PBMCs from the blood collected from a tuberculosis (TB) patient were given to four lab technicians to perform ELISPOT assay for interferon γ (IFNγ). While all steps recommended for ELISPOT were followed, the first step was performed differently by the four lab technicians, as detailed below.

A. Lab technician 1 coated each well with 250,000 formaldehyde-treated cells and stimulated the cells with TB-specific antigen.
B. Lab technician 2 coated each well with 250,000 cells and did not stimulate the cells with TB-specific antigen.
C. Lab technician 3 depleted T cells from PBMCs completely, coated the wells with monocyte-enriched PBMCs, and stimulated them with TB-specific antigen.
D. Lab technician 4 coated each well with 250,000 cells and stimulated the cells with TB-specific antigens.

Which of the lab technicians’ assays will yield a correct ELISPOT result for interferon γ?

Correct option is D

The correct answer is (d) Lab technician 4.

Lab technician 4 followed the correct ELISPOT assay protocol by using 250,000 Peripheral Blood Mononuclear Cells (PBMCs) and stimulating them with TB-specific antigens. This setup ensures the activation of T-cells present in PBMCs, which in turn secrete Interferon gamma (IFNγ) if they recognize the TB antigens. The presence of IFNγ spots indicates an immune response, validating the assay.

Let's evaluate the actions of other technicians:

• Technician 1 used formaldehyde-treated cells which are fixed and non-viable, thus unable to secrete IFNγ.

• Technician 2 did not stimulate with antigen, so T-cells would not get activated and hence won't release IFNγ.

• Technician 3 removed T-cells from PBMCs, leaving only monocytes which are not primary producers of IFNγ in this context.

Therefore, only technician 4 retained all components (intact PBMCs including T-cells) and stimulated with antigen—fulfilling the conditions necessary for IFNγ production and accurate ELISPOT results.