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Useful For
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LPA (Lymphocyte Proliferation Assay) assessing T-cell function in patients on a 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.
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Method name and description
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LPA (Lymphocyte Proliferation Assay) performed by flow cytometry,after 7 days of incubation activity.
This technique is based on lymphocytes capability to respond to an antigen (specific response) that has the ability to induced memory lymphocytes in vitro. It occurs only if the patient has been immunized to that antigen, either by having recovered from an infection with the micro-organism containing that antigen, or by having been vaccinated. Therefore, some normal individuals may not respond to a given antigen, but most people will respond to at least one of several common microbial antigens.
Peripheral blood mononuclear cells (PBMC’s) are separated by centrifugation on Ficol-Hypaque density gradient and labeled with a fluorescent stain (CFSE) Carboxyfluorescein succinimidyl ester that is covalently linked to interior of the cell.
Lymphocytes are set up in micro-titer plate, cultures for a period of 7 days with and without mitogen/antigen. As cells divide, the fluorescent label gets divided equally between the daughter cells, halving their fluorescence.
Mitogens used for this assay are Phytohemagglutinin (PHA), Concanavalin A (Con A) which stimulates T lymphocytes, and Pokeweed mitogen (PWM), which stimulate both lymphoid populations.
The proliferation is evaluated by flow cytometry, using 4-color experiment:
1.CD45 APC H7 to label CD45
2.CD3 APC to label CD3
3. CFSE (FITC) to label PBMC’s.4 Cell Viability Solution (perCP) for cell viability.
A proliferation index% is calculated based on stimulation of the generation.
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Reporting name
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LPA-PHA
LPA-Con A
LPA-Pok
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Clinical information
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This method to determin the impaired T-cell function by culturing human peripheral blood mononuclear cells (PBMC) in vitro with mitogenic plant lectins (mitogens) such as phytohemagglutinin (PHA),Concanavalin A (Con A) and pokeweed mitogen (PWM) and antigens including, Candida Albicans, PPD, anti-CD3 antibody and Diptheria Toxoid/Tetanus Toxoid.Viability of cells measured within the same assay.
In this method,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.
In case of Insufficient peripheral blood mononuclear cells (PBMCs) are isolated from the patient's sample due to low WBC counts or specimen volume received, some stimulants may not be tested at the discretion of the laboratory to ensure the most reliable results.
Mitogens are very potent stimulators of T-cell activation and proliferation independent of their antigenic specificity. 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.
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Aliases
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LPA (Lymphocyte Proliferation Assay)
Lymphocyte Phytohemagglutiin (PHA)
Lymphocyt Function Test
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Specimen type / Specimen volume / Specimen container
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Specimen type: A fresh whole blood sample.
Container Tube and Specimen Volume:
- Heparin Tube (Lithium or Sodium) 5 mL,Whole Blood (Minimum: 3ml)
- or ACD (Acid Citrate Dextrose) tube of 6 ml.
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Collection instructions / Special Precautions / Timing of collection
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Pre-arrangement with the laboratory (4439-8857) Sunday thruThursday 7:00am-3:00PM.
Specimens should be processed within 24 hours of sampling. Transport Immediately after collection in room temperature.
Date and time of collection is required.
A fresh Two-three controls blood, from a normal healthy subject Mother, Father or Donor is required.
Do not use blood anticoagulant EDTA or citric acid.
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Relevant clinical information to be provided
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Sufficient clinical information relevant to the requested test should be provided
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Storage and transport instructions
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The specimen should be sent to the laboratory as soon as possbile after collection.
The sample should be stored at room temperature within 24 hours of blood collection.
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Specimen Rejection Criteria
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Insufficient quantity of blood.
Clotted
Wrong collection tube or anticoagulant.
Outdated specimens.
Improper sample storage.
Samples Collected without pre-arrangement with the laboratory will be rejected.
Insufficient peripheral blood mononuclear cells (PBMCs) are isolated from the patient's sample due to low WBC counts or specimen volume received,
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Biological reference intervals and clinical decision values
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| Results are reported as response to antigen or mitogen as: |
| Interpretation |
Patient Stimulation Index. |
| Normal |
≥ 75 % |
| Mild Reduction |
50 -75 % |
| Moderate Reduction |
25 – 50 % |
| Severe Reduction |
≤ 25 % |
All datas and gating will be reviewed and evaluated by the Laboratory Director and or designee.
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Factors affecting test performance and result interpretation
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Insufficient peripheral blood mononuclear cells (PBMCs) are isolated from the patient's sample due to low WBC counts or specimen volume received.
control values are outside the expected range.
Incubation conditions (e.g., temperature, CO2 level, humidity,contamination).
Event that the test system is inoperable due to assay problems.
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Turnaround time / Days and times test performed / Specimen retention time
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Turnaround time: 10 working day.
Before sample collection the test should have prior approval by the Lab Head of Section and should be pre-arrange with the laboratory. Immunology Flow Cytometry (4439-8857 or Immunology main laboratory. Sunday thru Thursday 7:00am-3:00PM.
Entire specimen is used in preparation of the assay.
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