What is the most likely cause if cells type negative for all HLA antigens in a complement-dependent cytotoxicity assay?

In a complement-dependent cytotoxicity assay, the role of complement is crucial for the detection of HLA antigens on the surface of cells. When cells are typed for HLA antigens, specific antibodies bind to the corresponding antigens present on the cell surface. The binding of these antibodies activates the complement system, leading to cell lysis if the antigen-antibody reaction is correct.

If cells type negative for all HLA antigens, it could indicate a failure in the assay process, and one likely cause would be that the rabbit complement is inactivated. Complement can become inactivated due to improper storage, exposure to certain temperatures, or pH changes. If the complement is not functional, it cannot facilitate the lysis of cells that have bound antibodies, resulting in all cells appearing negative in the assay regardless of whether HLA antigens are present.

The other options are less plausible in this context. For example, adding too much supravital dye may not directly affect the binding of antibodies or the complement activity; dead leukocytes would not produce a negative typing result if they were indeed expressing HLA antigens before death; and an overly concentrated antisera may not necessarily result in a negative typing outcome, particularly if proper controls are in place