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Test ID: LPA3P Lymphocyte Proliferation to Anti-CD3/Anti-CD28 and Anti-CD3/Interleukin-2 (IL-2) by Flow Cytometry, Blood

Reporting Name

Lymphocyte Proliferation, aCD3

Useful For

A second-level test after lymphocyte proliferation to mitogens (specifically phytohemagglutinin) has been assessed. The anti-CD3 proliferation panel is not a first-level test for assessing lymphocyte (T-cell) function


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


Evaluation of T-cell function in patients receiving immunosuppressive or immunomodulatory therapy


Evaluation of T-cell function in the context of identifying neutralizing antibodies in patients receiving therapeutic anti-CD3 antibody immunosuppression for solid organ transplantation or autoimmune diseases, such as type 1 diabetes

Clinical Information

T-cell stimulation in vitro is used extensively in the diagnostic immunology arena for facilitating T-cell proliferation and evaluation of T-cell function in a variety of clinical contexts.(1,2) The widely used method for assessing lymphocyte proliferation has 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; and 3) it does not provide any information on the contribution of apoptosis or 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, decrease in proliferation of only a subset of T cells, or an apparent decrease in total lymphocyte proliferation due to T-cell lymphopenia and underrepresentation of T cells in the peripheral blood mononuclear cell 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 sample.


While mitogens such as phytohemagglutinin (PHA) activate T cells by binding to cell membrane glycoproteins, including the T-cell receptor (TCR)-CD3 complex, there are a number of mitogenic or comitogenic antibodies, including those directed against the CD3 coreceptor that can stimulate T-cell proliferation. Typically, anti-CD3 antibodies provide an initial activation signal, but do not induce significant proliferation, and the addition of a costimulatory antibody (anti-CD28) provides the stimulus for robust proliferation.(3) An exogenous T-cell growth factor, such as interleukin-2 (IL-2), may also be used as an alternate to anti-CD28 costimulation, and in patients with suspected IL-2 receptor-associated signaling defects, it may be more helpful than the use of anti-CD28. IL-2, an autocrine cytokine, has been demonstrated to be critical in T-cell proliferation.(4,5) The interaction of IL-2 with the IL-2 receptor (IL-2R) plays a central role in regulation of T-cell proliferation.(4) Triggering of the TCR leads to synthesis of IL-2 in certain T-cell subsets and induction of high-affinity IL-2Rs in antigen- or mitogen-activated T cells, and the binding of IL-2 to IL-2R ultimately leads to T-cell proliferation. The use of exogenous IL-2 in association with anti-CD3 allows discrimination of whether T cells, which cannot proliferate to other mitogenic signals, can respond to a potent growth factor such as IL-2. Stimulation of T cells with soluble antibodies to anti-CD3 (and the associated TCR complex) causes mobilization of cytoplasmic calcium and translocation of protein kinase C from the cytoplasm to the cell membrane. This stimulation also causes induction of phosphatidylinositol metabolism and subsequent IL-2 production for proliferation.(6) T-cell activation induced by anti-CD3 antibody requires prolonged stimulation of protein kinase C, which apparently can be achieved by the concomitant use of the anti-CD28 antibody for costimulation without addition of other mitogenic stimuli, such as phorbol myristate acetate (PMA).(7)


This assay uses 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. The Click-iT-EdU assay has been shown to be an acceptable alternative to the 3H-thymidine assay for measuring lymphocyte/T-cell proliferation.(8)


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.(9) 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-aminoactinomycin D (7-AAD) and annexin V.


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, is 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, the 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.


Abnormal test results to anti-CD3/aCD28/interleukin-2 (IL-2) 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 underrepresentation of T cells within the peripheral blood mononuclear cells (PBMC) 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. The evaluation of T-cell proliferation to anti-CD3/IL-2 is likely to be helpful in assessing T-cell function in patients with refractory responses to other mitogenic and antigenic stimuli, specifically in the context of IL-2-receptor signaling defects, enabling greater mechanistic insight into the origins of T-cell dysfunction.


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 any mitogenic stimulus, including anti-CD3/anti-CD28, 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 identification 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.(10) In our evaluation of 43 pediatric samples (of all ages) with adult normal controls, only 21% and 14% were below the tenth percentile of the adult reference range for the mitogens, pokeweed mitogen (PWM) and phytohemagglutinin (PHA), respectively. Comparisons between pediatric and adult data have not been performed for anti-CD3/aCD28 due to unavailability of prospective blood samples from healthy or patient pediatric donors for purposes of analytical validation.


