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Clinical and Diagnostic Laboratory Immunology, January 1999, p. 89-95, Vol. 6, No. 1
CIRID at University of California,
Received 29 June 1998/Returned for modification 25 August
1998/Accepted 12 October 1998
Cytokines and soluble immune activation markers that reflect
cytokine activities in vivo are increasingly being measured in plasma,
serum, and other body fluids. They provide useful diagnostic and
prognostic information as well as insight into disease pathogenesis. Assays of neopterin, Cytokines are involved in the
pathology of a wide range of disorders (2). Measurements of
levels of cytokines and/or the soluble markers of immune activation
that are products of cytokine activities (3, 24, 26, 46, 49,
51) are useful for understanding pathogenesis and as diagnostic
and prognostic indicators in many diseases, including those induced by
human immunodeficiency virus (HIV) infection (15, 16, 18, 19, 25,
36, 42, 45, 56). The use of appropriately collected and stored
test samples of body fluids and of sensitive and accurate methods is critical to these analyses (14, 30, 48, 54). However, complications and difficulties in these assays have been reported (7, 21, 33, 55, 58), and a number of factors have been shown
to affect the quality and validity of the measurements (4, 10, 37,
38, 57). Differences in levels were found in studies that
compared the major techniques, enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), and bioassays (3, 13). Among the factors that have been implicated as causing the differences are compounds known to bind to cytokines, such as serum albumin and
At present, immunoassays are the most widely used techniques, although
there are known pitfalls of the procedure (28) as well as
additional limitations for cytokine measurements (43, 44).
In immunoassays, the pairs of antibodies used in different assay kits
have been shown to have unique recognition patterns for different forms
(monomeric or polymeric) or different epitopes of the antigen molecule
(7, 38, 44). Differences in the profiles have been shown to
cause differences of up to 100-fold in apparent cytokine levels of
identical samples (12, 29, 39, 40). Several World Health
Organization-sponsored collaborative studies for cytokine
standardization have shown that at least a part of such variations was
due to differences in standards used in the assays (5, 20, 47,
50). In addition, the importance of sample collection,
processing, and storage in affecting the validity of the measurements
and levels of cytokines in biological fluids has been demonstrated
(48, 54).
The present study was undertaken to identify factors that influence the
results of immunologic assays for seven cytokines, soluble cytokine
receptors, and soluble immune activation markers. The findings indicate
that reagent sources and the reference standards used have a major
impact on the results. The effects of additional factors, such as
measurement of levels in serum versus in plasma and the conditions of
sample storage, were also investigated. The normal reference ranges in
our laboratory for these analytes are reported.
Samples.
Serum and plasma samples were obtained from healthy
volunteers and HIV-negative and HIV-positive subjects participating in the Multicenter AIDS Cohort Study (MACS) of the natural history of AIDS
at the University of California, Los Angeles (UCLA), or in AIDS
Clinical Trial Unit studies at the UCLA AIDS Clinical Research Center.
Blood was collected and processed following our established lab
protocol. Blood was collected by venipuncture into 15-ml sterile
vacutainers either containing heparin or EDTA as anticoagulant for
plasma samples or without anticoagulant for serum samples. The
anticoagulant of choice for determination of levels of cytokines in
plasma was EDTA. If heparin was used as anticoagulant, each batch of
heparin was confirmed by testing to be pyrogen free to avoid
unintentional stimulation of blood cells. Also, only pyrogen-free
collection tubes were used. Collected blood was kept at 4°C (serum)
or at room temperature (plasma). Serum and plasma samples were
separated from blood within 1 to 3 h following blood collection:
serum was separated by centrifugation at 500 × g for
10 min, and plasma was separated at 350 × g for 15 min
at room temperature. Separated samples were aliquoted and stored at
Quantitation of levels of cytokines and soluble activation
markers in plasma.
Statistical analysis.
The statistical analyses were done by
calculating the Pearson correlation coefficients and performing paired
t tests with Statview and SAS software. A sigma plot was
used for the graphs.
Effects of different reagent sources.
Plasma samples from
HIV-seropositive patients with a range of CD4 levels were evaluated for
their contents of cytokines and soluble immune activation markers by
reagents from two or more suppliers (Table
1). The two commercial kits used for
TNF- Comparison of standards from different manufacturers.
