Evaluation of leukocyte stabilisation in TransFix®-treated blood samples by flow cytometry and transmission electron microscopy

• Published in: Journal of immunological methods Vol. 295; no. 1; pp. 67 - 78
• Main Authors: Canonico, B., Zamai, L., Burattini, S., Granger, V., Mannello, F., Gobbi, P., Felici, C., Falcieri, E., Reilly, J.T., Barnett, D., Papa, S.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.12.2004; Elsevier
• Subjects: Adult; Apoptosis; Biological and medical sciences; Cell Count; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell permeability; Fixatives - pharmacology; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunophenotyping; Leukocytes - cytology; Leukocytes - drug effects; Leukocytes - ultrastructure; Microscopy, Electron, Transmission; Molecular immunology; Permeability - drug effects; Phenotype; Techniques; Tissue Fixation - methods; TransFix; Ultrastructure; PBS; Flow cytometry; SSC; FI; Cell permeability; PBCs; Phenotype; Ultrastructure; FSC; PI; UK NEQAS; TEM; FC; TransFix; Apoptosis; Leukocyte; Cell death; Transmission; Electron microscopy; Blood; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2004.09.013

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.