Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells
ABSTRACT Background:We evaluated the effects of the TransFixTM short-term stabilization technique on leukocyte subpopulations in both optimal and adverse storage temperatures and on different cellular concentrations. Particularly, we analyzed DNA cell content and membrane structure also for erythrocytes using a multiparametric approach.Methods:We studied biomolecular and morphological aspects of transfixed cells, by means of SEM, TEM, Western blotting, and by flow cytometry (FC). Furthermore, FC, Tunel, and electrophoresis were applied to evaluate DNA behavior.Results:We confirm preservation of scatter characteristics and immunophenotyping, extending such evaluations to cells stored in suboptimal conditions (25°C and 37°C) and in high density. Data demonstrate for lymphomonocytic cells an optimal conservation, slightly decreasing at higher temperatures for both 1/5 and 1/10 ratio (TransFix™/sample), with enhanced autofluorescence. Eosinophils, basophils, and neutrophils are shown to preserve differently over time. The three different cellular concentrations evaluated (30,000–120,000 cell/μl) demonstrate substantial stability in FI values. Furthermore DNA content analysis attests the absence of any apoptotic pattern. Transfixed red cell protein profile as well as their morphological features appears almost unaltered.Conclusions:Cytometric performance is suboptimal in aged unfixed specimens because of apoptosis that affects light scatter properties. Our findings highlight that lymphomonocytic cells are well stabilized even at suboptimal temperature and cell density. TransFix™ is able to abolish any apoptotic features and acts as an optimal blood preservative for appropriate preanalytical stabilization. © 2010 Clinical Cytometry Society
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ABSTRACT: In this report, we have evaluated the effects of a TransFix®-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix®-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.Journal of Immunological Methods 01/2005; · 2.23 Impact Factor
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ABSTRACT: Melatonin influences circadian rhythms and acts as antioxidant and free radical scavenger. UV irradiation triggers multiple cellular events which lead to cell death, in particular to apoptosis; this process involves reactive oxygen species. Apoptotic machinery involves several pathways, in which mitochondria play crucial roles. In this work we have evaluated by means of cytometric, biochemical and ultrastructural approaches, if incubation of U937 promonocytic leukemia cells with melatonin may affect apoptotic behavior induced by UV-B. The cell line was treated with 1 mm melatonin before and after UV-B exposure. Melatonin pretreatment significantly reduced the number of apoptotic cells, as revealed by FITC Annexin-V and propidium iodide assays (P < 0.005), as well as attenuated mitochondria alterations, as shown by ultrastructural morphology, Mito Tracker and JC-1 staining, and cytochrome c (cyt c) release (P < 0.005). On the contrary, incubation with melatonin after UV-B exposure significantly protect U937 cells from UV-B induced alterations, showing a possible delay of the apoptotic machinery (as revealed by the presence of earlier stages of apoptosis and significant cyt c release). Our results suggest that, in our experimental model, melatonin may play a role as noncytotoxic anti-apoptotic compound and, at least in part, may protect U937 cells from UV-B induced mitochondria dysfunction/damage.Journal of Pineal Research 04/2006; 40(2):158-67. · 7.30 Impact Factor
- Experimental Cell Research - EXP CELL RES. 01/1991; 195(1):237-246.