Test Catalog

Test Id : LPAGF

Lymphocyte Proliferation to Antigens, Blood

Useful For
Suggests clinical disorders or settings where the test may be helpful

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

 

This test is not intended for assessment of maternal engraftment.

Reflex Tests
Lists tests that may or may not be performed, at an additional charge, depending on the result and interpretation of the initial tests.

Test Id Reporting Name Available Separately Always Performed
AGSTM Additional Flow Stimulant, LPAGF No, Bill Only No

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

If insufficient peripheral blood mononuclear cells (PBMC) are isolated from the patient's sample due to low white blood cell counts or specimen volume received, selected dilutions or stimulants may not be tested at the discretion of the laboratory to ensure the most reliable results. Testing with one stimulant will always be performed. When adequate specimen is available for both stimulants to be tested, an additional test ID will be performed at an additional charge.

Method Name
A short description of the method used to perform the test

Flow Cytometry

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

Yes

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

Lymphocyte Proliferation, Antigens

Aliases
Lists additional common names for a test, as an aid in searching

Blastogenesis Antigens

Immune Competence

Lymphocyte Blastogenesis Antigen

Lymphocyte Phytohemagglutiin

Lymphocyte Transformation

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

If insufficient peripheral blood mononuclear cells (PBMC) are isolated from the patient's sample due to low white blood cell counts or specimen volume received, selected dilutions or stimulants may not be tested at the discretion of the laboratory to ensure the most reliable results. Testing with one stimulant will always be performed. When adequate specimen is available for both stimulants to be tested, an additional test ID will be performed at an additional charge.

Specimen Type
Describes the specimen type validated for testing

WB Sodium Heparin

Ordering Guidance

This test should not be ordered for patients younger than 3 months of age unless there is a clinical history of candidiasis. For more information see Cautions. 

Shipping Instructions

Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. Collect 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 collection.

 

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

Necessary Information

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

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

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

Container/Tube: Green top (sodium heparin)

Specimen Volume:

<3 months: 1 mL

3-24 months: 3 mL

25 months-18 years: 5 mL

>18 years: 20 mL

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

Additional Information: For serial monitoring, the recommendation is to collect the specimen at the same time of day. For more information see Cautions.

 

Blood Volume Recommendations Based on Absolute Lymphocyte Count (ALC)

Antigen only

ALC x 10(9)/L

Blood volume for minimum Candida albicans (CA) and tetanus toxoid (TT) Only

Blood volume for full assay

<0.5

>18.5 mL

>40 mL

0.5-1.0

18.5 mL

40 mL

1.1-1.5

8.5 mL

20 mL

1.6-2.0

6.0 mL

12 mL

2.1-3.0

4.5 mL

10 mL

3.1-4.0

3.0 mL

6 mL

4.1-5.0

2.5 mL

5 mL

>5.0

2.0 mL

4 mL

 

Mitogen and antigen

ALC x 10(9)/L

Blood volume for minimum of each assay

Blood volume for full assay

<0.5

>28 mL

>60 mL

0.5-1.0

28 mL

60 mL

1.1-1.5

12 mL

30 mL

1.6-2.0

8.5 mL

20 mL

2.1-3.0

6.5 mL

15 mL

3.1-4.0

4.5 mL

10 mL

4.1-5.0

3.5 mL

8 mL

>5.0

2.5 mL

6 mL

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

<6 years: 1 mL

6-18 years: 2 mL

>18 years: 6 mL

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Gross hemolysis Reject
Gross lipemia Reject

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

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

Useful For
Suggests clinical disorders or settings where the test may be helpful

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

 

This test is not intended for assessment of maternal engraftment.

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

If insufficient peripheral blood mononuclear cells (PBMC) are isolated from the patient's sample due to low white blood cell counts or specimen volume received, selected dilutions or stimulants may not be tested at the discretion of the laboratory to ensure the most reliable results. Testing with one stimulant will always be performed. When adequate specimen is available for both stimulants to be tested, an additional test ID will be performed at an additional charge.

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

Determining impaired T-cell function by culturing human peripheral blood mononuclear cells (PBMC) in vitro with recall antigens, including Candida albicans (CA) and tetanus toxoid (TT), has been part of the diagnostic immunology repertoire for many years.(1,2) The widely used method for assessing lymphocyte proliferation to antigens 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 sample.

 

Antigens, like CA and TT, have been widely used to measure antigen-specific recall (anamnestic) T-cell responses when assessing cellular immunity. In fact, it may be more revealing about cellular immune compromise than assessing the response of lymphocytes to mitogens because the latter can induce T-cell proliferative responses even if those T cells are incapable of responding adequately to antigenic (physiologic) stimuli. Therefore, abnormal T-cell responses to antigens are considered a diagnostically more sensitive, but less specific, test of aberrant T-cell function.(2)

 

Antigens used in recall assays measure the ability of T cells bearing specific T-cell receptors (TCR) to respond to such antigens when processed and presented by antigen-presenting cells. The antigens used for assessment of the cellular immune response are selected to represent antigens, seen by a majority of the population, either through natural exposure (CA) or as a result of vaccination (TT).

