What tests are used to assess immune cell function?

Immune Cell Function Tests

Flow cytometry is the primary method for assessing immune cell function, with specialized panels including ELISPOT, cytotoxicity assays, and intracellular cytokine staining providing comprehensive evaluation of immune cell subsets and their functionality. 1

Core Immune Cell Function Tests

Flow Cytometry-Based Tests

• Lymphocyte subset analysis: Enumerates CD4+ and CD8+ T cells, B cells, and NK cells 2, 1

  • CD4+ T-cells are identified as CD3+CD4+
  • CD8+ T-cells are identified as CD3+CD8+
  • B cells are identified as CD19+
  • NK cells are identified as CD3-(CD16 and/or CD56)+

• Functional assays:

  • ELISPOT: Identifies number of functional antigen-specific cells; can test multiple analytes simultaneously 2
  • Intracellular cytokine staining: Measures cytokine production by specific cell subsets 2
  • Cytotoxicity assays: Measures cell-mediated killing ability 2
    • Granzyme B ELISPOT
    • 51Cr release assay
    • Flow cytometry-based cytotoxicity assays (measuring target cell death)

• Multi-parameter analysis:

  • Polyfunctional T cell analysis: Measures multiple cytokines simultaneously 2
  • CD107a expression: Marker of degranulation in cytotoxic cells 2

Specialized Testing Approaches

Comprehensive Panels

• Three-color panels: Enumerate CD4+ and CD8+ T-cells, validate CD45 gate, assess tube-to-tube variability 2
• Four-color panels: Add CD45 to identify lymphocytes based on CD45 and side scatter 2
• Advanced spectral cytometry: Up to 50-color panels for comprehensive immune profiling 3

Specific Cell Function Tests

• T-cell function tests: Lymphocyte proliferation response to mitogens and antigens 1
• NK cell function: Combined assessment of cytotoxicity and phenotype 2
• B-cell function: Antibody production capacity, subset distribution 4

Quality Control Requirements

• Positive methodologic control: Use whole-blood specimen from control donor or validated commercial materials 2
• Positive control for reagents: Test labeling efficiency of new reagent lots 2
• Flow cytometer quality control: Daily verification of optical alignment 2
• Standardization: Sum of %CD3+ (T-cells), %CD19+ (B-cells), and %CD3-(CD16/CD56)+ (NK-cells) should equal lymphocyte purity ±5% 2

Practical Considerations

• Specimen handling:

  • Process within 48 hours (preferred) or maximum 72 hours after collection 2
  • Reject hemolyzed, frozen, or clotted specimens 2
  • Store stained samples in dark under refrigeration (39°-50°F) 2

• Reporting requirements:

  • Report data as percentage of total lymphocytes and absolute counts 2
  • Correct for lymphocyte purity of the gate 2
  • Report both percentages and absolute counts when available 2

Common Pitfalls and Caveats

• Target selection: Clinical efficacy correlates better with anti-tumor cell activity than with peptide-specific responses alone 2
• Assay limitations: MHC tetramer frequencies may not correlate with clinical outcomes even when IFNγ ELISPOT responses do 2
• Sample limitations: Cryopreservation can affect certain immune cell functions; fresh samples are preferred for certain assays 5
• Age considerations: B cells show significant changes in numbers and subset distribution over lifespan, requiring age-specific reference ranges 4

Flow cytometry remains the gold standard for immune cell function assessment due to its ability to analyze multiple parameters at the single-cell level while processing large numbers of cells efficiently 4. The choice of specific assays should be guided by the clinical question and suspected immune dysfunction.