A preliminary study of an alternative method for evaluating skin sensitizing potential of chemicals.

BACKGROUND: In order to comply with the European legislation concerning the risk assessment of skin sensitizers, considerable progress has been made in developing alternative methods, such as human cell line activation test (h-CLAT). H-CLAT is based on cytometric measurement of fluorescence emitted by anti-CD54 and anti-CD86 antibodies in THP-1 cells. Following this method, a range of substances have been analyzed; the emitted fluorescence, generally at low intensity, has caused problems concerning the interpretation of results. AIM: Find an alternative parameter to h-CLAT for evaluating the sensitizing potential of chemicals. MATERIALS AND METHODS: Cells have been analyzed with flow cytometry after treatment with sensitizing compounds administered at non-cytotoxic concentrations. RESULTS: Sensitizers were able to inducealterations in cell morphology to a more 'condensed' one allowing the identification of cells under microscope as a 'sensitized' subpopulation. These variations cause similar modifications in 'scattering' parameters, making cells easily monitorable by flow cytometry. No changes have been observed in cells treated with non-sensitizers or in untreated cells. CONCLUSION: This method based on the analysis of forward scatter and side scatter parameters, can be used as an alternative method for identifying sensitization potential of chemical compounds.

CONTEXTE: Pour se conformer à la législation européenne en matière d'évaluation des risques des sensibilisants cutanés, beaucoup des progrès ont été fait dans le développement de méthodes alternatives comme le test d'activation de la lignée cellulaire humaine (H-CLAT). H-CLAT se base sur la mesure cytométrique de la fluorescence émise par les anticorps anti-CD54 et anti-CD86 dans les cellules THP-1. Suivant cette méthode, une gamme de substances a été analysée; la fluorescence émise, généralement de faible intensité, a posé des problèmes d'interprétation des résultats. OBJECTIF: Trouver un paramètre alternatif à h-CLAT pour évaluer le potentiel sensibilisant des produits chimiques. MATÉRIAUX ET MÉTHODES: Les cellules ont été analysées par cytométrie en flux après le traitement avec des composés sensibilisants administrés à des concentrations non cytotoxiques. RÉSULTATS: Les sensibilisateurs ont pu induire des altérations de la morphologie cellulaire en une morphologie plus condensée, permettant l'identification des cellules au microscope comme sous-population sensibilisée. Ces variations entraînent des modifications similaires des paramètres de "scattering", rendant les cellules facilement contrôlables par cytométrie en flux. Aucun changement a été observé dans les cellules traitées avec des agents non sensibilisants ni dans les cellules non traitées. CONCLUSION: Cette méthode basée sur l'analyse des paramètres de "forward" et "side scatter" peut être utilisée comme méthode alternative pour identifier le potentiel de sensibilisation des composés chimiques.

Protective effect of black relative to white race against non-alcoholic fatty liver disease in patients with severe obesity, independent of type 2 diabetes.

Severe obesity (body mass index ⩾35 kg m-2) and type 2 diabetes (T2D) are potent and additive risk factors for non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH). Scant available evidence indicates that black relative to white patients with severe obesity are less susceptible to NAFLD, but it is unclear if T2D abolishes this apparent racial disparity. Therefore, we compared biopsy-proven NAFLD and its progression between black (n=71) and white (n=155) patients with severe obesity stratified by presence or absence of T2D. Although prevalence of T2D was similar between races (37%, P>0.9), whites were significantly more likely than blacks to have NAFLD, NASH and advanced fibrosis (defined as bridging fibrosis and/or cirrhosis). Importantly, T2D was associated with increased odds of NAFLD, NASH and advanced fibrosis (defined as bridging fibrosis or cirrhosis) in whites only (P<0.05). In turn, a higher proportion of blacks than whites with T2D were free of NAFLD (58 versus 22%, P<0.01). These preliminary findings question translation of the powerful interconnection between T2D and NAFLD in whites with severe obesity to blacks and point to an important role of race in the pathophysiology and treatment of these diseases.

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.

The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones.

Mathematical modeling of growth and death dynamics of mouse embryonic stem cells irradiated with γ-rays.

