Granulocyte storage and antigen stability

BACKGROUND: Current methods for the detection of granulocyte antibodies req uire panels of freshly isolated cells. This makes these assays time-consumi ng, costly, and technically difficult. STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the bi nding of antibodies to granulocytes was modified for use with a flow cytome ter, and methods were tested to store granulocytes for use in that assay. G ranulocytes were stored at 4 degrees C for 7 days under three conditions: I -percent formaldehyde-fixed cells were stored in Hanks' balanced salt solut ion (HBSS); untreated cells were stored in tissue culture medium (RPMI-1640 ); and cells were fixed and stored with a commercial white cell-storage sol ution (Cyto-Chex Reagent). Antigen stability was evaluated by using monoclo nal antibodies (MoAbs) and alloantibodies. Serologic studies were done by a n indirect immunofluorescence assay and assessed by flow cytometric analysi s. RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI-1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell-storage solution remained. All antigens were detectable by th e MoAbs and alloantisera on Day 7. However, nonspecific staining by the flu orescein isothiocyanate (FITC)-conjugated secondary antibody hindered inter pretation of test results on Day 4. Nonspecific staining occurred over time and was associated with increased cell permeability during storage. Two so urces of nonspecific staining were identified. The first source was the FIT C-conjugated secondary antibody; it was eliminated by switching to a phycoe rythrin conjugate. The second source was factors in human serum; it was res olved by examining only viable, impermeable cells identified by using 7-ami noactinomycin-D. CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of nonspecific staining due to enhanced membrane permeability of dying cells.