 

This assay uses a method that directly measures the S-phase proliferation of lymphocytes through the use of Click chemistry. Cell viability, apoptosis, and death can also be measured by flow cytometry using 7-aminoactinomycin D (7-AAD) and annexin V. The Click-iT-EdU assay has shown to be an acceptable alternative to the 3H-thymidine assay for measuring lymphocyte/T-cell proliferation.(3)

 

The degree of impairment of antigen-specific T-cell responses can vary depending on the nature of the cellular immune compromise. For example, some, but not all, patients with partial DiGeorge syndrome, a primary cellular immunodeficiency, have been reported to have either decreased or absent T-cell responses to CA and TT.(4) Similarly, relative immune compromise, especially to TT, has been reported in children with vitamin A deficiency, but the measurements have been largely of the humoral immune response. Since this requires participation of the cellular immune compartment, it can be postulated that there could be a potential impairment of antigen-specific T-cell responses as well.(5)

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

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%

Interpretation
Provides information to assist in interpretation of the test results

Abnormal test results to antigen 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, there could be a dilution effect with underrepresentation of T cells within the peripheral blood mononuclear cell (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. In the case of antigen-specific T-cell responses to tetanus toxoid (TT), there can be absent responses due to natural waning of cellular immunity, if the interval between vaccinations has exceeded the recommended period, especially in adults. In such circumstances, it would be appropriate to measure TT-specific T-cell responses 4 to 6 weeks after a booster vaccination.

 

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 antigens can be regarded as a more sensitive, but less specific, test for the diagnosis of infection susceptibility.

 

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. It is reasonable to expect robust T-cell-specific responses to TT in children without cellular immune compromise, as a result of repeated childhood vaccinations. The response to Candida albicans can be more variable depending on the extent of exposure and age of exposure. 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.

 

Without obtaining formal pediatric reference values, there 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.

Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

There is no clinical utility to assessing antigen responses in infants younger than 3 months old due to limited antigen exposure and vaccination. The only exception would be infants who develop candidiasis prior to 3 months of age.

 

When interpreting results, note 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.

 

Lymphocyte proliferation responses to antigens (and mitogens) are significantly affected by time elapsed since blood collection. Results have been shown to be variable for specimens assessed between 24 and 48 hours post-blood collection. Therefore, lymphocyte proliferation results must be interpreted with due caution and results should be correlated with clinical context. Specimens more than 24-hours old may yield spurious results.

 

Diminished results may be obtained in cultures that contain excess neutrophils or nonviable cells.(6)

 

Timing, and consistency in timing, of blood collection is critical when serially monitoring patients lymphocyte subsets (specifically T cells in this context) and their diurnal variation can potentially affect the magnitude of the proliferative response, especially in patients who already have severe T-cell lymphopenia. 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. Circadian variations in circulating T-cell counts negatively correlate 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.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

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, Howlett C, Jabara HH, Bonilla FA: Analysis of in vitro lymphocyte proliferation as a screening tool for cellular immunodeficiency. Clin Immunol. 2009;131:41-49

3. Yu Y, Arora A, Min W, Roifman CM, Grunebaum E: 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

4. Davis CM, Kancheria VS, Reddy A, et al: Development of specific T cell responses to Candida and tetanus antigens in partial DiGeorge syndrome. J Allergy Clin Immunol. 2008,122:1194-1199

5. Semba RD, Muhilal, Scott AL, et al: Depressed immune response to tetanus in children with vitamin A deficiency. J Nutr. 1992;122:101-107

6. Fletcher MA, Urban RG, Asthana D, et al: Lymphocyte proliferation. In: Rose NR, de Macario EC, Folds JD, et al, eds. Manual of Clinical Laboratory Immunology. 5th ed. ASM Press; 1997:313-319

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

8. 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

Method Description
Describes how the test is performed and provides a method-specific reference

Peripheral blood mononuclear cells in RPMI 1640 medium supplemented with L-glutamine and 20% human AB serum are added to wells that contain either medium plus 20% AB serum alone (unstimulated) or varying concentrations of Candida albicans and tetanus toxoid antigens. Cells are analyzed by flow cytometry for day 0 viability as outlined below. After 6 days of incubation, EdU (thymidine analog) is added to all wells, where it becomes incorporated into the synthesizing DNA during a final 18- to 24-hour incubation period. A daily experimental normal control is included with each batch of patient samples to serve as an internal control.

 

On day 7 following the second incubation, the cells are 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, and CD69+ activated T cells. Results are reported for the percent viable cells on day 0, as well as percent proliferating cells within each group of lymphocytes and T cells.(Package insert: 4; Invitrogen Click-iT-EdU. Life Technologies; v10 11/2008; unpublished Mayo method)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Friday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

11 to 14 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

Entire specimen is used in preparation of the assay

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

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
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

86353

86353 (if appropriate)

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports