Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells.
BACKGROUND
We evaluated the effects of the TransFix(TM) 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 degrees C and 37 degrees 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/microl) 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.
Canonico
Barbara
B
Department of Human, Environment and Nature Sciences, University of Urbino Carlo Bo, Urbino, Italy. barbara.canonico@uniurb.it
Betti
Michele
M
Luchetti
Francesca
F
Battistelli
Michela
M
Falcieri
Elisabetta
E
Ferri
Paola
P
Zamai
Loris
L
Barnett
David
D
Papa
Stefano
S
D017304:Annexin A5 / Q000378:metabolism; D002452:Cell Count; D048430:Cell Shape; D004247:DNA / Q000378:metabolism; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology* / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D016130:Immunophenotyping; D020287:In Situ Nick-End Labeling; D007962:Leukocytes / Q000166:cytology* / Q000648:ultrastructure; D011419:Propidium / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D013696:Temperature
D017304:Annexin A5; D011933:Reagent Kits, Diagnostic; D003609:Dactinomycin; D011419:Propidium; C025942:7-aminoactinomycin D; D004247:DNA; D016650:Fluorescein-5-isothiocyanate