Measurement of Cytokine Production UNIT 7.

18B
Using Whole Blood
Whole blood (WB) ex vivo stimulation is a useful tool to investigate cytokine responses
to a variety of stimuli, including bacterial endotoxin (LPS), antigens, allergens, and
antibiotics. It is also useful in determining the effects that potential inhibitors (e.g., phar-
macological agents) may have on inflammatory processes. The culture of WB approxi-
mates the state of circulating cells in vivo and contains physiological concentrations of
factors that influence immune cell function more closely than does the culture of periph-
eral blood mononuclear cells (PBMC). WB culture may be the most appropriate milieu
in which to study ex vivo cell activation and cytokine production in vitro.

A WB ex vivo stimulation assay is a two-step process: (1) antigenic or mitogenic stim-

ulation of cells in whole blood and (2) analysis of either plasma or cells for resulting
cytokine production using a variety of methods such as RT-PCR, ELISA, flow cytome-
try or multiplex analyses, such as the fluorescent bead-based Luminex x-Multi Analyte
Profile (MAP) technology.

This unit describes the techniques required to perform WB ex vivo stimulation. The
optimal concentration of antigen, the incubation times used to stimulate whole blood
cultures, and the steps taken to prepare each antigen for ex vivo stimulation often require
optimization for each assay. Conditions that have been found useful for stimulation of
cells with a select group of antigens and mitogens are noted throughout the Basic Protocol.

CAUTION: When working with human blood or potential infectious agents, biosafety
practices must be followed (see Chapter 7 Introduction).

NOTE: When performing WB ex vivo stimulation experiments, all tubes, pipets, and
reagents used in the handling and subsequent stimulation of whole blood must be free of
endotoxin, since it may have a profound effect on the level and type of cytokine response
measured.

EX VIVO STIMULATION OF WHOLE BLOOD BASIC

PROTOCOL
This protocol requires the use of relatively fresh whole blood samples (i.e., samples
obtained within 24 hr of analysis) collected into an appropriate anti-coagulant, preferably
sodium heparin (see Critical Parameters). Whole blood is stimulated with a minimal
volume of antigen/mitogen diluted in RPMI 1640 (typically 1% of the total volume of
blood to be stimulated, e.g., 5 µl of antigen/mitogen solution per 500 µl of blood), to
minimize further dilution of the whole blood sample.

Materials
Antigen or mitogen (see Table 7.18B.1)
Inhibitors (e.g., dexamethasone)
RPMI 1640 medium supplemented with 2 mM L-glutamine, room temperature
Whole blood sample collected into sodium heparin anti-coagulant (typically 15
USP sodium heparin/ml of blood)
Polypropylene 1.5-ml microcentrifuge tubes or 24-well Costar polystyrene tissue
culture plates, or 5-ml 12 × 75–mm Falcon polypropylene round-bottom culture
tubes
37◦ C, 5% CO2 humidified incubator
Immunologic
Studies in
Humans
Contributed by Cary W. Thurm and John F. Halsey 7.18B.1
Current Protocols in Immunology (2005) 7.18B.1-7.18B.12
Copyright 
C 2005 by John Wiley & Sons, Inc.
Supplement 66
Table 7.18B.1 Antigen/Mitogen Concentrations

Antigen Final concentration Incubation time Cytokines analyzed

LPS 4–50 ng/ml 4–8 hr TNF-α, IL-1β, IL-6, IL-8

PHA 0.5–10 µg/ml 4–24 hr TNF-α, IL-1β, IL-6, IL-8, IL-2, IL-4, IFN-γ, IL-10
Candida albicans 40 µg/ml 4–24 hr TNF-α, IL-1β, IL-2, IL-6, GM-CSF, IFN-γ
soluble extract
Tetanus toxoid 0.1–1.0 Lf/ml 2–48 hr TNF-α, IL-1β, IL-2, IL-6, GM-CSF, IFN-γ
CpG DNA 10 µM 4–8 hr TNF-α, IL-1β, IL-6, IL-8
Zymosan 100–500 ng/ml 4–24 hr TNF-α, IL-1β, IL-6, IL-8
Anti-CD3 10 µg/ml 4–24 hr TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10