Following ionizing radiation, mouse embryonic stem cells (mESCs) undergo both apoptosis and block at G2/M phase of the cell cycle. The dynamics of cell growth and the transition through the apoptotic phases cannot be directly inferred from experimental data, limiting the understanding of the biological response to the treatment. Here, we propose a semi-mechanistic mathematical model, defined by five compartments, able to describe the time curves of untreated and γ-rays irradiated mESCs and to extract the information therein embedded. To this end, mESCs were irradiated with 2 or 5 Gy γ-rays, collected over a period of 48 h and, at each time point, analyzed for apoptosis by using the Annexin V assay. When compared to unirradiated mESCs, the model estimates an additional 0.2 probability to undergo apoptosis for the 5 Gy-treated cells, and only a 0.07 (not statistically significantly different from zero) when a 2 Gy-irradiation dose is administered. Moreover, the model allows us to estimate the duration of the overall apoptotic process and also the time length of its early, intermediate, and late apoptotic phase.

In-vitro effects of the tyrosine kinase inhibitor imatinib on glioblastoma cell proliferation.

Glioblastoma (GBL) is the most malignant brain tumour in adults, causing the death of most patients within 9-12 months of diagnosis. Treatment is based on a combination of surgery, radiation therapy, and chemotherapy. With these treatment modalities, however, responses are extremely poor, so identification of novel treatment strategies is highly warranted. Platelet-derived growth factors (PDGF) and their receptors are commonly coexpressed in GBL, suggesting that stimulation of autocrine PDGF receptors may contribute to their growth. Interest in these receptors as drug target for glioblastoma treatment has increased with the clinical availability of the PDGFR kinase inhibitor antagonist imatinib mesylate (STI571). In this study, T98G and A172 human GBL cell lines were analysed for their sensitivity to treatment with imatinib. In particular, we focussed our attention on analysis of DNA distribution by flow cytometry at different times of incubation with different imatinib concentrations (1-30 microM: ). Our results show that imatinib induces growth arrest in T98G and A172 cells in the G(0)/G(1) phase of the cell cycle, at all the concentrations tested, as early as 24 h after treatment. However we have also seen, by means of annexin V staining, that at 20 and 30 microM: concentrations, in concomitance with a significant growth arrest in the G(0)/G(1) phase, there is an increase of apoptotic cells 48 h after treatment, suggesting that imatinib at low concentrations (1-10 microM: ) could act as a cytostatic agent whereas at high concentrations (20, 30 microM: ) it mainly behaves as a cytotoxic agent.

Relationship between Vac A toxin and ammonia in Helicobacter pylori-induced apoptosis in human gastric epithelial cells.

VacA toxin is one of the most important virulence factors produced by H. pylori even though neither its role nor its action mechanisms are completely understood. First considered as a toxin inducing only cell vacuolation, VacA causes apoptosis of gastric epithelial cells by targeting mitochondria. A hotly debated question about VacA action is its relationship with ammonia, which is produced in vivo by H. pylori urease. While ammonia is strictly required for VacA-dependent vacuolation, its role in VacA-induced apoptosis is much less defined. This study was thus aimed to investigate the relationship between VacA toxin and ammonia in H. pylori-induced mitochondrial damage and apoptosis of human gastric epithelial cells in culture by means of flow cytometry. Our results show that, unlike cell vacuolation, in MKN 28 cells neither apoptosis nor dissipation of mitochondrial transmembrane potential induced by VacA require ammonia. Nevertheless, ammonia significantly potentiates both these VacA-induced effects, but independently of the swelling of VacA-containing endosomes (i.e., vacuolation). Our findings make unlikely the hypothesis that ammonia-dependent swelling and rupture of endosomal vesicles in which VacA is sequestered after cell internalization may allow the toxin to reach mitochondria and trigger apoptosis.

Extra-corporeal liver BNCT for the treatment of diffuse metastases: what was learned and what is still to be learned.

Almost eight years ago, in December 2001, we performed for the first time in the world thermal neutron irradiation on an isolated liver of a patient. The organ was affected by diffuse metastases of a colon carcinoma and had been previously loaded with a (10)B compound. In July 2003, the same procedure was applied again on a patient for the treatment of unresectable and incurable hepatic metastases of a carcinoma of the rectum. Both patients are dead at present. Now we can analyze in depth the clinical history of these patients and evaluate the effectiveness of this therapy. From this exciting experience we learned much, and we also found out about complications till then unknown, which need to be studied and addressed experimentally. Unfortunately we can base our conclusions just on the experience we had with these two patients. We could have been much more detailed and firm in our statements if the number of clinical cases was larger. The BNCT Pavia project has been suspended, but it is more than likely to resume in a short time. Good findings were many. The procedure is feasible; the original concept of complete immersion of the diseased liver in a homogeneous neutron field proved effective and winning. The tumor masses resulted completely necrotic and unknown metastases too appeared radically treated; healthy hepatic tissue was preserved from both morphological and functional points of view; no symptoms of cirrhosis appeared even four years after treatment. For the long term surviving patient, quality of life was excellent. Other findings require to be tackled in depth. The "post-irradiation syndrome" we observed in both patients, with identical symptoms and biochemical derangements, creates a dramatic--even though totally reversible--clinical condition, that is the probable cause of death for our second patient, suffering from cardiomyopathy, 33 days after treatment. For the first patient, recurrences were a late yet fatal complication, for which even a further surgical revision was ineffective. We offer some hypotheses about their origin and possible prevention.

