Test Catalog

Test Id : LPMGF

Lymphocyte Proliferation to Mitogens, 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

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
MGSTM Additional Flow Stimulant, LPMGF No, (Bill Only) No

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

To ensure the most reliable results, if insufficient peripheral blood mononuclear cells 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.

 

Testing with one stimulant will always be performed. When adequate specimen is available for both stimulants to be tested, the second stimulant will be evaluated 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, Mitogens

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

(PHA) Phytohemagglutinin

Blastogenesis Mitogens

Immune Competence

In vitro T-Cell Function

Lymphocyte Blastogenesis Mitogens

Lymphocyte Transformation

Mitogen Studies

Mitogens

Phytohemagglutinin (PHA)

Pokeweed (PWM)

PWM (Pokeweed)

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

To ensure the most reliable results, if insufficient peripheral blood mononuclear cells 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.

 

Testing with one stimulant will always be performed. When adequate specimen is available for both stimulants to be tested, the second stimulant will be evaluated at an additional charge.

Specimen Type
Describes the specimen type validated for testing

WB Sodium Heparin

Shipping Instructions

Specimens must 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.

 

Specimens arriving on the weekend may be canceled.

Necessary Information

1. Date and time of collection are required.

2. 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: 20 mL

See tables for information on recommended volume based on absolute lymphocyte count

Pediatric Volume:

<3 months: 1 mL

3-24 months: 3 mL

25 months-18 years: 5 mL

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

Additional Information: For serial monitoring, it is recommended that specimen collection be performed at the same time of day.

 

Table. Blood Volume Recommendations Based on Absolute Lymphocyte Count (ALC)

Mitogen only

ALC x 10(9)/L

Blood volume for minimum phytohemagglutinin (PHA) only

Blood volume for minimum PHA and pokeweed mitogen (PWM)

Blood volume for full assay

<0.5

>6.5 mL

>8.5 mL

>22 mL

0.5-1.0

6.5 mL

8.5 mL

22 mL

1.1-1.5

3.0 mL

4.0 mL

10 mL

1.6-2.0

2.0 mL

2.5 mL

7 mL

2.1-3.0

1.5 mL

2.0 mL

6 mL

3.1-4.0

1.0 mL

1.5 mL

4 mL

4.1-5.0

0.8 mL

1.0 mL

3 mL

>5.0

0.5 mL

0.8 mL

2 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

See Specimen Required

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

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

To ensure the most reliable results, if insufficient peripheral blood mononuclear cells 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.

 

Testing with one stimulant will always be performed. When adequate specimen is available for both stimulants to be tested, the second stimulant will be evaluated at an additional charge.

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

The method of determining impaired T-cell function by culturing human peripheral blood mononuclear cells (PBMC) 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 (3)H-thymidine incorporated into the DNA of proliferating cells. The disadvantages with the (3)H-thymidine method of lymphocyte proliferation are:

1. The technique is cumbersome due to the use of radioactivity.

2. It does not distinguish between different cell populations responding 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 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 distinguished 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[2+]) in T cells, which is essential for T-cell proliferation. While PHA is a strong T-cell mitogen, PWM is a weak T-cell mitogen, but it induces B-cell activation and proliferation as well.

 

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. In the assay, an alkyne-modified nucleoside is supplied in cell-growth media for a defined 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-aminoactinomycin D (7-AAD) and annexin V.

 

The Click-iT-EdU assay has shown to be an acceptable alternative to the (3)H-thymidine assay for measuring lymphocyte/T-cell proliferation.(5,6)

 

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-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(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.

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 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%

Interpretation
Provides information to assist in interpretation of the test results

Abnormal mitogen stimulation test results 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 under-representation of T cells within the peripheral blood mononuclear cell 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.

 

No single laboratory test can identify or define impaired cellular immunity on its own.

 

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.(9) In an in-house 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 and phytohemagglutinin, 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.

 

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 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.

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

When interpreting results, note that the range of lymphocyte proliferative responses observed in healthy, immunologically competent individuals is large. The reference values (based on healthy donors) provided will be helpful in ascertaining the magnitude of the patient 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 older than 24 hours may yield spurious results.

 

Diminished results may be obtained in specimens that contain excess neutrophils or nonviable cells.(10)

 

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

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 Apr;131(1):41-49. doi: 10.1016/j.clim.2008.11.003

3. Lis H, Sharon N: Lectins: Carbohydrate-specific proteins that mediate cellular recognition. Chem Rev. 1998 Apr;98(2):637-674. doi: 10.1021/cr940413g

4. Salic A, Mitchison TJ: A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA. 2008 Feb;105(7):2415-2420. doi: 10.1073/pnas.0712168105

5. 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 Feb;123(2):S87. doi: 10.1016/j.jaci.2008.12.307

6. Clarke ST, Calderon V, Bradford JA: Click chemistry for analysis of cell proliferation in flow cytometry. Curr Protoc Cytom. 2017 Oct;82:7.49.1-7.49.30. doi: 10.1002/cpcy.24

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 Jun;34(6):512-516

9. Hicks MJ, Jones JK, Thies AC, Weigle KA, Minnich LL: Age-related changes in mitogen-induced lymphocyte function from birth to old age. Am J Clin Pathol. 1983 Aug;80(2):159-163. doi: 10.1093/ajcp/80.2.159

10. 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

11. Knight V, Heimall JR, Chong H, et al: A toolkit and framework for optimal laboratory evaluation of individuals with suspected primary immunodeficiency. J Allergy Clin Immunol Pract. 2021 Sept;9(9):3293-3307.e6. doi: 10.1016/j.jaip.2021.05.004

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 5% human AB serum are incubated unstimulated or stimulated with varying concentrations of pokeweed (PWM)- and phytohemagglutinin. A daily experimental normal control is included with each batch of patient samples to serve as an internal control.

 

Following incubation, the cells are assessed for proliferation and stained with the following markers: CD45+ lymphocytes, CD3+ T cells, CD69+ activated cells, and CD19+ B cells (PWM only). Results are reported for the percent viable cells on day 0, as well as the percentage of proliferating cells of total lymphocytes, T cells, and B cells (PWM only).(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.

8 to 11 days

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

Not retained. 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)

 

LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
LPMGF Lymphocyte Proliferation, Mitogens 69018-0
Result Id Test Result Name Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
32318 Viab of Lymphs at Day 0 33193-4
32320 Max Prolif of PHA as % CD3 57741-1
32319 Max Prolif of PHA as % CD45 69038-8
32323 Max Prolif of PWM as % CD19 69037-0
32322 Max Prolif of PWM as % CD3 69020-6
32321 Max Prolif of PWM as % CD45 69019-8
32317 Interpretation 69052-9
32324 Mitogen Comment 48767-8

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