1. Dissolve and/or dilute the antigen or mitogen as well as any inhibitors in RPMI
1640 supplemented with 2 mM L-glutamine and bring to room temperature. For each
antigen/mitogen in the assay, prepare a solution at 100 times (100×) the intended
final concentration in the assay. Prepare volume sufficient for the number of samples
(i.e., number of samples to be stimulated times 5 µl/sample = minimum volume to
prepare).
Do not use serum-supplemented medium. It may contain cytokine/growth factor products
(e.g., transforming growth factor-β), which may modify the cytokine response profile.
Antibiotics are also omitted due to potential interference with stimulation assays.
Some researchers have noted that antigen/mitogen-stimulated cytokine levels are progres-
sively reduced as whole blood is diluted. Consequently, whole blood should be diluted as
little as possible in order to achieve maximum sensitivity for short-term cultures (<24 hr
of stimulation). Antigens, mitogens, or inhibitors that cannot be dissolved at 100× con-
centration should be dissolved/diluted at the highest concentration obtainable. However,
the authors have found that minimal dilution of whole blood in L-glutamine-supplemented
RPMI (or other compatible tissue culture media) may be required to stabilize whole-blood
samples that are not freshly collected (i.e., ≥24 hr post-collection) if the stimulation ex-
periment involves incubation periods ≥24 hr (see Fig. 7.18B.1).
The optimal concentration of the antigens, mitogens or inhibitors to be used for whole
blood stimulation should be determined in pilot experiments in advance (see Table
7.18B.1). In general, the optimal concentration of antigen/mitogen is that which induces a
detectable amount of the cytokine(s) of interest within the shortest period of ex vivo stim-
ulation. In longitudinal studies, a sufficient amount of the reagents of a single lot should
be obtained. Stock solutions may be prepared in sterile RPMI 1640. To maintain sterility,
filtration through a 0.22-µm filtration device may be necessary. These stocks should be
dispensed into sterile vials, in a volume suitable for one assay, and held frozen at −20◦ C
until use.
Special considerations might be required for preparation of antigen/mitogen/inhibitor
solutions due to solubility and salt concentrations (see Troubleshooting). Also the stability
of some antigens, mitogens, and inhibitors may be affected by freezing. The investigator
may need to conduct stability studies comparing freshly made stimulant solutions and
those that have been frozen.

2. Label culture tubes consistent with the guidelines of the study being conducted. At
a minimum, reserve tubes for “stimulated” (mitogen/antigen as specified by investi-
gator) and “nonstimulated” (RPMI 1640 only).
Measurement Ex vivo whole blood stimulation can be done in a variety of culture vessels, the choice
of Cytokine of which is primarily dictated by the methodology used for analyzing cytokine responses.
Responses Using Sterile, endotoxin-free 1.5-ml polypropylene microcentrifuge tubes, 12 × 75–mm round-
Whole Blood
bottom (Falcon) polypropylene culture tubes, and 24-well treated culture dishes have
7.18B.2
Supplement 66 Current Protocols in Immunology
Figure 7.18B.1 (A) Effects of incubation time and dilution on anti-CD3 stimulation. (B) Effects of incubation
time and dilution on PHA responses.

