A miRNA Signature in Human Cord Blood Stem and Progenitor Cells as Potential Biomarker of Specific Acute Myeloid Leukemia Subtypes.

MicroRNAs (miRNAs) are important regulators of several cellular processes. During hematopoiesis, specific expression signatures have been reported in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation. Here we explored the expression of miRNAs in umbilical cord blood stem (HSC) and progenitor cells (HPC) and compared it to unilineage granulocyte and granulo-monocyte differentiation as well as to primary blasts from patients with acute myeloid leukemia (AML). CD34 + CD38- ad CD34 + CD38 + cells were profiled using a global array consisting of about 2000 miRNAs. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative biologically enriched functions and networks. At least 15 miRNAs to be differentially expressed between HSC and HPC cell population, a cluster of 7 miRNAs are located in the q32 region of human chromosome 14 (miR-377-3p, -136-5p, 376a-3p, 495-3p, 654-3p, 376c-3p and 381-3p) whose expression decreased during the early stages of normal myelopoiesis but were markedly increased in a small set of AML. Interestingly, miR-4739 and -4516, two novel microRNA whose function and targets are presently unknown, showed specific and peculiar expression profile during the hematopoietic stem cells differentiation into unilineages and resulted strongly upregulated in almost all AML subsets. miR-181, -126-5p, -29b-3p and -22-3p resulted dis-regulated in specific leukemias phenotypes. This study provides the first evidence of a miRNA signature in human cord blood stem and progenitor cells with a potential role in hematopoietic stemness properties and possibly in leukemogenesis of specific AML subtypes.

Treatment of recalcitrant ulcers with allogeneic platelet gel from pooled platelets in aged hypomobile patients.

We evaluated growth factor contents and clinical efficacy of allogeneic platelet gel (PG) prepared with standard blood banking procedures from routine platelet concentrates (PCs) obtained from buffy coats. The PGs were used to treat 11 hypomobile very elderly patients unable to undergo autologous blood processing and previously ineffectively treated with expensive advanced medications for 8-275 weeks. PGs were prepared by platelet activation with human thrombin or commercial batroxobin. Median and range growth factor contents (ng/mL) were: platelet derived growth factor (PDGF-AB/-BB) 112 (31-157) and 20 (3.8-34); transforming growth factor (TGF-ß1/-ß2) 214 (48-289) and 0.087 (0.03-0.28); basic-fibroblast growth factor (b-FGF) 0.03 (0.006-0.214); vascular endothelial growth factor (VEGF) 1.15 (0.18-2.46); epidermal growth factor (EGF) 4.50 (0.87-6.64); insulin-like growth factor (IGF-l) 116 (72-156). In the clinical study, 222 PGs were used within 2 h of activation to treat 14 chronic skin ulcers in the 11 patients. No improvement was seen in 3 patients with 24, 27 and 30 cm(3) ulcers who could be treated for no more than 4, 7 and 8 weeks due to progressively worsening clinical conditions, while 11 ulcers with 3.2 cm(3) median size (range 0.2-3.6) in the remaining 8 patients showed 91 ± 14 % reduction after a median of 12 weeks (range 1-20). Cost of PG treatment (19,976 euro) amounted to about 10% of the ineffective advanced medication hospital reimbursement fees (191,236 euro). This study supports efficacy and feasibility of allogeneic PG to treat recalcitrant ulcers in very elderly hypomobile patients for whom autologous blood processing may be difficult.

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Animal model for liver cell banking from non-heart beating donors after prolonged ischaemia time.

BACKGROUND AND AIM: Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. METHODS: Hepatocytes were isolated from the liver within 15 min after death (t=15 min) and after 4, 8 and 12h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. RESULTS: The number of cells per gram of tissue was similar at 15 min, 4 and 8h, while it was significantly decreased at 12h. About 0.2 x 10(6) viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8h of ischaemia per gram of tissue. CONCLUSIONS: Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.

Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Clinical Oncology.

