WHOLE BLOOD LYMPHOCYTE PROLIFERATION ASSAY

Monday, May 10, 2004

Description
WHOLE BLOOD LYMPHOCYTE PROLIFERATION ASSAY

Procedure
PRINCIPAL AND/OR PURPOSE OF THE TEST
Lymphocyte proliferation assays (LPA) have been widely used to help
describe the abnormalities associated with diverse congenital
immunodeficiencies as well as those considered to be secondary to
infectious diseases, cancer, aging, malnutrition, substance abuse, stress,
surgery, shock, and autoimmune diseases. In the context of the research
agenda of the AIDS clinical trial group (ACTG), the whole blood (WB) LPA
provides an appropriate and practical means of assessing and monitoring
cellular immune status in drug trials, and in measuring specific response
to antigens in vaccine trials, and is one which requires realitively
small amounts of peripheral blood. This attribute is of particular value
for pediatric drug trials. The WB assay is preferred if it is desirable
to determine the proliferation of the blood cells without removing or
washing away the soluble substances in the whole blood which may influence
function of the cells (ie., hormones, cytokines, neuropeptides). The WB
assay described relies upon the incorporation of a radioactive nucleoside
([ H]-thymidine) into cellular DNA. This system makes use of the fact 3
that activated lymphocytes are inclined to divide and that cellular
division requires DNA synthesis. If provided with an exogenous source of
nucleosides, lymphocytes will transport these molecules into the cell,
charge them with triphosphates, and subsequently incorporate them into DNA
during chromsomal replication. At the biochemical level it is the
conversion of soluble nucleosides into insoluble DNA polymers upon which
the assay is based. Following incubation with labeled nucleoside,
lymphocytes are harvested onto glass fiber filters. Nonincorporated
nucleoside is washed away, whereas the label incorporated into DNA remains
attached to the solid support. The amount of radionuclide present is then
determined by liquid scintillation counting.
2. SPECIMEN REQUIRED AND COLLECTION METHOD, ANTICOAGULATES, ANY SPECIAL
PATIENT PREPARATION OR RESTRICTIONS
2.01 Heparinized Whole Blood (green top) is the required anticoagulant
for LPA.
2.02 Tripotassium EDTA (lavender top) is the required anticoagulant for
leukocyte count and differential analysis, which is required for
analysis of the results of this assay.
2.03 Whole blood samples should be held at ambient temperature (18-22 C) o
prior to analysis. If possible, samples should be assayed within 8
hours of draw. However, samples can be analyzed up to 24 hours after
draw (6). However, when logistics of a protocol require that samples
be assayed at >8 and <24 hours, all samples from patients and
controls collected for that protocol should be treated in a similar
manner. A study of the whole blood response to mitogen of 44 normal
controls (Figure 1) showed an average loss of 14% in the responses
observed after 24 hours as compared to fresh samples.
2.04 Specimen will be rejected if the blood is lysed, clotted, older than
24 hours or if it has been refrigerated.
3. REAGENTS, STANDARDS, CONTROLS AND MEDIA USED, AS WELL AS SUITABLE VENDORS.
OTHER VENDORS ARE ACCEPTABLE IF VALIDATED.
3.01 RPMI 1640 with L-glutamine (Gibco BRL, Gaithersburg, MD)
3.02 Penicillin and streptomycin (P/S), 100X liquid (5000 units/ml and
5000Fg/ml respectively).
Defrosted solution should be aliquoted and stored at -20 C. o
3.03 Tritiated thymidine ([3H]-TdR) (NEN Products, Dupont).
3.04 Tissue culture grade, sterile, round bottom 96-well plates with
tight fitting lids (Corning Costar, Cambridge, MA).
3.05 Biodegradable, nontoxic scintillation fluid (Cytoscint, ICN, Costa
Mesa, CA)
3.06 Sterile, disposable pipette tips.
4. STIMULANTS
Recall antigens that could find use in clinical studies are numerous, and
selection should be dictated by the clincial or research problem at hand.
For example, proliferation assays with proteins and glycoproteins
associated with HIV-1 may be useful in detecting cellular immune responses
to this virus in infected individuals . If response to an antigen to which
most people are exposed is to be measured as a marker of general cellular
immune competency, Candida albicans and/or tetanus toxoid are two which
are frequently selected. In ACTG protocols, the specific stimulants will
be specified.
4.01 Plant mitogens which have been proven useful in studies with human
subjects include phytohemmagglutinin (PHA), pokeweed mitogen (PWM)
and concanavalin A (Con A). All three can be obtained from Sigma
Chemical Co., St. Louis, MO.
