Test ID: LPMAF
Lymphocyte Proliferation Panel for Mitogens and Antigens

Evaluating patients suspected of having diminished cellular immune function

Evaluating patients with primary and secondary immunodeficiency diseases that affect T lymphocytes, including combined immunodeficiency diseases (eg, severe combined immunodeficiency, cellular immunodeficiency diseases, and some patients with humoral immunodeficiency diseases (eg, common variable immunodeficiency)

Evaluating functional T-cell recovery post-hematopoietic stem cell transplant or immunosuppressive therapy for solid-organ transplantation or in other clinical contexts

Flow Cytometry

Specimen must arrive within 24 hours of draw and by 10 a.m. on Friday. Send specimen Sunday through Thursday only. Draw and package specimen as close to shipping time as possible.

Container/Tube: Green top (sodium heparin)

Specimen Volume:

<3 months: 3 mL

3-24 months: 5 mL

25 months-5 years: 6 mL

6-18 years: 8 mL

>18 years: 30 mL

Collection Instructions:

1. Send specimen in original tube. Do not aliquot.

2. Ship specimen overnight in an Ambient Mailer-Critical Specimens Only (Supply T668).

Additional Information:

1. Date and time of draw and ordering physician name and phone number are required.

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

Several classes of ligands are capable of inducing blastogenesis and stimulating proliferation of lymphocytes in vitro, including plant mitogens (phytohemagglutinin [PHA], pokeweed [PWM], and concanavalin A [Con A]), bacterial products and superantigens (potent bacterial toxins that at low concentrations have the ability to activate large numbers of T cells), and phorbol esters. Cellular proliferation follows a complex series of signals that begins with engagement of lymphocyte surface receptors by a mitogenic or antigenic ligand. Subsequent signals, including gene activation and secretion of cytokines, result in synthesis of DNA and cell division. Measurement of mitogen-induced lymphocyte proliferation in vitro provides a semiquantitative assessment of total cell-mediated immunity.(1)

The proliferative responses to PHA and Con A involve T lymphocytes, and the response to PWM involves both T and B lymphocytes in a T-dependent manner. Diminished proliferative responses to lectin mitogens occur in a variety of primary and secondary immunodeficiency diseases including diseases that affect T lymphocytes, B lymphocytes, and T and B lymphocytes.(2)

Specific antigen recognition involves T-cell receptor recognition of specific peptide in the context of the appropriate MHC molecule on an antigen-presenting cell. T cells activate and proliferate in response to specific antigenic stimulus. The recall antigens (eg, Candida albicans and tetanus toxoid) are used to assess antigen-specific T-cell responses.

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 a.m. and noon, with no change between noon and afternoon. Natural killer (NK) cell counts, on the other hand, are constant throughout the day.(3) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(4-6) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(4) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening (7), and during summer compared to winter.(8) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

LYMPHOCYTE PROLIFERATION TO ANTIGENS

Viability of lymphocytes at day 0: > or =75.0%

Maximum proliferation of Candida albicans as % CD45: > or =5.7%

Maximum proliferation of Candida albicans as % CD3: > or =3.0%

Maximum proliferation of tetanus toxoid as % CD45: > or =5.2%

Maximum proliferation of tetanus toxoid as % CD3: > or =3.3%

LYMPHOCYTE PROLIFERATION TO MITOGENS

Viability of lymphocytes at day 0: > or =75.0%

Maximum proliferation of phytohemagglutinin as % CD45: > or =49.9%

Maximum proliferation of phytohemagglutinin as % CD3: > or =58.5%

Maximum proliferation of pokeweed mitogen as % CD45: > or =4.5%

Maximum proliferation of pokeweed mitogen as % CD3: > or =3.5%

Maximum proliferation of pokeweed mitogen as % CD19: > or =3.9%

Diminished responses to lectin mitogens and/or antigens may be consistent with a primary or secondary immunodeficiency disease. Abnormal results are not specific for a particular disease, and the magnitude of the abnormality is not necessarily related to the degree of immunodeficiency. In the case of antigen-specific proliferative responses, it is possible to have low or absent responses if a long interval has passed since the original or booster vaccination (tetanus toxoid).

The range of proliferative responses observed in healthy immunologically competent individuals is large.

During T-cell reconstitution post-hematopoietic stem cell transplant or immunosuppression, functional T-cell recovery is likely to develop gradually. Therefore, in the case of mitogenic responses, it may be more relevant to assess the patient's individual data rather than use the reported comparison to the normal healthy control.

Lymphocyte proliferation to lectin mitogens and antigens can be affected by alcohol, numerous therapeutic drugs (eg, anxiety, bereavement, and depression), and acute illnesses (eg, acute viral infections).

Specimens >24 hours old may give spurious results.

Diminished results may be obtained in cultures that contain excess neutrophils or nonviable cells.(1)

Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See data under "Clinical Information."

1. Fletcher MA, Urban RG, Asthana D, et al: Lymphocyte proliferation. In Manual of Clinical Laboratory Immunology. 5th edition. Edited by NR Rose, EC de Macario, JD Folds, et al. Washington DC. ASM Press, 1997, pp 313-319

2. Bonilla FA, Bernstein IL, Khan DA, et al: Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol 2005;94:S1-S63

3. 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

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

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

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

7. 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

8. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-551

Monday through Friday

Do not send specimen after Thursday.

86353 x 2