR) expression was identified in 45% of CTC-positive patients. DISCUSSION: In conclusion, CTCs are detected in one out of three patients with RM-HNC. CTC detection is a strong prognostic parameter and may be predictive of treatment efficacy. The frequency of EGFR expression in CTCs seems to be lower than that expected in the primary tumor.

Changes in circulating endothelial cells count could become a valuable tool in the diagnostic definition of acute graft-versus-host disease.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is burdened by life-threatening complications, with graft-versus-host disease (GvHD) being the major cause of morbility and mortality. Recently, clinical and physiopathologic evidences showed that vascular endothelium can be a target of GvHD in the early phase and circulating endothelial cells (CECs) represent surrogate markers of endothelial damage. METHODS: Using the CellSearch System (Veridex LLC, Raritan, NJ), CECs were counted before (T1), after conditioning regimen (T2), at engraftment (T3), at GvHD onset (T4), and after steroid treatment (T5) in 40 patients (7 Hodgkin's Disease, 13 Acute Myeloblastic Leukemia, 5 Acute Lymphoblastic Leukemia, 8 Multiple Myeloma, 3 Chronic Lymphocytic Leukemia, 1 Non-Hodgkin Lymphoma, 1 Chronic Myeloid Leukemia, 2 Severe Aplastic Anemia) undergoing allo-HSCT. RESULTS: The median CEC per milliliter at T1 was 20 (n=33, range 4-718), in comparison to a value of 2 (range, 1-14) in controls (P<0.001). At T3, CEC per milliliter were 47 (range, 16-148) in GvHD patients and 92 (range, 23-276) in patients without GvHD (P=0.006). This difference remained significant in multivariate analysis (odds ratio, 0.97; 95% confidence interval, 0.96-0.99; P=0.02). At GvHD onset, the relative increase of CEC counts from time of engraftment (T4 vs. T3) was 44% (range, -43% to 569%) in GvHD patients versus 0% (range, -49% to 2%) in patients without GvHD (P=0.003), being confirmed as significant in multivariate analysis (odds ratio, 1.04; 95% confidence interval, 1.0-1.08; P=0.04). CONCLUSION: Changes in CEC count can represent a promising marker to monitor endothelial damage in patients undergoing allo-HSCT and could become a valuable tool in the diagnostic definition of GvHD.

Intra-erythrocyte infusion of dexamethasone reduces neurological symptoms in ataxia teleangiectasia patients: results of a phase 2 trial.

BACKGROUND: Ataxia Teleangiectasia [AT] is a rare neurodegenerative disease characterized by early onset ataxia, oculocutaneous teleangiectasias, immunodeficiency, recurrent infections, radiosensitivity and proneness to cancer. No therapies are available for this devastating disease. Recent observational studies in few patients showed beneficial effects of short term treatment with betamethasone. To avoid the characteristic side effects of long-term administration of steroids we developed a method for encapsulation of dexamethasone sodium phosphate (DSP) into autologous erythrocytes (EryDex) allowing slow release of dexamethasone for up to one month after dosing. Aims of the study were: the assessment of the effect of EryDex in improving neurological symptoms and adaptive behaviour of AT patients; the safety and tolerability of the therapy. METHODS: Twenty two patients (F:M=1; mean age 11.2 ± 3.5) with a confirmed diagnosis of AT and a preserved or partially supported gait were enrolled for the study. The subjects underwent for six months a monthly infusion of EryDex. Ataxia was assessed by the International Cooperative Ataxia Rating Scale (ICARS) and the adaptive behavior by Vineland Adaptive Behavior Scales (VABS). Clinical evaluations were performed at baseline and 1, 3, and 6 months. RESULTS: An improvement in ICARS (reduction of the score) was detected in the intention-to-treat (ITT) population (n=22; p=0.02) as well as in patients completing the study (per protocol PP) (n=18; p=0.01), with a mean reduction of 4 points (ITT) or 5.2 points (PP). When compared to baseline, a significant improvement were also found in VABS (increase of the score) (p<0.0001, ITT, RMANOVA), with statistically significant increases at 3 and 6 months (p<0.0001). A large inter-patient variability in the incorporation of DSP into erythrocytes was observed, with an evident positive effect of higher infusion dose on ICARS score decline. Moreover a more marked improvement was found in less neurologically impaired patients. Finally, a 19 month-extension study involving a subgroup of patients suggested that Erydex treatment can possibly delay the natural progression of the disease.EryDex was well tolerated; the most frequent side effects were common AT pathologies. CONCLUSIONS: EryDex treatment led to a significant improvement in neurological symptoms, without association with the typical steroid side effects. TRIAL REGISTRATION: Current Controlled Trial 2010-022315-19SpA.

