An update on methods for cryopreservation and thawing of hemopoietic stem cells.

The aim of this article is to review a number of variables that may affect the cryopreservation of minimally manipulated products containing allogeneic or autologous hemopoietic progenitor cells (HPC) used for transplantation, with particular reference to processing, type and addition of cryoprotectant, cell concentration, volume, freezing procedure, cooling rate, storage, thawing, and quality management. After defining final product's requirements in compliance with norms, laws and regulations, it is crucial to define the critical control points of the process. New approaches of processing were developed in the last few years such as automatic devices for volume reduction and high cell concentration in the frozen product. DMSO at 10% final concentration is still the most used cryoprotectant for HPC cryopreservation. Although controlled rate freezing is the recommended method for HPC cryopreservation, alternative methods may be used. Last generation vapor storage vessels ensure temperature stability better than older tanks. Their use may reduce risks of cross-contamination. Finally we review advantages and disadvantages of thawing procedures that may be carried out in the laboratory or at the patient's bedside.

Risk of hepatocellular carcinoma in relation to ABO blood type.

BACKGROUND: Mortality and incidence rates of hepatocellular carcinoma (HCC) parallel the geographical distribution of hepatitis B and C viruses among the general population, however genetic factors modulate individual cancer risk. AIMS: ABO blood type, as a genetic marker, has previously been associated with the risk of several malignancies; we aimed to evaluate whether an association exists with HCC. METHODS: This is a retrospective case-control study based on ABO distribution in 194 patients with HCC, compared with 215 decompensated cirrhotics without HCC listed for liver transplantation, and 90,322 healthy blood donors. RESULTS: In patients with HCC, prevalence of blood type O was 35%, vs. 44% in cirrhotics (OR: 0.67, 95% CI 0.45-0.99; p=0.046) and 45% in blood donors (OR: 0.65, 95% CI 0.48-0.88; p=0.004). CONCLUSIONS: ABO blood type non-O is associated with higher risk of hepatocellular carcinoma, compared to cirrhotics without HCC and healthy subjects.

Extensive Characterization of Platelet Gel Releasate From Cord Blood in Regenerative Medicine.

Platelet gel derived from peripheral blood is widely applied in many clinical fields of surgery as biomaterial containing growth factors with high proliferative properties. In 2010, we studied and patented a platelet gel derived from cord blood. In this study, due to the crucial role of the factors released by the platelet gel, we first extended the characterization of its releasate. Using a wide proteomic array and splitting the two components of the releasate, that is, platelets and plasma, we have been able to study their growth factor content. Interestingly, we discovered high levels of hormones and molecules able to support tissue growth in the cord blood platelet gel releasate and, in addition, higher concentrations of several angiogenic factors if compared with the peripheral blood counterpart. On the contrary, the latter was much richer in inflammatory factors. The second aim of our work was to study the effects on cell culture, immunophenotype, and function of mesenchymal stem cells exposed to these two platelet gel releasates as substitute for the animal serum. Since our findings nicely show that the use of the peripheral versus the cord blood platelet gel releasate can differently influence the mesenchymal stem cell commitment, we can suggest that in addition to its peculiar angiogenic properties cord blood platelet gel releasate shows excellent proliferative properties as cell culture supplement.

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

Identification of new hematopoietic cell subsets with a polyclonal antibody library specific for neglected proteins.

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.

Immunological testing for malaria and blood donor deferral: the experience of the Ca' Granda Polyclinic Hospital in Milan.

BACKGROUND: Current European regulations require a deferral period of 6 months or 3 years, depending on the risk of exposure, for prospective blood donors at risk of malaria. This period may be reduced to 4 months if an immunological or molecular genomic test is negative at each donation, but Italian regulations have not adopted this provision. As cases of transfusion-transmitted malaria have been recorded in medical literature in blood donors deferred for 3 years and not tested, the Immunohematology and Transfusion Centre of the Ca' Grande Polyclinic Hospital in Milan decided to introduce immunological testing for all donors at risk of malaria. MATERIALS AND METHODS: Four hundred and twelve blood donors at risk of malaria, who had lived in a malarial area during the first 5 years of life or for more than 6 consecutive months, were tested for malarial antibodies using an enzyme immunoassay kit. The kit (Malaria EIA, Newmarket, UK) uses four recombinant antigens specific for P. falciparum and P. vivax and with cross-reactivity for P. ovale and P. malariae. The kit detects total immunoglobulin antibodies against P. falciparum and P. vivax and shows 80% cross-reactivity with P. ovale and 67% with P. malariae. Antibody-positive samples were further checked by an immunochromatographic test for P. falciparum, P. vivax, P. ovale and P. malariae antigens and by haemoscopy (thin film and thick smear). RESULTS: Italian citizens accounted for 16.8% (69/412) of the whole group of donors examined. We found that 8.7% of the donors who were classified as being at risk of malaria were positive for total immunoglobulin antibodies. Only one Italian citizen resulted positive for the test. The positive candidates were deferred from blood donation. None of the antibody-positive donors was confirmed positive by the immunochromatographic test and by haemoscopy. CONCLUSION: The introduction of a malarial screening test in the assessment of blood donor eligibility may increase the safety of blood donations, but could further reduce blood availability. If immunological testing were to be accepted nationally as a valid method of assessing the risk of malaria, more than 90% of the donors who are currently deferred for 3 years could be accepted 4 months after their last visit to an endemic area, thus increasing the availability of blood.