all been used to perform the ex vivo stimulation assay with very little difference in the
cytokine responses generated (see Fig. 7.18B.2). Thus, for assays in which the ultimate
cytokine response will be analyzed in plasma, stimulation may be performed in 1.5-ml
microcentrifuge tubes to minimize handling and to facilitate subsequent harvesting of
plasma from stimulated blood cultures. For assays in which leukocytes will be analyzed
for cytokine production (e.g., flow cytometric analysis, see UNIT 6.24). 12 × 75-mm round-
bottom polystyrene culture tubes that do not contribute to selective loss of cells (i.e., plastic
adherence) may be more appropriate.
The authors routinely also collect plasma from the whole-blood sample at the onset of the
experiment for a baseline control, which can be used to determine the concentration of
the cytokines of interest existing in the whole blood at the onset of the experiment.
3. Pipet 0.5 ml of whole blood into the culture tubes.
The volume of blood to be stimulated does not appear to be a critical factor in whole Immunologic
blood culture. Typically, 0.5 ml of whole blood is used. In house studies of whole Studies in
Humans
blood stimulation with LPS indicated that similar results were obtained with samples
7.18B.3
Current Protocols in Immunology Supplement 66
 Figure 7.18B.2 Comparison of culture vessels. Representative whole blood sample stimulated
for 4 hr with 50 ng/ml or RPMI media.

Figure 7.18B.3 Effects on volume on ex vivo stimulation. The indicated volume of a representa-
tive sample of whole blood was stimulated for 4 hr with 50 ng/ml LPS.

ranging from 0.25 to 1.0 ml of blood analyzed for IL-1β, IL-6, and TNF-α production (see
Fig. 7.18B.3).

4. Add 5 µL of the 100× antigen/mitogen solution or RPMI 1640 medium (nonstim-

ulated control) to the indicated whole blood samples. Mix samples gently but thor-
oughly to disperse.
If the investigator is examining the affects of inhibitors on the cytokine response to an
antigen or stimulant, the inhibitor should also be added at this time. However, pilot exper-
iments should be conducted to determine the order of addition of inhibitor and antigen or
mitogen to the WB culture. For example, the researcher may find that preincubation of the
Measurement whole blood with inhibitor is necessary before adding the antigenic/mitogenic stimulants.
of Cytokine
Responses Using 5. Incubate the samples in a 37◦ C, 5% CO2 humidified incubator for the time specified
Whole Blood
by the particular project (e.g., 1, 2, 4, 24, or 48 hr).
7.18B.4
Supplement 66 Current Protocols in Immunology
 6. After incubation, gently mix the samples. Then analyze samples for cytokine expres-
sion using a number of different methods.
Cells can be fixed, permeabilized and immunostained for analysis of intracellular
cytokines by flow cytometry (see UNIT 6.24). RT-PCR analysis for cytokine gene ex-
pression using mRNA isolated from the WB cultures may be done. Alternatively,
secreted cytokines can be analyzed from plasma harvested from the WB culture. Plasma
should immediately be transferred to fresh, sterile microcentrifuge tubes and then be
stored at −70◦ C if they are not immediately analyzed for cytokine content.