4.02 A T lymphocytes specific stimulant is monoclonal antibody to the CD3
receptor. This can be obtained from Coulter, Hialeah, FL or from
Becton Dickinson, San Jose,CA.
4.03 Recall antigens are also useful. Candida albicans antigen is
obtained from Greer Laboratories, Lendir, NC. Tetanus toxiod is
supplied by Connaught Laboratories, Swift Water, PA. HIV-1 peptides
can be obtained from NIAID Reagent Program, Rockville, MD.
4.04 Preparation of antigen/mitogen working solutions:
Antigen/mitogen solutions are prepared in RPMI-0 for the whole blood
assay or in RPMI-10% for the PBMC assay. For each antigen/mitogen in
the assay, prepare a solution at twice (2X) the intended final
concentration in the assay wells. Prepare sufficient volume in order
to fill the requisite number of assay wells (# assay wells X
100Fl/well = minimum volume to prepare). Usual 2X working
concentrations are as follows:
Candida albicans 20 Fg/ml
Tetanus toxoid 2.5 Fg/ml
PHA 20 Fg/ml
PWM 10 Fg/ml
Anti-CD3 1 Fg/ml
It is suggested, however, that each laboratory deterimine optimum
antigen or mitogen concentrations. In longitudinal studies, it is
wise to obtain a sufficient amount of a single lot of each
stimulant. Stock solutions may be prepared in sterile RPMI 1640.
These stocks should be dispensed into sterile vials, in suitable
volume for one assay, and held frozen at -20 C until use. o
5. INSTRUMENTATION
5.02 CO incubator with > 95% humidity. 2
5.03 Cell harvester and glass fiber filter papers.
5.04 Beta counter.
5.06 Calibrated pipetters and multichannel pipetters.
5.07 Refrigerated centrifuge.
6. STEP-BY STEP DIRECTIONS
6.01 EDTA blood tube is used for determination of leukocyte count and
differential analysis. This assessment should be done within 6 hours
of blood collection. If the hematology is performed in a laboratory
separate from the immunology laboratory, the data must be available
in order to calculate the LPA results.
6.02 Prepare RPMI supplemented with P/S. Filter through 0.2 filter.
RPMI P/S may be stored at 4 C for no longer than 1 week. 0
6.03 Dispense 100 Fl/well of each antigen/mitogen concentration being
tested. For control wells in whole blood assays, dispense 100 Fl of
RPMI P/S. All test concentrations and control wells are to be done
in triplicate. Prepared plates may be placed in air tight wrap and
stored at -80 C until needed. On assay day, thaw antigen/mitogen o
culture plates at 37 C in incubator. Label the plate with patient 0
identification, date plated, date to be pulsed and date to be
harvested.
6.04 Dilute heparinized blood (1:5) with RPMI P/S. Dispense diluted blood
100 Fl/well to all the wells containing mitogens/antigens or culture
medium.
6.05 Assay Setup
Wrap plates in aluminum foil, plastic wrap or plastic bag, and
incubate at 37 C in a 95% humidified atmosphere containing 5% CO . o
2
This is day 1.
6.06 Harvesting and Counting
On the morning of last day of incubation prepare a working solution
of 20 Fci/ml triatiated thymidine in RPMI P/S. Prepare sufficient
volume to dispense 25 FL/well. (# assay wells X 25FL/well = minimum
volume to prepare.). Pulse plates with 25 Fl (0.5 Fci) of [3H]-TdR.
Usually, mitogens such as PHA and conA are incubated with cells for
72 hours, PWM and anti-CD3 can be incubated for 3 days to 6 days.
Recall antigens are incubated 6 to 7 days. After six hours, harvest
on glass fiber filters using a cell harvester. If harvesting cannot
be done immediately after the six-hour pulse, plates must be stored
at 4 C until harvested. After harvesting, filters are dried o
overnight at room temperature and punched-out into scintillation
vials, or the filter sheets are processed for counting in a
betaplate counter. Add appropriate quantity of scintillation fluid
to the vials and leave filters for at least three hours in
scintillation fluid prior to counting. Vials are counted on a beta
scintillation counter at laboratory-determined settings.
7. CALCULATIONS
7.01 Data can be expressed and calculated in two different ways following
determination of mean values of the triplicates:
7.01a Net counts or cpm = (cpm experimental - cpm background
unstimulated)
For whole blood assays: Mean of the net cpm is transformed to
mean cpm/100,000 lymphocytes. The number of lymphocytes
present in the culture is determined from total leukocyte
count times percent lymphocytes. Both of these values are
obtained from the complete blood count with differential.
7.01b Stimulation Index (SI) = (cpm experimental/cpm background
unstimulated)

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WHOLE BLOOD LYMPHOCYTE PROLIFERATION ASSAY