title: Third-color CD45 staining of paraformaldehyde-fixed peripheral blood lymphocytes.
abstract: Third-color CD45 gating is a useful procedure in various clinical applications (Borowitz et al.: Am J Clin Pathol 100:534-540, 1993; Steltzer et al.: Ann NY Acad Sci 677:267-280, 1993) allowing for the selective immunophenotyping of abnormal cell populations. In laboratories where dual-color protocols are in place, the need for CD45 gating may not become evident until after cells have been exposed to fixatives. This brief study demonstrates that third-color CD45 staining and gating is possible with paraformaldehyde fixed lymphocytes, eliminating the need to restain fresh cells with the entire antibody panel, and with a significant saving of time and money. Gates must be modified from those obtained by fresh cell staining in order to include both bright and dim CD45 populations. In a standard protocol, third-color CD45 staining intensity was decreased 10-fold post-fixation with paraformaldehyde except, unexpectedly, for CD8+ cells. The latter phenomenon could be explained if the spatial arrangement of CD45 and CD8 is such that the CD45 epitope is protected from cross-linking by paraformaldehyde fixation in CD8+ cells, but not others.
authors.affiliation: Department of Pathology, Flow Cytometry Lab, New York Medical College, Elmsford, New York, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Pizzolo
authors.forename: M R
authors.initials: MR
authors.lastname: Melamed
mesh_terms: D016827:CD8 Antigens / Q000032:analysis / Q000276:immunology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000032:analysis* / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*; D011108:Polymers / Q000737:chemistry*; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D016827:CD8 Antigens; D011108:Polymers; D005557:Formaldehyde; D017493:Leukocyte Common Antigens; C003043:paraform