Optimal storage conditions for preserving granulocyte viability as monitored by Annexin V binding in whole blood.
It is important in the laboratory to develop techniques to preserve leucocyte viability in blood specimens for subsequent flow cytometric analysis. This article describes a new simple whole blood lysis method using Annexin V FITC staining which can be used to define both early and late apoptosis of granulocytes in heterogeneous cell populations, without the need for additional stains or to purify the cells (which may result in loss of the cells of interest). The differential Annexin V binding assay was in good agreement with the light microscopy reference method and showed excellent correlation with 7-aminoactinomycin D (7-AAD) staining. It was not affected by problems of morphological interpretation and artifactal changes of granulocyte deformability noted using light microscopy, or the technical difficulties encountered due to red cell contamination using the 7-AAD method. Using this new differential Annexin V staining method, we determined the optimum conditions that maintain granulocyte viability for subsequent flow cytometric analysis and are now employed in our laboratory. These conditions were lithium heparin (Hep) anticoagulated whole blood specimens kept at 4 degrees C with the addition of nutrient medium. Specimens that are anticoagulated with acid citrate dextrose (ACD) or ethyl-diacetyl-tetraacetic acid (EDTA) should also be treated similarly to preserve granulocyte viability and to overcome problems associated with identification of cell populations by flow cytometry.
Hodge
G L
GL
Haematology Department, Adelaide Women's and Children's Hospital, North Adelaide, Australia.
Flower
R
R
Han
P
P
D000328:Adult; D017304:Annexin A5 / Q000097:blood*; D000925:Anticoagulants / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D001793:Blood Preservation; D002470:Cell Survival / Q000187:drug effects; D003609:Dactinomycin / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D006098:Granulocytes / Q000166:cytology*; D006801:Humans; D008027:Light; D008722:Methods; D009504:Neutrophils / Q000166:cytology; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D013194:Staining and Labeling; D013696:Temperature
D017304:Annexin A5; D000925:Anticoagulants; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D