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1.  Evaluation of an easy and affordable flow cytometer for volumetric haematopoietic stem cell counting 
Blood Transfusion  2014;12(3):416-420.
Accurate estimation of haematopoietic stem cell (HSC) counts by flow cytometry may be difficult in laboratories in which sophisticated equipment and staff with specific expertise are not available. Affordable flow cytometers that can perform basic functions may help to overcome these difficulties. In this study we compared HSC and leucocyte counts determined by volumetric and bead-based protocols performed with the small, low-cost Accuri® C6, with those obtained with two gold-standard instruments, the four-colour FACSCalibur® and the eight-colour FACSCantoII®, our reference flow cytometers.
Materials and methods
With the three cytometers we tested, in parallel, 111 consecutive samples from cord blood, peripheral blood from patients with myelofibrosis and myeloproliferative syndromes, fresh and thawed HSC collected by apheresis and bone marrow products. The findings were compared with one-way ANOVA, Bland-Altman analysis and linear regression.
The results of HSC and leucocyte enumeration by the three devices were strongly correlated (r2>0.99; p<0.0001). ANOVA performed on different subgroups of samples did not reveal significant differences between HSC count determined by the C6 bead-based and reference flow cytometers in any of the subgroups. Regarding the C6 volumetric protocol, a statistically significant difference was observed only in the cord blood subgroup. Time for instrument set-up, calibration and analysis was slightly longer with Accuri® C6 (40 min) than with FACSCantoII® (30 min).
Accuri® C6 is a reliable instrument for HSC enumeration in fresh samples, using both volumetric and bead-based approaches, although the volumetric protocol on cord blood samples needs to be improved. The Accuri® C6 is easy to use, does not require profound knowledge of flow cytometry and could be employed in an urgent setting. Its performance may be improved by more efficient calibration and shorter analysis time.
PMCID: PMC4111825  PMID: 24887218
haematopoietic stem cells; volumetric flow cytometry; apheresis
2.  Implementation and outcomes of a transfusion-related acute lung injury surveillance programme and study of HLA/HNA alloimmunisation in blood donors 
Blood Transfusion  2012;10(3):351-359.
Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality. Antibodies against human leucocyte antigens (HLA) and human neutrophil antigens (HNA) are often detected in the implicated donors. We investigated the incidence and aetiology of TRALI in Lombardy. Moreover, we determined the rate of HLA and HNA alloimmunisation and the HNA genotype in a cohort of local blood donors.
Materials and methods.
During a 2-year observational study in eight blood transfusion services, suspected TRALI cases were collected and characterised by means of HLA and HNA antibody screening of implicated donors, donor/recipient cross-matching and HLA/HNA molecular typing. In addition, 406 Italian donors were evaluated for alloimmunisation and in 102 of them HNA gene frequencies were determined.
Eleven cases were referred to the central laboratory, of whom three were diagnosed as having TRALI, seven as having possible TRALI and one as having transfusion-associated circulatory overload. Seven TRALI cases were immune-mediated whereas in three we did not find either alloantibodies in implicated donors or a positive reaction in the cross-match. The most frequently implicated blood component was red blood cells (in 5 males and in 1 female), whereas four cases of TRALI were associated with transfusion of fresh-frozen plasma (in 3 females and in 1 male). The frequency of reported TRALI/possible TRALI cases was 1:82,000 for red blood cells and 1:22,500 for fresh-frozen plasma. No cases were observed for platelets. Overall, the frequency of HLA or HNA alloimmunisation in blood donors was 29% for females and 7% for males. The latter could be related, at least in part, to natural antibodies. HNA gene frequencies showed that HNA-1b is more frequent than HNA-1a in our sample of donors.
The recently adopted national policy to prevent TRALI, i.e. using only plasma donated by males, would have had a positive impact in our setting.
PMCID: PMC3417735  PMID: 22395353
transfusion-related acute lung injury; HLA and HNA alloimmunisation; HNA frequencies
3.  Evidence of Distinct Tumour-Propagating Cell Populations with Different Properties in Primary Human Hepatocellular Carcinoma 
PLoS ONE  2011;6(6):e21369.
Background and Aims
Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).
After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ−/− mice.
The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features.
Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.
PMCID: PMC3121782  PMID: 21731718
4.  Correlation of Circulating CD133+ Progenitor Subclasses with a Mild Phenotype in Duchenne Muscular Dystrophy Patients 
PLoS ONE  2008;3(5):e2218.
Various prognostic serum and cellular markers have been identified for many diseases, such as cardiovascular diseases and tumor pathologies. Here we assessed whether the levels of certain stem cells may predict the progression of Duchenne muscular dystrophy (DMD).
Methods and Findings
The levels of several subpopulations of circulating stem cells expressing the CD133 antigen were determined by flow cytometry in 70 DMD patients. The correlation between the levels and clinical status was assessed by statistical analysis. The median (±SD) age of the population was 10.66±3.81 (range 3 to 20 years). The levels of CD133+CXCR4+CD34- stem cells were significantly higher in DMD patients compared to healthy controls (mean±standard deviation: 17.38±1.38 vs. 11.0±1.70; P = 0.03) with a tendency towards decreased levels in older patients. Moreover, the levels of this subpopulation of cells correlated with the clinical condition. In a subgroup of 19 DMD patients after 24 months of follow-up, increased levels of CD133+CXCR4+CD34- cells was shown to be associated with a phenotype characterised by slower disease progression. The circulating CD133+CXCR4+CD34- cells in patients from different ages did not exhibit significant differences in their myogenic and endothelial in vitro differentiation capacity.
Our results suggest that levels of CD133+CXCR4+CD34- could function as a new prognostic clinical marker for the progression of DMD.
PMCID: PMC2377332  PMID: 18493616

Results 1-4 (4)