Standard
preparations provided by one supplier were tested with the reagents and
standards from a second supplier. Two comparisons are summarized in
Table 2. IFN-
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Variables That Affect Assays for Plasma Cytokines
and Soluble Activation Markers
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
2-microglobulin, soluble interleukin-2
receptor, and soluble tumor necrosis factor receptor type II as well as of the cytokines tumor necrosis factor alpha and gamma interferon (IFN-
) were evaluated by using serum and plasma samples of human immunodeficiency virus (HIV)-positive and HIV-negative subjects. Many
factors were found to influence the outcomes of these assays. Substantial differences in apparent levels of analytes were frequently found when enzyme-linked immunosorbent assay (ELISA) kits from different manufacturers were used. In some cases, differences were
found in the standards provided by separate manufacturers. Furthermore,
the analytic results from different lots of ELISA kits supplied by
single manufacturers differed by as much as 50%. The need for
uniformity in the standards for quantitative assays was clearly
illustrated. International reference standards are available for
cytokines but not for soluble cytokine receptors or soluble activation
markers. Marker levels in serum or in plasma were similar except those
for IFN-. Most of the analytes were stable under several storage
conditions. Thus, batch testing of frozen stored samples is feasible.
The findings indicate that for longitudinal studies, the levels of
cytokines and immune activation markers in plasma or serum should be
measured by using preverified reagents from one manufacturer. The
quality of laboratory performance can have an impact on clinical
relevance. Proficiency testing and external quality assurance programs
can help to develop the needed consensus.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
2-microglobulin (6, 27, 32, 53), autoantibodies (11,
23, 31), and soluble receptors that inhibit cytokine activity,
i.e., soluble tumor necrosis factor (TNF) receptor types I and II for
TNF- (sTNF-RI and sTNF-RII, respectively) (1, 3, 52).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
70°C for subsequent batch testing.
2-Microglobulin (2M) was quantitated by
using microparticle enzyme immunoassay (MEIA) (Abbott Laboratories,
Abbott Park, Ill.) and enzyme immunoassay (EIA) kits (Pharmacia AB,
Uppsala, Sweden, and ORGenTec Diagnostika GmbH, Mainz, Germany) (see
Table 1). Neopterin was measured by competitive EIA kit (ICN, Costa Mesa, Calif.) and RIA kit (IMMUtest Neopterin; BRAHMS, Berlin, Germany). Soluble interleukin-2 receptor (sIL-2R) was determined by EIA
kits (Endogen Inc., Cambridge, Mass., and Immunotech, Marseilles, France). sTNF-RII was quantitated in plasma at 1:20 dilution by using
EIA kits (HyCult, Uden, The Netherlands, and Genzyme, Cambridge, Mass.). TNF- was measured by EIA kits (Medgenix, Fleurus, Belgium, and Innogenetics, Zwijndecht, Belgium). IL-10 was measured by EIA kits
(Immunotech and Endogen). Gamma interferon (IFN-) was determined by
EIA kits with and without CIRID modifications of the protocols of the
manufacturers (Immunotech, T-Cell Diagnostics, Endogen, Biosource
International, Genzyme, and R&D Systems). All other assays were
performed according to the manufacturers' instructions. CIRID
modifications of each manufacturer's protocol consisted mainly of
increasing the times of incubation to overnight and the number of
washes between steps to 10.
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
assays were quite comparable. However, substantial and
significant differences in levels in plasma were found when test kits
from different manufacturers were used for 2M, neopterin, sIL-2R,
sTNF-RII, IFN-, and IL-10 assays. These data suggest that the major
difference between the compared kits is probably in the standards
provided for quantitation. In addition to the differences in standards,
the IL-10 levels determined by the two kits were poorly correlated,
indicating that there probably are substantial differences in the pairs
of antibodies utilized in the two IL-10 assay kits. Generally, similar effects were found when plasma samples from HIV-seronegative donors were tested and analyzed (data not shown).
TABLE 1.
Differences due to reagent sources in cytokine and
soluble activation marker levels in plasma from HIV-positive subjects
results agreed well, but
those for 2M and sIL-2R were substantially different. The standards from observed concentrations of 2M (NIBSC [National Institute for
Biological Standards and Control] 80123200 standard) were 1.00 mg/liter with Abbott IMx assay and 1.36 mg/liter with ORGenTec assay.
