Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells

Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, University of Perugia, Italy
Cytometry Part B Clinical Cytometry (Impact Factor: 2.4). 07/2010; 78B(4):267 - 278. DOI: 10.1002/cyto.b.20510


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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Available from: Barbara Canonico, Sep 19, 2014
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    • "Notably, identical results are obtained in fresh blood assays where the anti-CD16 antibody is omitted because CD16 + nonocytes are however excluded by gating out CD115 + cells and CD16 + neutrophils can be easily recognized on the basis of their high SSC. Nonetheless, the use of this antibody is important for excluding contaminating neutrophils in stabilized and stored whole blood, where slight changes in the SSC of granulocytes are commonly observed [25]. The enumeration of circulating fibrocytes by using the singleplatform technology requires only 100 μl of venous blood. "
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