[A new setting of opportunistic cardiovascular screening: from blood donation to preventive cardiology. Preliminary results of the Cardiorisk program]. / Un nuovo modello di screening cardiovascolare di opportunità: dalla donazione di sangue alla cardiologia preventiva. Primi risultati del programma Cardiorisk.

BACKGROUND: Cardiovascular diseases remain the leading cause of mortality and disability in developed countries. Therefore, it is necessary to increase a policy of primary prevention. The most recent European guidelines recommend the use of the absolute risk profile as a tool to identify high-risk individuals, but also underline the need for interventions on the whole population. They also mentioned the concept of opportunistic screening for cardio- and cerebrovascular risk factors. METHODS: From September 2004 to December 2008, 13 619 consecutive blood donors were evaluated to determine the absolute risk profile by using the CUORE Project score. Inclusion criteria were age between 35 and 69 years, no evidence of cardiovascular disease, 12 h fasting, and informed consent. All blood donors underwent physical examination and blood tests. The absolute risk profile system includes 8 variables: age, gender, diabetes, smoking habit, systolic blood pressure, total and HDL cholesterol, and antihypertensive therapy. The population was classified into five risk categories (<5%; 5-10%; 10-15%; 15-20%; > or =20%). The results were analyzed according to age and gender. RESULTS: The mean risk score was 2.9 +/- 3 in men and 0.8 +/- 1.04 in women. Furthermore, the proportion of subjects at low risk was high even in the most advanced age groups in both sexes, differently from the general population. In particular, in young and female subjects the risk score did not exceed 20%. The proportion of men at high risk increased in adulthood, varying between 0.5% in the 50-59 age range to 4% in subjects > or =60 years. CONCLUSIONS: Our results demonstrate the feasibility of a primary cardiovascular prevention program in a new opportunistic setting, not assessed previously. The implementation of this program is a valuable tool not only to identify high-risk subjects but also to maintain a favorable risk profile in low-risk subjects over time.

Harvesting of autologous blood stem cells after a mobilising regimen with low-dose cyclophosphamide.

Although high-dose cyclophosphamide (HD-CTX) is commonly used as a mobilising regimen for autologous peripheral blood stem cell (PBSC) collection, significant morbidity and insufficient harvesting may complicate the procedure. Alternative regimens and lower doses of cyclophosphamide (CTX) have been investigated as possible ways of overcoming these difficulties. Low-dose CTX (1.5 g/m2) was administered to 102 lymphoma patients as an autologous PBSC mobilising regimen. The collection of 6 x 10(6) CD34+ cells/kg was chosen as the target of the apheresis sessions, whereas 3 x 10(6)/kg were considered the minimum necessary to perform autologous stem cell transplantation (ASCT) safely. The apheretic sessions were started a median of eight days after CTX administration; a median of two aphereses was required. More than 6 x 10(6) CD34+ cells/kg were collected from 78 patients, between 3 and 6 x 10(6)/kg from 19, and fewer than 3 x 10(6)/kg from 5, two of whom underwent bone marrow harvesting and one a successful second PBSC harvesting session using the same mobilising regimen. Eighty-two patients underwent autografting, six of whom received a second transplant after relapse (five using autologous PBSCs coming from the first apheretic course). Low-dose CTX proved to be a safe and effective regimen for autologous PBSC mobilization and also compared favourably with alternative regimens in terms of the rate of harvesting insufficiency. This does not imply that low-dose CTX is the best mobilising regimen for all patients, and the identification of prognostic factors predicting mobilising potential may help in choosing the best individualised regimen.