OBJECTIVE: To determine the most effective, evidence-based approach to the use of platelet transfusions in patients with cancer. OUTCOMES: Outcomes of interest included prevention of morbidity and mortality from hemorrhage, effects on survival, quality of life, toxicity reduction, and cost-effectiveness. EVIDENCE: A complete MedLine search was performed of the past 20 years of the medical literature. Keywords included platelet transfusion, alloimmunization, hemorrhage, threshold and thrombocytopenia. The search was broadened by articles from the bibliographies of selected articles. VALUES: Levels of evidence and guideline grades were rated by a standard process. More weight was given to studies that tested a hypothesis directly related to one of the primary outcomes in a randomized design. BENEFITS/HARMS/COST: The possible consequences of different approaches to the use of platelet transfusion were considered in evaluating a preference for one or another technique producing similar outcomes. Cost alone was not a determining factor. RECOMMENDATIONS: Appendix A summarizes the recommendations concerning the choice of particular platelet preparations, the use of prophylactic platelet transfusions, indications for transfusion in selected clinical situations, and the diagnosis, prevention, and management of refractoriness to platelet transfusion. VALIDATION: Five outside reviewers, the ASCO Health Services Research Committee, and the ASCO Board reviewed this document. SPONSOR: American Society of Clinical Oncology

The current risk of retroviral infections transmitted by transfusion in patients who have undergone multiple transfusions. Cooleycare Cooperative Group.

BACKGROUND: To conduct a multicenter, prospective survey within the program of the Cooleycare Cooperative Group to evaluate the rate of transfusion-transmitted infections with human immunodeficiency virus (HIV) and human T-lymphotropic virus (HTLV) in a cohort of patients who were homozygous for beta thalassemia and underwent multiple transfusions during the 6-year follow-up. PATIENTS AND METHODS: One thousand three hundred eighty-four patients with beta thalassemia from 36 centers were enrolled from December 1989 to March 1990. Serum samples were tested at regular intervals during the period from December 1989 to March 1996 for anti-HIV and anti-HTLV antibodies in 1 laboratory. Samples from 1073 and 1001 of the 1384 patients were available for evaluation also during the periods from December 1992 to March 1993 and December 1995 to March 1996, respectively. The risk of acquiring infection was calculated by the ratio between the number of patients who experienced seroconversion and the number of red blood cell units administered to the patients during the study period. RESULTS: The prevalence of HIV infection found in the period from December 1989 to March 1990 was 2.9% (40 of 1384 patients). During follow-up, 1 of 1001 patients showed anti-HIV seroconversion. The incidence of HIV infection was 1.7 per 10,000 person-years (upper boundary of the 95% confidence interval, 5 per 10,000). The risk of HIV infection was 1 in 190,000 U (upper boundary of the 95% confidence interval, 1 in 67,000). At baseline, 4 patients were infected with HTLV (3 with HTLV-1 and 1 with HTLV-2). No seroconversions were observed during follow-up; the risk of HTLV infection was less than 1 in 190,000 U. CONCLUSION: The application of reliable screening procedures for donor selection reduced the transmission of transfusion-associated HIV infection in 1989-1995 to fewer than 2 cases in 10,000 person-years or 1 case per 190,000 units of red blood cells.

HIV and HTLV infections in 1305 transfusion-dependent thalassemics in Italy. The COOLEYCARE Cooperative Group.

