Frozen-stored granulocytes can be used for an immunofluorescence test to detect granulocyte antibodies.
BACKGROUND
Donor- and/or recipient-derived granulocyte antibodies are considered to be the main cause of transfusion-related acute lung injury (TRALI), neutropenia, and febrile transfusion reactions. Several types of tests are performed to detect antibodies in donated blood and/or the serum of a transfusion recipient. Because granulocytes cannot endure the freezing-thawing process, they cannot be stored in liquid nitrogen (LN2). Therefore, testing is time-consuming, because freshly prepared granulocytes are needed for each testing. An attempt has been made to develop a method that uses granulocytes stored in LN2 for the granulocyte immunofluorescence test (GIFT).


METHODS
Freshly prepared granulocytes were suspended in a solution of 90-percent fetal bovine serum (FBS) plus 10-percent DMSO and then frozen and stored in LN2. In the case of GIFT, frozen-stored granulocytes were rapidly thawed, washed, and fixed with 1-percent paraformaldehyde (PFA) and then treated with MoAbs or serum containing antibodies that were reactive to granulocytes. After staining of granulocytes with FITC or PE, FACS analysis was performed.


RESULTS
A comparison of FACS profiles of freshly prepared granulocytes stained with MoAbs or serum with FACS profiles of frozen-thawed-fixed granulocytes showed that the surface antigen expression was restored. Comparable results from FACS profiles of freshly prepared and frozen-thawed-fixed granulocytes were obtained.


CONCLUSIONS
By being fixed with 1-percent PFA, frozen-stored-thawed granulocytes can be used in the GIFT. With this method, the testing time can be shortened. Moreover, because representative panel granulocytes can be stored in several aliquots, uniform granulocytes can be used as panel cells for each testing.
Nishimura
M
M
Department of Research, The Japanese Red Cross, Central Blood Center, Tokyo, Japan.
Mitsunaga
S
S
Juji
T
T
D000911:Antibodies, Monoclonal; D015925:Cryopreservation / Q000592:standards*; D005240:Feasibility Studies; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006098:Granulocytes / Q000276:immunology*; D015235:HLA-B Antigens / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D007518:Isoantibodies / Q000097:blood*; D007519:Isoantigens / Q000276:immunology
D000911:Antibodies, Monoclonal; D015235:HLA-B Antigens; D007106:Immune Sera; D007518:Isoantibodies; D007519:Isoantigens; C072753:neutrophil-specific antigen NA2