Lymphocyte enumeration: a comparison between a modified avidin-biotin-immunoperoxidase system and flow cytometry

Area of Science:

• Immunology
• Cell Biology

Background:

• Flow cytometry (FC) is a standard method for enumerating lymphocyte subpopulations.
• The avidin-biotin-peroxidase (ABC) procedure is an alternative immunodetection method.

Purpose of the Study:

• To compare the reliability and sensitivity of the indirect avidin-biotin-peroxidase (ABC) procedure against flow cytometry (FC) for enumerating lymphocyte subpopulations.
• To evaluate the ABC technique's potential as a cost-effective and simpler alternative to FC.

Main Methods:

• Lymphocyte subpopulations were enumerated using both conventional flow cytometry (FC) and an indirect avidin-biotin-peroxidase (ABC) procedure.
• Two counting methods were employed: comparing immunostained cells to total lymphocytes (Method I) and comparing immunostained cells to total cells (Method II).

Main Results:

• The ABC procedure detected a greater proportion of immunostained lymphocytes than FC across all subsets tested using Method I.
• Using Method II, ABC detected more total T cells than FC, with similar results for T helper and T suppressor cells.
• The ABC technique demonstrated comparable or superior sensitivity to FC for enumerating lymphocyte subsets.

Conclusions:

• The indirect avidin-biotin-peroxidase (ABC) technique is a valid and sensitive method for enumerating T lymphocyte subsets.
• The ABC method offers advantages over FC, including reduced cost, simplicity, reduced cell requirement, and a permanent staining record.
• The ABC technique is well-suited for widespread application in identifying lymphocyte membrane antigens.