Circulating tumour cells in locally advanced head and neck cancer: preliminary report about their possible role in predicting response to non-surgical treatment and survival.

BACKGROUND AND PURPOSE: The mechanism of dissemination of locally advanced head and neck cancer (LAHNC) is far to be resolved. Circulating tumour cells (CTC) have been identified as a prognostic factor in metastatic breast and prostate cancer. This prospective multi-centric analysis studied the possible role of CTC identification in LAHNC. MATERIALS AND METHODS: CTC were searched in 73 patients with LAHNC (oropharynx, n=39; nasopharynx, n=10; larynx, n=10; paranasal sinuses, n=6, of whom 3 with sinonasal undifferentiated carcinoma, SNUC; hypopharynx, n=5; oral cavity, n=3). All of them (apart from SNUC) had squamous cell cancers. The relationship between CTC positivity and other clinical prognostic factors has been investigated. Response to treatment and survival has been related with changes in CTC number during the treatment. RESULTS: CTC were frequently identified in oro- and hypopharyngeal cancer and in SNUC. They were more frequent in stage IV than in stages I-III disease (18% versus 6%, p=NS (not significant)). Partial or complete response (CR) was related with the absence or disappearance of CTC during treatment (p=0.017). A decrease in the CTC number or their absence throughout the treatment seems also related with non-progressive disease, after both complete or incomplete remission and with the proportion of patients alive and NED (no evidence of disease) (p=0.009). CONCLUSIONS: These preliminary data suggest a possible role of CTC determination in head and neck cancer. Additional and longer follow up data need to be collected to confirm these findings.

Circulating tumor cells as predictors of prognosis in metastatic breast cancer: clinical application outside a clinical trial.

AIMS AND BACKGROUND: Circulating tumor cells have a prognostic role in metastatic breast cancer. The aim of the study was to confirm the ability of circulating tumor cells, detected by the US Food and Drug Administration approved Cell Search assay, to predict the outcome of patients treated in a community general hospital. PATIENTS AND METHODS: A prospective mono-institutional study was conducted at the Department of Medical Oncology at Spedali Civili, Brescia, Italy, from January 2009 to September 2010. A total of 93 consecutive patients with metastatic breast cancer were enrolled. Patients underwent a blood sample collection to detect circulating tumor cells at baseline and, subsequently, at the first follow-up examination (after 3-4 weeks from the beginning of a systemic therapy). A third sample was drawn at disease progression (at the beginning of a subsequent new course of therapy). The prognostic cutoff value of circulating tumor cells was fixed at 5 cells/7.5 ml of blood. RESULTS: At baseline, median overall survival and progression-free survival in the subgroup ≥5 circulating tumor cells/7.5 ml of blood were significantly shorter (5 months and 3 months, respectively) than in the subgroup with <5 circulating tumor cells (8 months and 7 months, respectively) (P = 0.003 and P <0.001). At the first follow-up, the subgroup with more than 5 circulating tumor cells/7.5 ml of blood had a median overall survival of 4 months versus 8 months in the subgroup with <5 circulating tumor cells (P <0.001) and a median progression-free survival of 3 months versus 7 months respectively (P <0.001). At multivariate analysis, the level of circulating tumor cells at the first follow-up and at baseline remained significant as a predictor of progression-free and overall survival. The number of metastatic sites was significantly associated with overall and progression-free survival and correlated with the number of circulating tumor cells. CONCLUSIONS: Our study confirms the role of circulating tumor cells as predictors of prognosis in metastatic breast cancer patients treated in general clinical practice.

Strategies for the transfusion of subjects with complex red cell immunisation: the Bank of rare blood donors of the Region of Lombardy.

