Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry.
BACKGROUND
Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible.


METHODS
Three-color flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3(+), CD4(+), CD8(+)), B cells (CD19(+)), and natural killer cells (CD16(+)/56(+)) in WB and PBMCs using variations of a standard stain/fix WB staining procedure (Optilyse) that included staining following fixation and freezing of fixed samples before or after staining.


RESULTS
Comparable lymphocyte subset percentages in WB or PBMCs were observed regardless of Optliyse method used (all Ps >/= 0.8). However, differences in fluorescence intensity for several markers were observed across procedures. Compared with the standard stain/fix procedures, fix/stain decreased the mean fluorescence intensities for CD4, CD8, CD19 and CD16/56 in WB and PBMCs (P </= 0.03 for these markers P = 0.105 for CD8 in PBMCs). Further decreases in mean fluorescence intensity were seen with the fix/stain/freeze procedure. The stain/fix/freeze yielded intensities largely comparable to those seen with standard stain/fix procedure (P >/= 0.13), suggesting that, when the markers of interest are known at the time of field collection, implementation of this procedure might be desirable. Fix/freeze/stain resulted in diminution of intensity in general, but they tended to be more modest than those seen for fix/stain/freeze and therefore might be applicable to field studies in instances when the specific markers of interest cannot be defined upfront.


CONCLUSIONS
Freezing of fixed WB and PBMCs before or after cell surface staining is a reliable method for preserving specimens in field sites for later determination of lymphocyte subset percentages, which are commonly assessed in immunodeficient and cancer patients.
Pinto
Ligia A
LA
NCI-Frederick/SAIC-Frederick, Frederick, Maryland, USA. lpinto@ncifcrf.gov
Trivett
Matthew T
MT
Wallace
Dora
D
Higgins
Jeanette
J
Baseler
Michael
M
Terabe
Masaki
M
Belyakov
Igor M
IM
Berzofsky
Jay A
JA
Hildesheim
Allan
A
D001793:Blood Preservation; D002469:Cell Separation; D015925:Cryopreservation / Q000379:methods*; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D016131:Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives