Test ID: LPMGF
Lymphocyte Proliferation to Mitogens, Blood

Assessing T-cell function in patients on immunosuppressive therapy, including solid-organ transplant patients

Evaluating patients suspected of having impairment in cellular immunity

Evaluation of T-cell function in patients with primary immunodeficiencies, either cellular (DiGeorge syndrome, T-negative severe combined immunodeficiency [SCID], etc) or combined T- and B-cell immunodeficiencies (T- and B-negative SCID, Wiskott Aldrich syndrome, ataxia telangiectasia, common variable immunodeficiency, among others) where T-cell function may be impaired

Evaluation of T-cell function in patients with secondary immunodeficiency, either disease related or iatrogenic

Evaluation of recovery of T-cell function and competence following bone marrow transplantation or hematopoietic stem cell transplantation

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: 1 mL

3 months-5 years: 2 mL

6-18 years: 3 mL

>18 years: 10 mL

Collection Instructions:

1. Send specimen is 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. Specify "Mitogen" to differentiate from "Antigen" testing. This information is required.

3. If both antigens and mitogens are desired, order LPMAF/60593 Lymphocyte Proliferation Panel for Mitogens and Antigens.

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

The method of determining impaired T-cell function by culturing human peripheral blood mononuclear cells (PBMCs) in vitro with mitogenic plant lectins (mitogens) such as phytohemagglutinin (PHA) and pokeweed mitogen (PWM) has been part of the diagnostic immunology repertoire for many years.(1,2) The widely used method for assessing lymphocyte proliferation has hitherto been the measurement of 3H-thymidine incorporated into the DNA of proliferating cells. The disadvantages with the 3H-thymidine method of lymphocyte proliferation are:
1. The technique is cumbersome due to the use of radioactivity

2. It does not allow discrimination of responding cell populations in response to stimulation

3. It does not provide any information on contribution of activation-induced cell death to the interpretation of the final result

Further, decreased lymphocyte proliferation could be due to several factors, including overall diminution of T-cell proliferation or decrease in proliferation of only a subset of T cells, or an apparent decrease in total lymphocyte proliferation due to T-cell lymphopenia and under-representation of T cells in the PBMC pool. None of these can be discriminated by the thymidine uptake assay, but can be assessed by flow cytometry, which uses antibodies to identify specific responder cell populations. Cell viability can also be measured within the same assay without requiring additional cell manipulation or specimen.

Mitogens are very potent stimulators of T-cell activation and proliferation independent of their antigenic specificity.(3) It has been suggested that mitogens can induce T-cell proliferative responses even if they are incapable of responding adequately to antigenic (physiologic) stimuli. Therefore, abnormal T-cell responses to mitogens are considered a diagnostically less sensitive but more specific test of aberrant T-cell function. Lectin mitogens have been shown to bind the T-cell receptor, which is glycosylated through its carbohydrate moiety, thereby activating quiescent T cells. Mitogenic stimulation has been shown to increase intracellular calcium (CA++) in T cells, which is absolutely essential for T-cell proliferation. While PHA is a strong T-cell mitogen, PWM is a weak T-cell mitogen, but it also induces B-cell activation and proliferation as well.  

For this assay, we use a method that directly measures the S-phase proliferation of lymphocytes through the use of Click chemistry. In the Invitrogen Click-iT-EdU assay, the Click chemistry has been adapted to measure cell proliferation through direct detection of nucleotide incorporation. In the assay, an alkyne-modified nucleoside is supplied in cell-growth media for a defined time period and is incorporated within cells. The cells are subsequently fixed, permeabilized, and reacted with a dye-labeled azide, catalyzed by copper. A covalent bond is formed between the dye and the incorporated nucleotide, and the fluorescent signal is then measured by flow cytometry.(4) Specific proliferating cell populations can be visualized by the addition of cell-specific antibodies. Cell viability, apoptosis, and death can also be measured by flow cytometry using 7-AAD and Annexin V.

The Click-iT-EdU assay has already been shown to be an acceptable alternative to the 3H-thymidine assay for measuring lymphocyte/T-cell proliferation.(5)  

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. Natural killer (NK)-cell counts, on the other hand, are constant throughout the day. Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration. In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells. It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening, and during summer compared to winter. These data, therefore, indicate that timing, and consistency in timing, of blood collection is critical when serially monitoring patients for lymphocyte subsets.

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%

Abnormal test results to mitogen stimulation are indicative of impaired T-cell function if T-cell counts are normal or only modestly decreased. If there is profound T-cell lymphopenia, it must be kept in mind that there could be a "dilution" effect with under-representation of T cells within the peripheral blood mononuclear cells (PBMCs) population that could result in lower T-cell proliferative responses. However, this is not a significant concern in the flow cytometry assay, since acquisition of additional cellular events during analysis can compensate for artificial reduction in proliferation due to lower T-cell counts.