INTRODUCTION: Selecting units of rare blood for transfusion to patients with complex immunisation is one of the most critical processes of a Transfusion Centre. In January 2005 the 'Rare Blood Components Bank - Reference Centre of the Region of Lombardy' w as established with the following goals: 1) identifying regional rare blood donors; 2) creating a regional registry of rare donors; 3) organising a regional bank of liquid and frozen rare blood units; 4) setting up a regional Immunohaematology Reference Laboratory (IRL) to type donors and resolve complex cases. METHODS: The key elements in establishing the Bank were periodic meetings organised by the directors and representatives of the regional Departments of Transfusion Medicine and Haematology (DTMH) and the institution of three working groups (informatics, regulations, finance). RESULTS: The regional IRL was set up, the relevant operating procedures were distributed region-wide, software features were defined and later validated upon activation, and the funds assigned were allocated to various cost items. The number and characteristics of the donors to be typed were identified and 14 regional DTMHs started to send samples. Overall, 20,714 donors were typed, for a total of 258,003 typings, and 2,880 rare donors were identified. Of these, 97% were rare donors because of combinations of antigens (2,139 negative for the S antigen and 659 negative for the s antigen) and 3% (n=82) because they were negative for high-frequency antigens. In the first 2 years of activity, the IRL carried out investigations of 140 complex cases referred from other Centres and distributed 2,024 units with rare phenotypes to 142 patients. CONCLUSIONS: The main goal achieved in the first 24 months from the start of the project was to set up a regional network able to meet the transfusion needs of patients with complex immunisation.

High prevalence of SEN virus infection in patients on maintenance hemodialysis: frequent mixed infections with different variants and evidence for nosocomial transmission of the virus.

OBJECTIVE: The SEN virus (SENV) represents a recently described group of DNA viruses, two members of which (SENV-D and SENV-H) are linked with posttransfusion hepatitis. Since patients on hemodialysis have a high risk of being infected by blood-borne viruses, we investigated the prevalence of seven SENV isolates in two distinct units of our hospital. METHODS: The presence of SENV was investigated in 171 hemodialysis patients and in 163 controls by using a polymerase chain reaction based methodology, with which the specificity of amplified products was detected by hybridization with probes specific for each variant. Polymerase chain reaction products from 4 patients were sequenced. RESULTS: The overall detection of SENV DNA as well as of SENV-D, SENV-E, and SENV-G was significantly higher in one of the two units, and there was a higher degree of homology in the sequences prepared from patients of the same unit. Furthermore, we demonstrated that mixed infections with multiple SENV were common. No relationship was observed between presence of SENV and sex, age, duration of hemodialysis, previous transfusions or transplantation, hepatitis infection, and routine liver test results. CONCLUSION: Our results indicate that patients who undergo hemodialysis can be at high risk of SENV transmission and suggest an intraunit transmission of specific SENV variants.

Uncontrolled-rate freezing of peripheral blood progenitor cells allows successful engraftment by sparing primitive and committed hematopoietic progenitors.

BACKGROUND AND OBJECTIVES: Uncontrolled-rate freezing (URF) techniques, which are fast and easy, could represent an attractive alternative to controlled-rate cryopreservation procedures which are time consuming and require high-level technical abilities. It was the aim of the present study to evaluate, on a routine basis, whether URF might spare primitive hematopoietic progenitors and maintain engrafting capacity. DESIGN AND METHODS: One-hundred and nineteen peripheral blood progenitor cells (PBPC) collections from 104 patients with hematologic malignancies were cryopreserved in bags, with an URF procedure, in a cryoprotectant solution consisting of PBS, HSA and 10% DMSO and stored in liquid nitrogen. PBPC bags were tested before cryopreservation and at thawing for primitive (LTC-IC) and committed hematopoietic progenitors (CFU-Mix, BFU-E, CFU-GM) by means of long- and short-term culture assays, respectively. In addition, PBPC bags were evaluated for CD34+ cell numbers. RESULTS: Although thawing was associated with a statistically significant reduction of the absolute number of nucleated cells, recovery of LTC-IC, CFU-Mix, BFU-E, CFU-GM and CD34+ cells was not affected by the freezing/thawing procedures. No adverse effects were reported at thawing and only mild transient reactions were recorded in 22 patients during reinfusion of cryopreserved PBPC. All the patients underwent myeloablative therapy followed by reinfusion of PBPC, and prompt and rapid hematopoietic recovery was obtained in all patients. INTERPRETATION AND CONCLUSIONS: Our freezing procedure is fast and easy, and allows rapid hematopoietic recovery after myeloablative therapy by sparing primitive and committed hematopoietic progenitors. Our study strongly supports technical improvements aimed at cost reduction and feasibility of routine freezing procedures.