Check for false positives by running a media negative control.
Dust or microbes have entered your sample.
Keep reagents as sterile and clean as possible.
Ensure your cell culture technique is aseptic.
Reagents can be filtered using a low protein binding syringe 0.2 µm pore size.
Cells left on the membrane will give irregular-shaped spots.
Ensure all the cells are washed from the membrane with PBS Tween 20 before secondary antibody incubation.
PBMC preparation needs to be efficient to prevent contamination.
PBMC preparation needs to be efficient. Wash the plate well after cell culture stage.
Mitogens and other factors in the serum may be stimulating the cells.
Heat inactivate the serum.
Also see “Poorly defined spots” regarding plate movement
Secondary antibodies have aggregated.
Any secondaries used in your experiment can aggregate if stored improperly.
Filter the secondary antibody.