COMMENTARY
Background Information
The use of WB ex vivo stimulation as-
says for analysis of cytokines was originally
introduced as a replacement for analyzing
endotoxin-induced TNF-α responses of iso-
lated monocytes (Desch et al., 1989). Since
then, whole blood stimulation has been used
in numerous instances to investigate cytokine
release in response to various stimuli. The as-
say has been used to assess cytokine responses
to specific antigens, such as purified tuberculin
protein derivative (PPD) (Bellete et al., 2002),
cytomegalovirus (CMV; Nomura et al., 2000)
as well as responses to allergens (Benlounes
et al., 1999; Miles et al., 2003), and antibiotics
(Kurdowska et al., 2001). Lipopolysaccharide
(LPS) and phytohemagglutinin A (PHA) are
frequently used as powerful stimulants for the
secretion of cytokines from monocytes and
lymphocytes, respectively. These agents are
routinely used as standardized stimuli to assess
ex vivo acute immunomodulatory properties of
test agents, such as the effects of azathioprin
or dexamethasone on LPS-stimulated WB cul-
tures (Langezaal et al., 2001). Ex vivo stimula-
tion assays for measuring cytokine responses
are becoming increasingly useful, primarily
due to the easy access of samples from healthy
donors and patients and the minimal process-
ing of samples required. Cytokine patterns in
response to antigen(s) ex vivo have been shown
to reflect the nature of the immune response in
vivo (Heinzel et al., 1991). When coupled with
techniques for analyzing cytokine produc-
tion, such as multiplex cytokine analysis (e.g.,
Luminex flow cytometric bead assays and cy-
tokine microarrays), ELISA, RT-PCR, or flow
cytometry, WB ex vivo stimulation assays are
a simple yet powerful method for assessing
immunological responses to antigens or mito-
gens. WB ex vivo stimulation assays can also
be used to analyze release of general media-
tors such as eicosanoids, NO, or degranulation
products.
Traditionally, in vitro stimulation assay
have been performed on purified peripheral
blood mononuclear cells (PBMC) that require
time-consuming preparative steps that limit the
ability to process large numbers of samples, or
are prohibitive where facilities, equipment and
expertise are a constraint. Additionally, the cy-
tokine responses of in vitro–stimulated PBMC
may not be reflective of the true nature of cy-
tokine responses generated in vivo due to the
loss of selected cell populations and physio-
logical concentrations of factors that influence
immune cell function, as well as the influ-
ence of artificial medium (which usually con-
tains fetal calf serum) on isolated cells. Sub-
stantial differences occur in the pattern of T
cell cytokine responses when cells are cultured
in serum-supplemented medium compared to
those in serum-free medium, primarily due to
the presence of platelet-derived growth factor
(PDGF; Daynes et al., 1991). Also, it has been
demonstrated that the addition of autologous
red blood cells to human PBMC cultures en-
hances production of IL-2, IL-6, IFN-γ, and
TNF-α (Kalechman et al., 1993). In addition,
cultures of PBMC generate more IL-1 and IL-
2, and relatively less TNF-α and IFN-γ com-
pared to whole blood cultures in relation to
the number of mononuclear cells present (De
Groote et al., 1992).
A variety of clinical and investigational uses
of WB stimulation assays have been suggested,
including the assessment of the cellular aspects
of autoimmune disease, the monitoring of drug
and vaccine efficacy, and immunotoxicity. Re-
cent studies have used ex vivo WB cultures
to assess TNF-α released from samples ob-
tained from volunteers receiving phosphodi-
esterase (PDE) 4 inhibitor orally (Gale et al.,
2002). Also, whole blood cultures have been
used to assess the effects of immunosuppres-
sive drugs on LPS-stimulated ex vivo cytokine
expression such as the effects of macrocyclic
hydroxamic acids (Xue et al., 2001), resor-
cyclic acid lactones (Rawlins et al., 1999), and
glucocorticoids (e.g., dexamethasone; see Fig. Immunologic
7.18B.4), Recent insight gained from analy- Studies in
sis of Toll-like receptors (TLRs) suggest that Humans

