Test ID: TBBS
T- and B-Cell Quantitation by Flow Cytometry

Serial monitoring of CD4 T-cell count in HIV-positive patients

Follow-up and diagnostic evaluation of primary immunodeficiencies, including severe combined immunodeficiency (SCID)

Immune monitoring following immunosuppressive therapy for transplantation, autoimmunity, and other immunological conditions where such treatment is utilized

Assessment of immune reconstitution posthematopoietic cell transplantation

Early screening of gross quantitative anomalies in lymphocyte subsets in infection or malignancies

Absolute quantitation of circulating B cells for diagnosis of CLL patients as indicated in the 2008 IWCLL guidelines

Flow Cytometry

Specimen must arrive within 48 hours of draw. Draw and package specimen as close to shipping time as possible.

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions: Send specimen in original tube. Do not aliquot.

Additional Information:

1. Date of draw is required.

2. For serial monitoring, we recommend that specimen draws be performed at the same time of day.

Forms: If not ordering electronically, submit a General Request Form (Supply T239) with the specimen.

Lymphocytes in peripheral blood (circulation) are heterogeneous and can be broadly classified into T cells, B cells, and natural killer (NK) cells. There are various subsets of each of these individual populations with specific cell-surface markers and function. This assay provides absolute (cells/mcL) and relative (%) quantitation for the main categories of T cells, B cells, and NK cells, in addition to a total lymphocyte count (CD45+). Each of these lymphocyte subpopulations have distinct effector and regulatory functions and are maintained in homeostasis under normal physiological conditions. Each of these lymphocyte subsets can be identified by a combination of one or more cell surface markers. The CD3 antigen is a pan-T cell marker, and T cells can be further divided into 2 broad categories, based on the expression of CD4 or CD8 coreceptors. B cells can be identified by expression of CD19, while NK cells are typically identified by the coexpression of CD16 and CD56.  

The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 am and noon with no change between noon and afternoon. NK-cell counts, on the other hand, are constant throughout the day.(1) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(2-4) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(2) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared to the evening (5) and during summer compared to winter.(6) These data therefore indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

Abnormalities in the number and percent of T (CD3), T-helper (CD4), T-suppressor (CD8), B (CD19), and NK (CD16+CD56) lymphocytes have been described in a number of different disease conditions. In patients who are infected with HIV, the CD4 count is measured for AIDS diagnosis and for initiation of antiviral therapy. The progressive loss of CD4 T-lymphocytes inpatients infected with HIV is associated with increased infections and complications. The Public Health Service has recommended that all HIV-positive patients be tested every 3 to 6 months for the level of CD4 T lymphocytes.

Lymphocyte subset quantitation is also very useful in the evaluation of patients with primary immunodeficiencies of all ages, including follow-up for newborn screening for severe combined immunodeficiency (SCID) and immune monitoring following immunosuppressive therapy for transplantation, autoimmunity, or any other relevant clinical condition where immunomodulatory treatment is used.

It is also helpful as a preliminary screening assay for gross quantitative anomalies in any lymphocyte subset, whether related to malignancies or infection.

The 2008 guidelines for diagnosis and treatment of Chronic Lymphocytic Leukemia (CLL) from the International Workshop on Chronic Lymphocytic Leukemia (7) recommends changing the diagnostic criteria for CLL from an absolute lymphocyte count (ALC) greater than 5 x 10(9)/L to a circulating B-cell count >5 x 10(9)/L (8,9) previously defined in the 1996 National Cancer Institute (NCI) guidelines for CLL. This flow cytometric assay enables accurate quantitation of circulating B cells using a single platform technology with absolute quantitation through the use of flow cytometry beads.

The appropriate age-related reference values will be provided on the report.

When the CD4 count falls below 500 cells/mcL, HIV-positive patients can be diagnosed with AIDS and can receive antiretroviral therapy.

When the CD4 count falls below 200 cells/mcL, prophylaxis against Pneumocystis jiroveci pneumonia is recommended.