Pharmacogenetics and pharmagenomics, trends in normal and pathological aging studies: focus on p53.

In spite of the fact that the aging organism is the result of complex life-long gene/environment interactions, making peculiar the susceptibility to diseases and the response to drugs, pharmacogenetics studies are largely neglected in the aged. Altered response to drugs, cardiovascular and metabolic alterations, cancer and dementia are among the age associated ailments. The latter two are the major contributors to illness burden for the aged. Aging, dementia and cancer share a critical set of altered cellular functions in the response to DNA damage, genotoxic stress, and other insults. Aging in higher animals may be influenced by the balance of cell survival versus death, a decision often governed by checkpoint proteins in dividing cells. The paper is mainly focused on one of such proteins, p53 which has been recently shown to be involved in aging and Alzheimer's Disease (AD). Within this reference frame we studied p53 in aged controls and demented patients finding that with aging there is an increase of mutant like conformation state of p53 in peripheral blood cells, which is more pronounced in AD patients. As a result of such conformational change, p53 partially loses its activity and may become unable to properly activate an apoptotic program when cells are exposed to a noxious stimulus. Moreover we found that the tertiary structure of p53 and the sensitivity to p53-dependent apoptosis are affected by low concentrations of soluble beta amyloid, the peptide that accumulates in AD brain but also present in peripheral tissues. It is possible that p53 conformers may occur in the presence of misfolded molecules such as, but not limited to, beta amyloid. In particular at neuronal level the altered function of cell cycle proteins may affect synaptic plasticity rather than cell duplication.

Karyotype analysis of the euploid cell population of a mouse embryonic stem cell line revealed a high incidence of chromosome abnormalities that varied during culture.

It is common knowledge that mouse embryonic stem cell (mESC) lines accumulate chromosomal changes during culture. Despite the wide use of mESCs as a model of early mammalian development and cell differentiation, there is a lack of systematic studies aimed at characterizing their karyological changes during culture. We cultured an mESC line, derived in our laboratory, for a period of 3 months investigating its chromosome complement at different times. About 60% of the metaphases analysed were euploid throughout the culture period but, from passage 13, only 50% of the euploid metaphases had a proper chromosome complement. The remaining 50% showed chromosome abnormalities, mainly gain or loss of entire chromosomes, both within the same passage and among different passages analysed. The very heterogeneous spectrum of abnormalities indicates a high frequency of chromosome mutations that arise continuously during culture. The heterogeneity of the aberrant chromosome constitution of 2n = 40 metaphases, observed at different passages of culture, might be due either to their elimination or to a shift towards the hypoeu- or hypereuploid population of those metaphases that accumulate further chromosome abnormalities. The stability of the frequency of eu-, hypoeu- and hypereuploid populations during culture might, however, be due to the elimination of those cells that carry a high mutational burden. Based on our results, we suggest that karyotype analysis of the euploid cell population of mESC lines is necessary when such lines are used in the production of chimeric mice, for their contribution to the germ line, or when they are differentiated into specific cell types.

Conformationally altered p53: a novel Alzheimer's disease marker?

The identification of biological markers of Alzheimer's disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinson's disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

Immunophenotypic, cytogenetic and functional characterization of circulating endothelial cells in myelodysplastic syndromes.