7.18B.5
Current Protocols in Immunology Supplement 66
 Figure 7.18B.4 Effects of dexamethasone on LPS-induced TNF-α expression. Whole blood from
three individuals stimulated with 4 ng/ml LPS in the presence of decreasing concentrations of
dexamethasone.
ex vivo stimulation of WB with bacterial anti-
gens such as CpG DNA, LPS, and zymosan
may be useful in assessing the immune com-
petency of TLR-mediated signaling pathways
in individuals with recurrent infections (Picard
et al., 2003). Thus, WB cell culture may be
used to study the biological effects of sub-
stances or drugs on immune cell activation and
cytokine secretion.
Critical Parameters
When using WB cultures for the evalua-
tion of functional cellular responses, an impor-
tant consideration is the effect of sample col-
lection and processing on subsequent cellular
stimulation and cytokine analysis. The labora-
tory should inspect the sample for evidence of
hemolysis. Hemolyzed specimens should not
be used because hemolysis indicates that the
specimen may have been subjected to extreme
conditions that may have damaged leukocytes
as well as red blood cells. When analyzing hu-
man blood specimens, it is important to note
that partially filled Vacutainer blood collec-
tion tubes may produce osmotic conditions
detrimental to cells. In general, conditions that
are potentially harmful to cell viability, such
as temperature extremes, should be avoided.
Also, clotted specimens should not be used be-
cause selective loss of certain cell populations
may occur during clotting.
Measurement
of Cytokine The second important factor is the type
Responses Using of anticoagulant used in collection, since this
Whole Blood
factor has been found to influence ex vivo
7.18B.6
Supplement 66
cytokine production in whole blood. Sodium
heparin is the best choice of anticoagulant.
It has the least influence on cytokine produc-
tion in whole blood (Holobaugh and McChes-
ney, 1990). In addition, recovery of cytokines
appears to be considerably better from hep-
aranized blood compared to other anticoagu-
lants (Thavasu et al., 1992). Lithium heparin is
less desireable because of the potential for low
level endotoxin contamination (Riches et al.,
1992). Other anticoagulants such as EDTA or
acid citrate dextrose (ACD) are not recom-
mended for WB ex vivo stimulation assays as
these anticoagulants chelate calcium, an essen-
tial requirement for cellular activation.
Ex vivo stimulation should be performed
on whole blood samples as soon as reason-
ably possible. In those instances where blood
cannot be immediately stimulated, samples
should be stored horizontally in the collec-
tion vacutainer at ambient temperature in sub-
dued light prior to ex vivo stimulation. For
best results, ex vivo stimulation should be
performed within 24 hr of collection. Typi-
cally, the percent of cytokine-producing cells
is reduced by ∼5% beyond 8 hr of collec-
tion. However, satisfactory results are achiev-
able within 24 hr of collection when the sam-
ple is stored at ambient temperature under
subdued light. In-house studies of whole blood
stability suggest that similar inflammatory cy-
tokine responses to LPS and PHA can be
achieved on whole blood samples up to 48 hr
of storage (see Fig. 7.18B.5). In addition,
Current Protocols in Immunology
Figure 7.18B.5 Stability of stored whole blood cytokine responses: A representative whole blood sample
stimulated 4 hr with 50 ng/ml LPS (A), 50 ng/ml PHA (B) or RPMI after storing sample at room temperature
under subdued light for the indicated time periods.

for blood samples approaching 24 hr post- to be assayed. Additionally, since cytokines

collection, the authors have found that min-
imal dilution of the blood sample in serum-
free medium (e.g., RPMI supplemented with
L-glutamine) stabilizes the sample and lends
to a more robust cytokine output particularly
when examining largely T cell–mediated re-
sponses (see Fig. 7.18B.1).
Analysis for cytokine production should be
done either immediatedly after separating the
plasma from the culture or store plasma from
stimulated samples at ∼ −70◦ C until they are
Current Protocols in Immunology
are sensitive to freeze-thawing, the number of
cycles of freeze-thawing must be held to an
absolute minimum.
Troubleshooting
If high background levels of cytokines are
observed in the non-stimulated control, the
presence of contaminating endotoxin should
be suspected. The use of a non-stimulated con- Immunologic
trol in WB ex vivo experiments is extremely Studies in
Humans
important, particularly in instances where
7.18B.7
Supplement 66
 Figure 7.18B.6 LPS-stimulated cytokine responses. Four representative whole blood samples stimulated for
4 hr with 50 ng/ml LPS.