OBJECTIVE: To evaluate the prevalence of antibodies to HIV-1/2 and HTLV-I/II in 1305 transfusion-dependent beta-thalassemics treated in 36 centres in Italy. DESIGN: Patient serum samples were collected during 1990 and tested in Milan. METHODS: Sera were screened using an enzyme-linked immunosorbent assay (ELISA) containing viral lysate antigens from HIV-1 and HIV-2, and a particle agglutination assay for the detection of antibodies to HTLV-I and HTLV-II. Repeatedly reactive samples were examined by Western blot (WB) assays containing recombinant and viral lysate antigens. Differential diagnosis was finally made by ELISA based on synthetic peptides. RESULTS: Samples from 36 of the 1305 patients (2.76%) contained anti-HIV-1 antibodies. In four patients seroconversion occurred after the implementation of anti-HIV-1 screening in blood donors in Italy (1985). Of the 36 HIV-1-antibody-positive samples, four were HIV-2 [corrected] WB indeterminate. These four samples were negative in assays based on specific synthetic peptides, suggesting cross-reactivity. Anti-HTLV-I antibodies were found in two patients from Sicily and one from Apulia, both southern Italian regions. Anti-HTLV-II antibodies were detected in another patient from Sicily. CONCLUSIONS: Antibodies to HIV-1, HIV-2, HTLV-I and HTLV-II were detected in 2.76, 0, 0.23 and 0.08% of patients, respectively. The residual risk of HIV-1 infection through blood transfusion after the implementation of anti-HIV-1 screening in blood donors in Italy was approximately 1:50,000 blood units; this is based on an approximate number of 200,000 blood units administered to our group of patients during 1986-1990 and the occurrence of four new anti-HIV-1 seroconversions. Seroconversions to HTLV-I/II suggest that these viruses are present in Italian blood donors.

An immunoenzymatic assay for the detection and quantitation of platelet antibodies: the platelet beta-galactosidase test (PGT).

An immunoenzyme assay for detection of platelet antibodies and suitable for routine use is described. Purified rabbit IgG anti-human IgG antibodies are conjugated to beta-galactosidase with meta-maleimidobenzoyl-hydroxysuccinimide ester as a bifunctional reagent and o-nitrophenyl-beta-galactopyranoside as a substrate to evaluate the enzymatic activity of the labeled antiglobulin. The sera of 26 patients suffering from various diseases (acute leukemia, aplastic anemia and systemic lupus erythematosus) and 40 control subjects were assayed with the enzyme-labeled reagent and, for comparison, with an indirect immunofluorescence technique. Half of these patients had never been transfused. Platelet antibodies were detected by both assays in all the transfused patients except one, and in 3 out of 13 non-transfused patients. The sera of all the control subjects were negative. Quantitation of platelet antibodies was obtained by a sensitive antiglobulin absorption technique. A method for standardization of the reagents allowing comparison of results obtained in the same patient at different times and suitable for long-term follow-up studies is also described.

A quality system for placental blood banking.

A Quality System for Placental Blood Banking aimed at the transplantation of haematopoietic stem cells to related and unrelated allogeneic recipients is described. It includes the organizational structure, procedures, processes and resources needed to implement quality management. The Quality System described in this article is based on ISO 9002, a model for quality assurance in production, installation and servicing developed in 1987 and revised in 1994 by the International Organization for Standardization. ISO 9002 includes 20 clauses that provide guidance for the implementation of the Quality System. The development of the Quality System is started by the Placental Blood Bank Medical Director with the definition of a General Quality Plan including: (1) the written description of the Mission, Objectives, Technical and Organizational Policies, and Staff Organization Chart; (2) the definition and acquisition of adequate financial, human and structural resources; (3) the appointment of a Quality System Head, who must identify the Placental Blood Banking process together with the Placental Blood Bank personnel; implement a documentation plan; identify quality indicators; start regular internal audit; report audit results to the Medical Director for review. Following staff training and qualification, the Quality System is launched. The Placental Blood Bank can then undergo audit by an external inspector and be finally certified for compliance to ISO 9002. The Quality System must be maintained and subjected to external audit at regular intervals so that certification is confirmed.

Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood.

In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.

Autologous blood predeposit for elective surgery: a program for better use and conservation of blood.

