What clinical situations prompt ordering B‑cell (CD19/CD20), T‑cell (CD3, CD4, CD8) and NK‑cell (CD16/CD56) flow cytometry tests?

Clinical Indications for B‑Cell, T‑Cell, and NK‑Cell Flow Cytometry

Order B‑cell (CD19/CD20), T‑cell (CD3, CD4, CD8), and NK‑cell (CD16/CD56) flow cytometry panels when evaluating suspected primary immunodeficiency, monitoring HIV disease progression, assessing immune reconstitution, investigating recurrent infections, or diagnosing lymphoproliferative disorders. 1

Primary Immunodeficiency Evaluation

Flow cytometry to enumerate total B cells, CD4 and CD8 T cells, and NK cells is a screening test for cellular immunity defects. 1

Specific Clinical Presentations Requiring Testing:

• Recurrent sinopulmonary infections with encapsulated bacteria suggest antibody deficiency requiring B‑cell enumeration and subset analysis 1
• Severe combined immunodeficiency (SCID) presents with severe bacterial, viral, fungal, and opportunistic infections in infancy, requiring comprehensive T‑cell, B‑cell, and NK‑cell quantification 1
• Autoimmunity, lymphoproliferation, or hemophagocytic lymphohistiocytosis (HLH) indicate immune dysregulation disorders necessitating lymphocyte subset analysis 1
• Chronic skin and mucous membrane fungal infections may reflect T‑cell defects requiring CD4 and CD8 enumeration 1

Advanced B‑Cell Assessment:

When basic B‑cell counts are abnormal, flow cytometry to enumerate B‑cell subsets (naive and switched memory cells) provides detailed characterization of antibody deficiency syndromes. 1 This is particularly useful when distinguishing between common variable immunodeficiency subtypes and other B‑cell disorders. 2

HIV Disease Monitoring

CD4+ T‑cell counts and CD4/CD8 ratios are the most critical parameters for HIV patients, directly correlating with immune function, disease progression risk, and clinical decision‑making for antiretroviral therapy and opportunistic infection prophylaxis. 3

HIV‑Specific Testing Algorithm:

• Obtain baseline CD4 count, CD4%, CD8 count, CD8%, and CD4/CD8 ratio at diagnosis 3
• Monitor CD4 count and viral load every 3–6 months in all HIV‑infected persons 3
• Initiate opportunistic infection prophylaxis when CD4 count falls below 200 cells/mm³ or CD4% below 14% 3
• CD4+ T‑cells must be identified as positive for both CD3 and CD4 to ensure accurate enumeration 1
• CD8+ T‑cells must be identified as positive for both CD3 and CD8 1

The CDC guidelines emphasize that monoclonal antibody panels must contain CD3/CD4/CD45 and CD3/CD8/CD45 combinations, with CD3 serving as a control for tube‑to‑tube variability. 1 All CD3 values should be within 3% of each other; if greater variability occurs, repeat the tube. 1

Lymphoproliferative Disorder Diagnosis

Flow cytometry plays an essential diagnostic role in T/NK‑cell lymphoproliferative disorders by identifying aberrant immunophenotypes and clonal populations. 4

NK/T‑Cell Lymphoma Immunophenotype:

The typical profile includes CD20‑, CD2+, cytoplasmic CD3ε+ (surface CD3‑), CD4‑, CD5‑, CD56+, with expression of cytotoxic granule proteins (TIA1, perforin, granzyme B). 5 This distinguishes malignant NK cells from normal NK cells, which are CD3‑negative (surface), CD56‑positive, and/or CD16‑positive. 5

Standard NK‑Cell Identification:

A minimum three‑color panel (CD3/CD16/CD56 or CD3/CD19/CD16/CD56) is required for basic NK‑cell identification, while a four‑color panel (CD45/CD3/CD19/CD16/CD56) is recommended for comprehensive lymphocyte analysis. 5, 6

Functional Assessment Indications

Functional assays (cytotoxicity, cytokine production) should be prioritized over absolute NK cell counts when monitoring patients with malignancies or EBV‑associated diseases. 6

When to Order Functional Testing:

• Reduced NK‑cell activity is associated with higher cancer incidence and faster disease progression, making functional assessment more clinically relevant than cell counts alone 6
• Flow cytometry–based assays evaluating NK cell cytotoxicity, IFN‑γ production, and activation markers (CD69, CD25) provide superior clinical decision‑making information compared with simple NK cell counts 6
• CD107a surface expression combined with Annexin V binding offers a sensitive measure of NK‑cell degranulation and cytotoxic activity 6

Technical Quality Control Requirements

Monoclonal antibodies labeling T‑cells (CD3), B‑cells (CD19/CD20), and NK‑cells (CD16/CD56) should account for all lymphocytes in the specimen as an internal quality control. 1, 5

Critical Technical Points:

• CD45/side‑scatter gates for lymphocytes must contain >95% lymphocytes to avoid contamination with monocytes or basophils 6
• NK cells exhibit bright CD45 fluorescence but slightly higher side‑scatter than other lymphocytes; careful gating is essential to capture all NK subsets 6
• Differences between replicate CD3 results should be ≤2% in three‑color panels 1
• Store stained samples in the dark at 39–50°F (4–10°C) for no longer than 24 hours unless validated for longer storage 1

Common Clinical Pitfalls

Do not rely on a single tube containing CD3/CD4/CD8 without additional tubes for lymphocyte gate validation, as this prevents assessment of gate purity and recovery. 1 The CDC explicitly states this approach is inappropriate because there is no method to validate the lymphocyte gate. 1

Age and sex significantly affect normal ranges: absolute numbers of total lymphocytes and CD3+/CD8+ T cells are higher in younger individuals (<50 years), while absolute and percentage values of CD3+/CD4+ T lymphocytes are significantly higher in women. 7 Adjust reference ranges accordingly to avoid misinterpretation.

Volatile anesthetics such as isoflurane and sevoflurane suppress NK‑cell cytotoxicity and cytokine production for several days after surgery, influencing the timing of functional assessments. 6 Avoid testing immediately post‑operatively if assessing baseline immune function.