Figure 7.18B.7 IL-6 response to CpG DNA stimulation. Four representative whole blood samples stimulated
for 24 hr with 10 µM CpG DNA, 10 µM non-CpG DNA, or RPMI.

immunocompetency is being assessed. Some
researchers have noted a high level of sponta-
neous IL-8 production in whole blood cultures.
As a result, pilot experiments may be neces-
sary to assess the spontaneous expression of
the cytokine of interest in non-stimulated cul-
tures (i.e., >8 hr) prior to examining the effects
of antigen/mitogen stimulation.
It is necessary for the researcher to examine
Measurement
of Cytokine the optimal stimulation time and concentration
Responses Using for each antigen/mitogen with respect to the
Whole Blood cytokine of interest. The ability to monitor cy-
7.18B.8
Supplement 66
tokine responses generated from ex vivo stim-
ulated WB cultures is dependent upon numer-
ous factors, such as the duration of stimulation,
the type and concentration of antigen/mitogen
used for stimulation, as well as the class of
cytokine measured.
For those antigens, mitogens and inhibitors
with limited solubility in RPMI medium, the
investigator may also attempt to dissolve the
agent in other solvents compatible with rou-
tine cell culture such as Hank’s balance salt
solution (HBSS), or minimal concentrations
Current Protocols in Immunology
Figure 7.18B.8 PHA-stimulated cytokine responses. Four representative whole blood samples
stimulated for 4 hr with 50 ng/ml PHA.

Table 7.18B.2 Range of Cytokine Responses to 24 Hr Ex Vivo Challengea

Stimulantb n Average (SD) 20th percentile 80th percentile

IL-10
PHA 20 898.2 (540.9) 482.5 1457.6
Anti-CD3 20 566.8 (453.4) 190.4 1004.4
IFN-γ
PHA 18 225.1 (193.9) 78.5 286.7
Anti-CD3 19 300.3 (502.2) 59.6 328.3
IL-4
PHA 16 58.8 (24.0) 36.6 79.5
IL-2
PHA 20 1311.9 (1160.6) 490.0 1929.8
Anti-CD3 20 417.4 (362.4) 167.9 565.2
TT 19 191.8 (362.5) 12.2 231.7
a Individual whole-blood samples (n) obtained from normal donors were diluted 1:4 with RPMI 1640 medium supple-
mented with 2 mM L-glutamine prior to ex vivo challenge with the indicated stimulant.
b PHA (5 µg/ml); anti-CD3 (0.4 µg/ml); TT (2.5 Lf/ml).

of DMSO, ethanol or acetone (typically final
concentrations of 0.1%). Under these circum-
stances, experiments should be conducted to
determine if any solvent effects on cytokine
expression occur in non-stimulated controls.
Also, dialysis of lyophilized antigen, mitogen,
or inhibitor solutions against RPMI after re-
hydration may be necessary as the presence of Immunologic
residual amounts of salt may affect the ultimate Studies in
cytokine response. Humans

7.18B.9
Current Protocols in Immunology Supplement 66
 Table 7.18B.3 Intra-Assay Precision of 24 Hr Ex Vivo Challengea

IL-10 (pg/ml) Average (SD) %CV

Sample 1
PHA 1554.2 1442.5 1622.5 1539.7 (90.9) 5.9
Anti-CD3 516.1 525.6 489.8 510.5 (18.5) 3.6
RPMI 0.8 1.2 0.5 0.8 (0.3) 37.8
Sample 2
PHA 2135.2 2719.8 2637.8 2497.6 (316.5) 12.7
Anti-CD3 545.6 470.6 395.5 470.6 (75.0) 15.9
RPMI 0.8 0.7 0.5 0.7 (0.2) 22.8
Sample 3
PHA 2494.3 2671.7 2786.8 2651.0 (147.3) 5.6
Anti-CD3 1061.3 1219.7 1163.6 1148.2 (80.3) 7.0
RPMI 2.0 3.9 3.9 3.3 (1.1) 33.3
a Individual whole-blood samples were diluted 1:4 with RPMI 1640 medium supplemented with 2 mM L-
glutamine prior to ex vivo challenge with the indicated stimulant. Three separate ex vivo challenge experiments
were performed on each sample at the same time by the same technician; the results presented are the IL-10
responses obtained from each of the separate challenges.
b PHA (5 µg/ml); anti-CD3 (0.4 µg/ml); TT (2.5 Lf/ml).

