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

  1. Barbara Canonico1,*,
  2. Michele Betti1,2,
  3. Francesca Luchetti1,
  4. Michela Battistelli1,
  5. Elisabetta Falcieri1,3,
  6. Paola Ferri1,
  7. Loris Zamai1,
  8. David Barnett4,
  9. Stefano Papa1

Article first published online: 10 MAR 2010

DOI: 10.1002/cyto.b.20510

Issue

Cytometry Part B: Clinical Cytometry

Cytometry Part B: Clinical Cytometry

Volume 78B, Issue 4, pages 267–278, July 2010

Additional Information

How to Cite

Canonico, B., Betti, M., Luchetti, F., Battistelli, M., Falcieri, E., Ferri, P., Zamai, L., Barnett, D. and Papa, S. (2010), Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells. Cytometry, 78B: 267–278. doi: 10.1002/cyto.b.20510

Author Information

  1. 1

    Department of Human, Environment and Nature Sciences, University of Urbino “Carlo Bo”, Urbino, Italy

  2. 2

    Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, University of Perugia, Italy

  3. 3

    IGM-CNR, Istituti Ortopedici Rizzoli, Bologna, Italy

  4. 4

    Department of Haematology, Royal Hallamshire Hospital, Sheffield, United Kingdom

*Centro di Citometria e Citomorfologia, University of Urbino “Carlo Bo,” Campus Scientifico, via Cà Le Suore, 61029 Urbino, Italy

  1. †

    How to cite this article: Canonico B, Betti M, Luchetti F, Battistelli M, Falcieri E, Ferri P, Zamai L, Barnett D, Papa S. Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells Cytometry Part B 2010; 78B: 267–278.

Publication History

  1. Issue published online: 21 JUN 2010
  2. Article first published online: 10 MAR 2010
  3. Manuscript Accepted: 25 DEC 2009
  4. Manuscript Revised: 20 NOV 2009
  5. Manuscript Received: 22 JUL 2009

Funded by

  • Italian National Research Council. Grant Number: CNR97.04135.CT04

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Keywords:

  • TransFix™;
  • apoptosis;
  • DNA;
  • cell surface;
  • membrane structure

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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