Lymphocyte subset counts should be appropriately interpreted in context of the clinical presentation and other immunological parameters and relevant laboratory test results.

For serial monitoring of lymphocyte subsets it is recommended that the patient be evaluated at the same time of the day to account for diurnal variation.

For follow-up of infants identified by newborn screening (NBS) for severe combined immunodeficiency (SCID) and severe T-cell lymphopenia, SCID should be considered as a potential diagnosis in infants with <300 autologous CD3 T cells/mcL. Infants with 300 to 1500 autologous CD3 T cells/mcL may have leaky SCID, Omenn syndrome, or variant SCID, depending on other clinical and molecular features. 

T-cell lymphopenia in infants identified by newborn screening for SCID is defined as autologous CD3T cells < or =1500 cells/mcL.

While this assay can be used to follow patients on B-cell-depleting therapy, like Rituximab or Ofatumumab, it may be more reasonable and financially viable to use CD20B/89584 CD20 on B Cells (includes CD45, CD19 and CD20 markers).

This assay should not used for diagnosing lymphocytic malignancies or evaluation of lymphocytosis of unknown etiology, though the latter may be identified through this assay in a screening assessment. In such cases, LCMS/3287 Leukemia Immunophenotyping by Flow Cytometry, Peripheral Blood or Bone Marrow will be recommended, which includes a hematopathology review. However, this assay can be used for absolute quantitation of B cells in CLL patients as indicated above.

1. Carmichael KF, Abayomi A: Analysis of diurnal variation of lymphocyte subsets in healthy subjects and its implication in HIV monitoring and treatment. 15th Intl Conference on AIDS, Bangkok, Thailand, 2004, Abstract B11052

2. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. 2009 May 21;113(21):5134-5143

3. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411

4. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Pyschosom Med 1997;59:42-50

5. Malone JL, Simms TE, Gray GC, et al: Sources of variability in repeated T-helper lymphocyte counts from HIV 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J AIDS 1990;3:144-151

6. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516

7. Hallek M, Cheson BD, Catovsky D, et al: Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on CLL updating the National Cancer Institute Working Group 1996 guidelines. Blood 2008; 111:5446-5456

8. Hanson CA, Kurtin PJ, Dogan A: The proposed diagnostic criteria change for chronic lymphocytic leukemia: unintended consequences? Blood 2009;113:6495-6496

9. Hillmen P, Cheson BD, Catovsky D, et al: Letter to Editor. Blood 2009;113:6497-6498

10. U.S. Department of Health and Human Services. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Available at URL: http://aidsinfo.nih.gov/guidelines

11. Thompson MA, Aberg JA, Hoy JF, et al: Antiretroviral treatment of adult HIV infection: 2012 recommendations of the International Antiviral Society –USA panel. JAMA 2012; 308:387-402

The T- and B-cell surface marker assay uses monoclonal antibodies to identify the various membrane antigens, and flow cytometry to enumerate the number of cells expressing these differentiation antigens. CD14 is used to exclude monocytes, thereby improving accuracy and enhancing the purity of the lymphocyte population. The results are reported as the percent of lymphocytes that are T cells (CD3+), T-helper (CD3+, CD4+), T-suppressor(CD3+,CD8+), natural killer (CD16+CD56+, CD3-), and B lymphocytes(CD19+), and the absolute number of each cell type per microliter of blood. The assay is a 7-color, no-wash procedure and the absolute counts are calculated from internal bead standards. The total CD45+ lymphocyte count (reported as thousand cells per microliter) and the CD4:CD8 ratio is also reported.(Hoffman RA, Kung PC, Hansen WP, Goedstien G: Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc Natl Acad Sci USA 1980;77:4914-4917; US Department of Health and Human Services: Guidelines for performance of CD4+ T-cell determinations in persons with human immunodeficiency virus infection. MMWR 46 no. RR-2: 1997, pp 1-29)

Monday through Sunday

86355-B cells, total count

86357-Natural killer (NK) cells, total count

86359-T cells, total count

86360-Absolute CD4/CD8 count with ratio