There is no absolute correlation between T-cell proliferation in vitro and a clinically significant immunodeficiency, whether primary or secondary, since T-cell proliferation in response to activation is necessary, but not sufficient, for an effective immune response. Therefore, the proliferative response to mitogens can be regarded as a more specific but less sensitive test for the diagnosis of infection susceptibility.  

It should also be kept in mind that there is no single laboratory test that can identify or define impaired cellular immunity, with the exception of an opportunistic infection.

Controls in this laboratory and most clinical laboratories are healthy adults. Since this test is used for screening and evaluating cellular immune dysfunction in infants and children, it is reasonable to question the comparability of proliferative responses between healthy infants, children, and adults. One study has reported that the highest mitogen responses are seen in newborn infants with subsequent decline to 6 months of age, and a continuing decline through adolescence to half the neonatal response.(6) In our evaluation of 43 pediatric specimens (of all ages) with adult normal controls, only 21% and 14% were below the tenth percentile of the adult reference range for pokeweed (PWM) and phytohemagglutinin (PHA), respectively. A comment will be provided in the report documenting the comparison of pediatric results with an adult reference range and correlation with clinical context for appropriate interpretation.

It should be noted that without obtaining formal pediatric reference values it remains a possibility that the response in infants and children can be underestimated. However, the practical challenges of generating a pediatric range for this assay necessitate comparison of pediatric data with adult reference values or controls.

Lymphocyte proliferation responses to mitogens and antigens are significantly affected by time elapsed since blood collection. Results have been shown to be variable for specimens assessed >24 and <48 hours postblood collection, therefore, lymphocyte proliferation results must be interpreted with due caution and results should be correlated with clinical context.

When interpreting results it should be kept in mind that the range of lymphocyte proliferative responses observed in healthy, immunologically competent individuals is large. The reference ranges provided will be helpful in ascertaining the magnitude of the normal response.

Lymphocyte proliferation to mitogens is known to be affected by concomitant use of steroids, immunosuppressive agents, including cyclosporine, tacrolimus (FK506), Cellcept (mycophenolate mofetil), immunomodulatory agents, alcohol, and physiological and social stress.

Specimens >24-hours old may give spurious results.

Diminished results may be obtained in cultures that contain excess neutrophils or nonviable cells.(7)

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

(Clinical pathologic correlative studies.)

1. Dupont B, Good RA: Lymphocyte transformation in vitro in patients with immunodeficiency diseases: use in diagnosis, histocompatibility testing and monitoring treatment. Birth Defects Orig Artic Ser 1975;11:477-485

2. Stone KD, Feldman HA, Huisman C, et al: Analysis of in vitro lymphocyte proliferation as a screening tool for cellular immunodeficiency. Clin Immunol 2009;131:41-49

3. Lis H, Sharon N: Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem Rev 1998;98:637-674

4. Salic A, Mitchison TJ: A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA 2008;105:2415-2420

5. Yu Y, Arora A, Min W, et al: EdU-Click iT flow cytometry assay as an alternative to 3H-thymidine for measuring proliferation of human and mice lymphocytes. J Allergy Clin Immunol 2009;123(2):S87

6. Hicks MJ, Jones JK, Thies AC, et al: Age-related changes in mitogen-induced lymphocyte function from birth to old age. Am J Clin Pathol 1983;80:159-163

7. 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

Peripheral blood mononuclear cells (500,000 cells/mL) in RPMI 1640 medium supplemented with L-glutamine and 5% human AB serum) are added in duplicate to 16 wells of a sterile, flat-bottom, 48-well culture plate that contains either medium plus 5% AB serum alone (unstimulated) or varying concentrations of mitogens pokeweed (PWM) (0.5, 5, and 10 micrograms/mL) and phytohemagglutinin (PHA) (0.5, 2.5, and 5 micrograms/mL). Cells are incubated for 72 hours, after which EdU (thymidine analog) is added to all wells where it becomes incorporated into the synthesizing DNA during a second incubation of 18 to 24 hours. A daily experimental normal control is included with each batch of patient samples to serve as an internal control.  

Following the second incubation, the duplicate wells are prepared separately, the first set for viability with viability stain 7-AAD, apoptosis stain Annexin V and lymphocyte marker CD45. The second set is stained for proliferation via a copper-catalyzed click chemistry reaction where the EdU, an alkyne, is covalently bonded to a fluorescent azide. Cells are also stained for the following markers: CD45+ lymphocytes, CD3+ T cells, CD69+ activated cells, and CD19+ B cells (PWM only). Results are reported for the percent viable, dead, and apoptotic lymphocytes, as well as percent proliferating cells within each group of lymphocytes, T cells, and B cells (PWM only) (Package insert: 4, Invitrogen Click-iT-EdU; unpublished Mayo method)

Monday through Friday

Do not send specimen after Thursday.

86353