Granulocyte storage and antigen stability

Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time-consuming, costly, and technically difficult.

The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4 degrees C for 7 days under three conditions: 1 -percent formaldehyde-fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI-1640); and cells were fixed and stored with a commercial white cell-storage solution (Cyto-Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis.

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 the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)-conjugated secondary antibody hindered interpretation of test results on Day 4. Non-specific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC-conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7-aminoactinomycin-D.

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 non-specific staining due to enhanced membrane permeability of dying cells.