Circulating endothelial cells (CECs) are associated with neoangiogenesis in various malignant disorders. Using flow cytometry, we studied CECs in 128 patients with myelodysplastic syndrome (MDS). MDS patients had higher CEC levels than controls (P<0.001), and an inverse relationship was found between CECs and international prognostic scoring system risk (r=-0.55, P<0.001). There was a positive correlation between marrow microvessel density and CECs, low-risk patients showing the strongest association (r=0.62, P<0.001). We calculated a progenitor-to-mature CEC ratio, which was higher in MDS patients than in healthy subjects (P<0.001), the highest values were found at diagnosis. CECs assessed by flow cytometry positively correlated with the ability to produce endothelial colony-forming cells in vitro (ECFCs; r=0.57, P=0.021), which was significantly higher in MDS patients than in controls (P=0.011). Fluorescence in situ hybridization analysis showed that a variable proportion of CECs (from 40 to 84%) carried the same chromosomal aberration as the neoplastic clone, while endothelial cells isolated from in vitro assays were negative. This study suggests that CECs reflect the abnormal angiogenesis found in MDS, especially in the early stages of the disease. The increased number of functional endothelial progenitor cells in MDS strengthens the rationale for therapeutic interventions aimed at restoring a normal interaction between hematopoietic progenitors and marrow microenvironment.

Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein- and p53-deficient and proficient human osteosarcoma cell lines.

OBJECTIVES: To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. MATERIAL AND METHODS: RB- and p53-proficient U2OS cells and the RB- and p53-deficient SAOS-2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. RESULTS: One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G(1)-S and G(2)-M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS-2 cells. No effect on cell cycle progression after actinomycin D-induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS-2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS-2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS-2 cells after the long-term actinomycin D treatment, whereas no modification was observed in U2OS cells. CONCLUSIONS: These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB- and p53-deficient cells. A daily-repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.

Effect of nitric oxide on lymphocytes from sporadic amyotrophic lateral sclerosis patients: toxic or protective role?

Markers of oxidative and nitrosative stress have been found in spinal cord, cortex, cerebrospinal fluid and plasma of patients affected by amyotrophic lateral sclerosis (ALS), a fatal disorder characterised by progressive motor neuron degeneration. In this study, we investigated the effect of the NO-releasing agent, diethylamine NONOate (NONO), on lymphocytes from patients affected by the sporadic form of ALS (SALS) and controls by flow cytometry. In the same experimental conditions we investigated the expression of the antioxidant proteins, Bcl-2 and SOD1. Incubation with NONO induced cell damage in control lymphocytes but did not further damage the already affected untreated SALS lymphocytes. The incubation with NONO induced a time-dependent decrease of Bcl-2 and SOD1 in control lymphocytes. Surprisingly, in SALS lymphocytes the NONO treatment increased the expression of these proteins, which in basal conditions was depressed compared to control lymphocytes.

Cytofluorimetric evaluation of DNA ploidy in lung cancer: a bronchoscopic study.

The study of the biological characteristics of lung cancer is gaining more and more interest both because of their potential role as prognostic indicators and for therapeutic reasons. The DNA content estimated by flow cytometry in surgical samples of non-small cell lung cancer (NSCLC) has already been demonstrated to be correlated with survival in these patients. From July 1990 to February 1992 we analyzed the DNA distribution of bronchoscopic biopsies from 88 patients with lung cancer (18 small cell lung cancer, SCLC, and 68 NSCLC, two unspecified histology). Twenty-eight tumors (34.6%) had a diploid DNA distribution, while 53 were aneuploid (65.4%). A correlation was found between DNA ploidy and survival. Evaluation of the DNA content in bronchoscopic samples in a large series of patients could determine the role of this analysis prior to surgery in NSCLC and its value as a marker with respect to prognosis and response to therapy in SCLC.

Dual excitation multi- fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition.

The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent "energy transfer" from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 micro g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.

Cell kinetics in a model of artificial skin. An immunohistochemical and flow cytometric analysis.

Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE) using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.

Automation of human sperm cell analysis by flow cytometry.

Semen sample analysis is routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. Our aim was to automate sperm analysis with the use of flow cytometry for evaluation of cell counts and typing and with the use of a new membrane-permeant nucleic acid stain for evaluation of sperm viability. Statistical analysis of the comparison between manual and automated methods for sperm counts was performed by the Bland and Altman method; the mean difference was 0.243 x 10(6) sperms/ mL. The precision of the flow cytometric analysis was evaluated with whole sperm; the between-run CV was 7.5% and the within-run CV was 2.5%. Data observed suggest that flow cytometric sperm analysis, with high precision and accuracy and low costs, can be proposed for routine use in clinical laboratories.

Quality control study of the Italian Group of Cytometry on flow cytometry DNA content measurements: II. Factors affecting inter- and intralaboratory variability.

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.