Flow cytometry standarizationand validation for immunohematology in cattle

Abstract

Flow cytometry has become the method of choice for studying the immune system and its response in mammals, including cattle. The development of a broad panel of precise monoclonal antibodies (MAb) to bovine leukocyte differentiation molecules now enables a detailed characterization of the immune response in cattle. However, before extending these studies, it is essential to ensure the consistency of laboratory results. Currently, indirect labeling is used in single and multiparameter analysis. This technique introduces a probability of experimental error due to the multiple steps involved in processing peripheral blood mononuclear cells (PBMC). The objective of the chapter is to describe the development and testing strategy for ensuring the reliability of dual-immunolabeling methods for PBMC. We detail the experiments used to define optimal secondary antibody dilutions; set up autofluorescence, nonspecific binding, and gating controls, implement gating strategies, and measure intra-laboratory consistency. Our results show that variation between paired samples was very low, confirming that reliable data can be obtained with careful sample preparation. We propose that incorporating statistical comparisons of repeated immunolabeling of the same samples using the same MAb can serve as an effective strategy for controlling experimental error in routine dual indirect flow cytometry. Defining controls and dilutions, measuring the variation between laboratory results, and controlling experimental error are key steps in the validation process.