Table 7.18B.4 Inter-Assay Precision of 24 Hr Ex Vivo Challengea

IL-10 (pg/ml) Average (SD) %CV

Sample 1
PHA 1539.7 1921.6 1730.6 (267.0) 15.6
Anti-CD3 510.5 421.7 466.1 (62.8) 13.5
RPMI 0.8 0.5 0.7 (0.3) 40.1
Sample 2
PHA 2497.6 1861.3 2179.5 (449.9) 20.6
Anti-CD3 470.6 383.5 427.0 (61.6) 14.4
RPMI 0.7 0.8 0.76 (0.1) 13.5
Sample 3
PHA 2651.0 2562.9 2606.9 (62.3) 2.4
Anti-CD3 1148.2 702.8 925.5 (314.9) 34.0
RPMI 3.3 5.0 4.1 (1.2) 29.9
a Individual whole-blood samples were diluted 1:4 with RPMI 1640 medium supplemented with 2
mM L-glutamine prior to ex vivo challenge with the indicated stimulant. Separate ex vivo challenge
experiments were performed on each by two technicians; the results presented are the average IL-10
responses obtained from each of the separate challenges performed by the two technicians.
b PHA (5 µg/ml); anti-CD3 (0.4 µg/ml); TT (2.5 Lf/ml).

Anticipated Results (LPS) and CpG DNA, which stimulate primar-

The level and profile of cytokine responses ily monocyte populations through toll-like re-
generated through ex vivo stimulation of whole ceptors (TLR), and the plant mitogen phyto-
Measurement
of Cytokine blood are dependent upon the nature and con- hemagglutinin (PHA), which non-specifically
Responses Using centration of the antigen or mitogen used stimulates T cell populations, generate cy-
Whole Blood as stimulant. Stimulants such as endotoxin tokine responses measureable in the plasma
7.18B.10
Supplement 66 Current Protocols in Immunology
harvested from whole blood cultures after a
relatively short period of stimulation (see Figs.
7.18B.6, 7.18B.7, and 7.18B.8, respectively).
Antigens that stimulate specific T cell popula-
tions, such as antigen extracts prepared from
Candida albicans, may require longer ex vivo
stimulation (i.e., 24 to 48 hr), depending on
the ultimate cytokine response measured as
a biological endpoint (see Fig. 7.18B.9). Al-
though large-scale studies have yet to be de-
scribed, the authors have found that the range
of cytokine responses observed in WB stimu-
lation to various stimuli can be rather broad
when examining normal populations (see
Table 7.18B.2). Nonetheless, as demonstrated
in Tables 7.18B.3 and 7.18B.4, whole-blood ex
vivo stimulation assays can provide rather con-
sistent and precise data with respect to cytokine
responses to specific stimuli. When WB stimu-
Figure 7.18B.9 Candida albicans antigen-stimulated cytokine responses. Representative whole
blood was incubated for the indicated time periods with 40 µg/ml Candida albicans antigen extract
or RPMI. (A) IL-6 response. (B) TNF-α response.
Current Protocols in Immunology
lation is used in conjunction with intracellular
cytokine and surface marker staining followed
by flow cytometric analysis (see UNIT 6.24), the
nature of the antigen-specific responses can be
further delineated with respect to the specific
cell populations activated.
All results in the attending figures were ob-
tained using Fluorokine MAP kits (R&D Sys-
tems) and the Luminex100 analyzer.
Time Considerations
The incubation times vary with the anti-
gen or mitogen to be used as stimulant, the
addition of inhibitors, as well as the subse-
quent method used for analyzing the ultimate
cytokine response(s). The major time commit-
ment involves incubating the whole blood with
the selected stimulant, which may range any-
where from 2 to 48 hr.
Immunologic
Studies in
Humans
7.18B.11
Supplement 66
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Contributed by Cary W. Thurm and
John F. Halsey
IBT Reference Laboratory
ProGene Biomedical
Lenexa, Kansas
Current Protocols in Immunology