Indications for B-Cell, T-Cell, and NK-Cell Testing
Order comprehensive B-cell (CD19/CD20), T-cell (CD3, CD4, CD8), and NK-cell (CD16/CD56) panels when evaluating suspected primary immunodeficiency, monitoring HIV disease progression, assessing immune reconstitution after transplant, investigating recurrent infections, or diagnosing lymphoproliferative disorders including lymphomas and leukemias. 1
Primary Immunodeficiency Disorders
Flow cytometry lymphocyte subset analysis serves as the screening test for cellular immunity defects and should be ordered when clinical features suggest immune dysfunction. 1
Specific Clinical Scenarios:
Recurrent sinopulmonary infections with encapsulated bacteria warrant B-cell enumeration and subset analysis to detect antibody-deficiency disorders such as common variable immunodeficiency or X-linked agammaglobulinemia. 1
Infants with suspected severe combined immunodeficiency (SCID) require comprehensive quantification of T-cells, B-cells, and NK-cells, as SCID can present with various patterns of lymphocyte deficiency. 1
Chronic skin or mucosal fungal infections suggest T-cell defects; CD4+ and CD8+ enumeration should be performed to identify T-cell lymphopenia or functional defects. 1
Autoimmunity, lymphoproliferation, or hemophagocytic lymphohistiocytosis (HLH) indicate immune-dysregulation disorders (such as ALPS or familial HLH) and necessitate full lymphocyte subset analysis. 1
Chediak-Higashi syndrome presents with a profound defect in NK cell function, demonstrating the importance of NK-cell testing in patients with recurrent infections and characteristic giant granules in leukocytes. 2
Advanced B-Cell Subset Analysis:
When basic B-cell counts are abnormal, enumerate naïve and switched memory B-cell subsets to further characterize antibody-deficiency syndromes and differentiate common variable immunodeficiency subtypes from other B-cell disorders. 1
HIV Disease Monitoring
The absolute CD4+ T-cell count and CD4/CD8 ratio are the most critical parameters for HIV patients, as these directly correlate with immune function, disease progression risk, and clinical decision-making for antiretroviral therapy and opportunistic infection prophylaxis. 1
HIV-Specific Testing Requirements:
Baseline CD4 count, CD4%, CD8 count, CD8%, and CD4/CD8 ratio should be obtained at diagnosis, with CD4 count and viral load monitored every 3-6 months in all HIV-infected persons. 1
Opportunistic infection prophylaxis should be initiated when CD4 count falls below 200 cells/mm³ or CD4% below 14%, with consideration for prophylaxis against other opportunistic infections based on specific CD4 thresholds. 1
CD4 counts are measured in HIV-associated lymphomas as part of the workup, particularly for AIDS-related lymphomas where immune status affects treatment selection. 3
Lymphoproliferative Disorders and Hematologic Malignancies
Immunophenotypic analysis is essential for differentiating the various subtypes of non-Hodgkin lymphoma and can be performed using flow cytometry and/or immunohistochemistry. 3
B-Cell Neoplasms:
The NCCN guidelines provide detailed algorithms for using B-cell markers (CD19, CD20, CD79a, PAX5) in conjunction with additional markers (CD5, CD10, BCL2, BCL6, cyclin D1, Ki67) to diagnose: 3
- Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL)
- Mantle cell lymphoma (MCL)
- Follicular lymphoma (FL)
- Diffuse large B-cell lymphoma (DLBCL)
- Burkitt's lymphoma (BL)
- Marginal zone lymphomas (splenic, nodal, extranodal MALT)
- Hairy cell leukemia (HCL)
- Lymphoplasmacytic lymphoma
Flow cytometry is particularly sufficient for diagnosing CLL when a lymph node is not easily accessible, especially when combined with appropriate ancillary techniques (PCR for IGHV gene rearrangements, FISH for major translocations). 3
T-Cell and NK-Cell Neoplasms:
T-cell antigens (CD2, CD3, CD5, CD7) and NK-cell markers (CD16, CD56) are used to diagnose mature T-cell and NK-cell neoplasms, with additional markers (CD4, CD8, CD30, cytotoxic granule proteins, EBV-EBER) providing subtype classification. 3
The NCCN algorithms guide diagnosis of: 3
- Peripheral T-cell lymphoma, NOS
- Anaplastic large cell lymphoma (ALK+ and ALK-)
- Extranodal NK/T-cell lymphoma, nasal type
- Mycosis fungoides/Sézary syndrome
- Enteropathy-associated T-cell lymphoma
- Hepatosplenic T-cell lymphoma
- Primary cutaneous T-cell lymphoproliferative disorders
The typical NK-cell immunophenotype for NK/T-cell lymphomas includes CD20-, CD2+, cytoplasmic CD3ε+ (surface CD3-), CD56+, with cytotoxic granule proteins (TIA1, perforin, granzyme B) usually expressed. 4
Prognostic Value in CLL:
Higher T:MBC (T-cell to malignant B-cell) and NK:MBC ratios at diagnosis are associated with early stage disease, mutated IGHV genes, and longer time to treatment in chronic lymphocytic leukemia, suggesting that measurable characteristics of the host immune system relate to disease progression. 5
Both T:MBC ratio and NK:MBC ratio are independent predictors of time to treatment in newly diagnosed CLL patients on multivariate analysis. 5
Castleman's Disease
Laboratory evaluation for Castleman's disease should include complete blood count with differential, inflammatory markers, and HHV-8 testing, with HIV testing also recommended. 6
Lymphoma associated with Castleman's disease requires immunophenotyping as part of the diagnostic workup, particularly when lymphoproliferation develops. 3
Technical Quality Control Considerations
Monoclonal antibodies targeting CD3 (T-cells), CD19/CD20 (B-cells), and CD16/CD56 (NK-cells) must collectively account for the entire lymphocyte population in the specimen, serving as an internal quality control measure. 3, 1
All CD3 measurements within a testing set should be within 3% of each other; greater variability mandates repeat testing of the affected tube. 3, 1
Common Pitfall to Avoid:
Never use a single tube containing CD3/CD4/CD8 without additional tubes for lymphocyte-gate validation, as this prevents assessment of gate purity and recovery and is deemed inappropriate by the CDC. 1