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Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates.

Robert G Caldwell1, Peggy Marshall1, Jared Fishel1

  • 1a Covance Laboratories , Madison , WI , USA.

Journal of Immunotoxicology
|January 21, 2015
PubMed
Summary

A new assay accurately measures key immune cells (T-lymphocytes, B-lymphocytes, NK cells) in non-human primates. This validated method provides essential reference ranges for cynomolgus monkeys, rhesus monkeys, and baboons.

Keywords:
Immunophenotypingcynomolgusflow cytometrylymphocytesnon-human primateperipheral bloodreference rangevalidation

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Area of Science:

  • * Immunology
  • * Flow Cytometry
  • * Non-human Primate Research

Background:

  • * Accurate enumeration of lymphocyte subsets is crucial for preclinical research and drug development.
  • * Existing methods for non-human primates may lack comprehensive validation or species-specific reference ranges.
  • * Development of a standardized immunophenotyping assay is needed for consistent results across studies.

Purpose of the Study:

  • * To develop and validate a robust peripheral blood immunophenotyping assay for cynomolgus monkeys.
  • * To establish reference ranges for lymphocyte subsets in cynomolgus monkeys, rhesus monkeys, and baboons.
  • * To provide longitudinal immunophenotyping data for cynomolgus monkeys over one year.

Main Methods:

  • * Development of a heterogeneous lymphocyte gating strategy using CD45 staining and side-scatter.
  • * Utilization of antigen-specific gating for relative lymphocyte subset determination.
  • * Application of a dual-platform methodology combining flow cytometry and hematology analyses for absolute subset concentrations.

Main Results:

  • * Successful validation of the immunophenotyping assay across key parameters including precision, linearity, and sample stability.
  • * Establishment of reference ranges for total T-lymphocytes, helper T-lymphocytes, cytotoxic T-lymphocytes, B-lymphocytes, and natural killer cells in multiple primate species.
  • * Presentation of 1-year longitudinal immunophenotyping data for cynomolgus monkeys, demonstrating assay reliability over time.

Conclusions:

  • * The developed assay provides a validated and reliable method for quantifying peripheral blood lymphocyte subsets in non-human primates.
  • * The established reference ranges are essential for accurate interpretation of immunophenotyping data in research settings.
  • * This methodology supports consistent and reproducible immunological assessments across various non-human primate species.