We report the results of a pilot study on predeposit autotransfusion for elective surgery. In 2 years 319 U, each consisting of 350 ml of blood, were donated by 206 patients. Each patient gave 1, 2, or 3 U according to hematocrit levels within 10 days of undergoing surgery on the gastrointestinal tract, lung, liver, kidney, brain, thyroid, arteries, or breast. A hemoglobin drop of about 0.8 gm/dl occurred after each blood donation. A total of 259 autologous and 128 homologous U were transfused to 166 patients during surgery or within postoperative day 4; 40 patients required no blood transfusion. About 70% of patients did not need homologous blood products in addition to autologous units. The most active team of surgeons and anesthesiologists covered about 40% of the patients' blood needs during the study period with autologous units. No patient experienced untoward reactions before, during, or after surgery. Surgeons' and anesthesiologists' responses to the program improved during the study as soon as the advantages of the procedure became clearer. However, gentle pressure from the Transfusion Center was essential for the program's expansion. The patients' acceptance was excellent. We conclude that predeposit autotransfusion is a safe and feasible procedure for transfusion treatment in elective surgery.

Ultrastructural features of CD34+ hematopoietic progenitor cells from bone marrow, peripheral blood and umbilical cord blood.

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomagnetically separated CD34+ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34+ cells from PBSC were morphologically very homogeneous and 94.7+/-4.5% of these cells were MPO-: these ultrastructural features are generally considered typical of immature cells. The CD34+ BM cells were instead more heterogeneous, with 24.6+/-7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.

Revisitation of the clinical indications for the transfusion of platelet concentrates.

Platelet transfusion is indicated when the expected benefits of increasing the number of functional platelets in the patient's circulation outweigh the potential risks generated by exposing the patient to allogeneic, manipulated and stored blood products such as platelet concentrates. Although reassuring evidence has been collected indicating that current risks associated with blood transfusion are lower than those of several voluntary and involuntary human activities, balancing benefits and risks of platelet transfusion may not be easy in a proportion of patients and in a number of conditions. To facilitate this task, guidelines have been developed, with particular attention to cancer patients. As witnessed by the most recent guidelines, over the last few years there has been a progressive, although not absolute, consensus on: (i) the routine use of platelets as a tool to prevent hemorrhage in oncohematology (the so called 'prophylactic approach') as opposed to limiting platelet transfusion to actual bleeding episodes (the so-called 'therapeutic approach') and (ii) lowering the trigger for prophylactic platelet transfusion in stable oncohematology recipients from 20 x 109 to 10 x 109 platelets/L. This has been accompanied by a reduction of platelet use per oncohematology patient of about 20%, an important outcome in view of the progressive increase of platelet demand due to more aggressive therapy in cancer patients. In selected clinical conditions, specific triggers ranging from 30 x 10(9) to 100 x 10(9) platelets/L have been recommended, with higher values when surgical procedures are required for the patient's treatment. Indications and trigger values proposed in the guidelines must be considered within the context of careful clinical evaluation of each patient, with a clear appreciation of the power of discrimination of automated platelet counters at low counts, and of the quality and local availability of platelet products for emergency.

Quality-related variables at hepatological websites.

BACKGROUND: The amount of hepatology-related information available on the Internet has substantially increased, but little is known about the characteristics and quality of the websites. AIM: The aim of this study was to describe analytically and evaluate critically the information concerning three diseases of hepatological interest: chronic hepatitis, hemochromatosis and Caroli's disease. METHODS: In accordance with a validated method, the three search terms were entered into four English language search engines and the first five links of each were considered (a total of 60 sites). The characteristics of the websites were described and their quality was evaluated by three independent reviewers who assigned scores of 1-5 for accuracy, reliability and depth. The relationships between the site characteristics and quality scores were analysed by means of multiple logistic regression. RESULTS: The overall rating score was sufficient (>3) in 51% (95% confidence interval: 38-65%) of cases. The majority of the sites (73%) were aimed at patients rather than at physicians. Commercial sponsorship was significantly more frequent among the chronic hepatitis sites (45%) than among the hemochromatosis (15%) or Caroli's disease sites (0%) (P = 0.002); 61% of the commercial sites did not include a financial disclosure. The only variable that independently related to poor quality was the presence of commercial sponsorship (odds ratio 18.1; 95% confidence interval: 1.7-192.5). CONCLUSIONS: Hepatological websites are characterised by poor quality and are mainly aimed at patients. Quality is negatively affected by commercial interests, which are often undeclared. Guidelines for the certification and surveillance of websites relating to liver diseases are highly advisable.