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 (including anti-CD3 stimulation) and antigens are significantly affected by time elapsed since blood collection. Results have been shown to be variable for specimens assessed greater than 24 and less than 48 hours post blood collection; therefore, lymphocyte proliferation results must be interpreted with due caution and results should be correlated with clinical context.

Testing Algorithm

Testing with 2 stimulants will always be performed. When adequate specimen is available, a third stimulant will be performed and billed separately. If isolated patient’s peripheral blood mononuclear cells (PBMCs) have a low WBC, selected dilutions or stimulants may not be used at the discretion of the laboratory to ensure the most reliable results.

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
ADSTM Additional Flow Stimulant No, (Bill Only) No

Report Available

5 to 8 days

Day(s) Performed

Monday through Friday

Specimens are required to be received in the lab weekdays and by 4 p.m. on Friday. No weekend processing.

Clinical Reference

1. Dupont B, Good RA: Lymphocyte transformation in vitro in patients with immunodeficiency diseases: use in diagnosis, histocompatability 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. Frauwirth KA, Thompson CB: Activation and inhibition of lymphocytes by costimulation. J Clin Invest 2002;109:295-299

4. Smith KA, Gillis S, Baker PE, et al: T-cell growth factor-mediated T cell proliferation. Ann NY Acad Sci 1979;332:423

5. Cantrell DA, Smith KA: The interleukin-2 T cell system: a new cell growth model. Science 1984;224:1212

6. Ledbetter JA, Gentry LE, June CH, et al: Stimulation of T cells through the CD3/T cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway. Mol Cell Biol 1987;7(2):650-656

7. Davis LS, Lipsky PE: T cell activation induced by anti-CD3 antibodies requires prolonged stimulation of protein kinase C. Cell Immunol 1989;118:208-221

8. 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

9. 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

10. 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

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

Method Name

Flow Cytometry

Specimen Type

WB Sodium Heparin

Shipping Instructions

Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. Draw and package specimen as close to shipping time as possible. Ship specimen overnight in an Ambient Shipping Box-Critical Specimens Only (T668) following the instructions in the box.


It is recommended that specimens arrive within 24 hours of draw.


Samples arriving on the weekend and observed holidays may be canceled.

Necessary Information

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

Specimen Required

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


Supplies: Ambient Shipping Box-Critical Specimens Only (T668)

Container/Tube: Green top (sodium heparin)

Specimen Volume:

<3 months: 1 mL

3 months-5 years: 3 mL

6-18 years: 5 mL

>18 years: 20 mL

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


Blood Volume Recommendations Based on Absolute Lymphocyte Count (ALC)



Blood Volume for Minimum aCD28 Only

Blood Volume for Minimum of aCD3, aCD28, and IL-2

Blood Volume for Full Assay


>15 cc

>28 cc

>50 cc


15 cc

28 cc

50 cc


6.5 cc

12 cc

24 cc


4.5 cc

8.5 cc

16 cc


3.5 cc

6.5 cc

12 cc


2.5 cc

4.5 cc

8 cc


1.8 cc

3.5 cc

6 cc


1.5 cc

2.5 cc

5 cc

Specimen Minimum Volume

1 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
WB Sodium Heparin Ambient 48 hours GREEN TOP/HEP

Reference Values

Viability of lymphocytes at day 0: ≥75.0%

Maximum proliferation of anti-CD3 as % CD45: ≥19.4%

Maximum proliferation of anti-CD3 as % CD3: ≥20.3%

Maximum proliferation of anti-CD3 + anti-CD28 as % CD45: ≥37.5%

Maximum proliferation of anti-CD3 + anti-CD28 as % CD3: ≥44.6%

Maximum proliferation of anti-CD3 + IL-2 as % CD45: ≥41.7%

Maximum proliferation of anti-CD3 + IL-2 as % CD3: ≥46.2%

Test Classification

This test was developed using an analyte specific reagent. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

86353 x 1-Anti-CD3 + anti-CD28 stimulation

86353 x 1-Anti-CD3 + IL2 stimulation

86353 x 1-Anti-CD3 stimulation (as indicated)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
LPA3P Lymphocyte Proliferation, aCD3 59063-8


Result ID Test Result Name Result LOINC Value
35203 Viab of Lymphs at Day 0 33193-4
35171 Max Prolif, soluble aCD3 as % CD45 81760-1
35172 Max Prolif, soluble aCD3 as % CD3 81756-9
35173 Max Prolif, soluble aCD28 as % CD45 81759-3
35174 Max Prolif, soluble aCD28 as % CD3 81758-5
35176 Max Prolif, soluble IL2 as % CD45 81755-1
35177 Max Prolif, soluble IL2 as % CD3 81757-7
35205 Interpretation 69965-2
35204 aCD3 Comment 48767-8
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