TABLE 2.
Comparison of different standards
from one manufacturer were
compared and found to differ significantly. The mean levels (± standard deviations) for 13 samples were 3,099 (± 3,201) pg/ml with
lot A and 1,465 (± 1,303) pg/ml with lot B.
Failure to detect cytokines in normal plasma.
In general,
cytokines could not be detected in the plasma of many healthy
individuals with kits available to our laboratory. IFN-, IL-10, and
TNF- could not be detected in 77%, 45%, and 7% of the healthy
subjects, respectively. However, our findings with soluble immune
activation markers indicated that all samples from healthy subjects had
measurable levels of 2M, neopterin, sIL-2R, and sTNF-RII.
Serum versus plasma measurements.
Both serum and plasma
samples from 16 to 52 HIV-seropositive individuals were tested for
2M, neopterin, sIL-2R, sTNF-RII, TNF-, IFN-, and IL-10 (Table
3). The mean levels of all analytes in
serum and those in plasma were very close (the coefficients of
variation [CVs] were less than 10%) except for IFN-, for which levels in plasma were higher than those in serum (CV was 39%), and for
IL-10, for which levels in serum were higher than those in plasma (CV
was 17%). Correlation analyses demonstrated a strong correlation
between plasma levels and serum levels of all analytes except IL-10,
for which the correlation was weak (Table 3). These data indicate that
there are no significant differences between levels of cytokines and
immune activation markers in plasma and those in serum except for
IFN and to some extent for IL-10. Thus, most of the levels could be
measured by using either of the two sample types.
|
Influence of freeze-and-thaw cycles on levels in plasma of
cytokines and soluble activation markers.
The effects of repeated
cycles of freezing of plasma and/or serum samples at 70°C and
thawing at room temperature were evaluated for five analytes (Fig.
1). One milliliter of each plasma and serum sample from three HIV-seropositive and four HIV-seronegative individuals was tested. Portions of each sample were pipetted into six
identical 1-ml cryovials, frozen at 70°C, and kept frozen for
20 h. Samples were brought to room temperature and kept over a 3- to 4-h period to thaw and then refrozen. Such freeze-thaw cycles were
conducted 1, 2, 3, 4, 5, and 10 times on sequential days for the sample
from each subject. After all freeze-thaw cycles were completed, the
aliquots from all seven subjects were tested on the same day and with
the same plate and reagent for 2M, neopterin, sIL-2R, sTNF-RII, and
TNF-. The results (Fig. 1) indicated that there were no significant
differences in mean levels of any of those analytes in plasma and serum
after up to 10 repeated freeze-thaw cycles. The CV between mean values
for the repeated cycles for each of the analytes was below 4%.
|
Influence of storage temperature on levels of cytokines and soluble
activation markers in plasma.
The stability of cytokines and
soluble activation markers in plasma and/or serum samples from six
HIV-seropositive and five HIV-seronegative individuals was evaluated
after storage for 20 days at room temperature (24°C), refrigerated
(4°C), or frozen (70°C). Samples from each subject were aliquoted
into three 1-ml cryovials and stored at the three temperatures. Twenty
days later, all samples were batch tested for 2M, neopterin,
sTNF-RII, sIL-2R, IFN-, and TNF-. No significant differences
among the mean levels at the three storage temperatures were seen for
the first five analytes (Fig. 2). The CVs
between levels at the three storage temperatures were below 14%.
However, the mean level of TNF- in samples kept at room temperature
was 55% lower than the mean levels in samples stored at 4 or 70°C
(CV was 38%).
|
Reference ranges for levels in plasma of cytokines and soluble
activation markers.
Donor characteristics may influence the
reference (normal) ranges for plasma cytokine and immune activation
marker levels. Data for two HIV-seronegative populations are presented
in Table 4. The mean levels of several
markers were found to be higher in the largely Caucasian seronegative
homosexual men (age range, 30 to 54 years) than in a medical center
employee population (age range, 24 to 65 years; 32% male and 68%
female) that included Asian, Hispanic, African-American, and Caucasian
donors. However, of the five parameters, only 2M and TNF- levels
were significantly different (P < 0.05) between the
two populations.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The purpose of this study was to determine the influence of various factors on the measurement of plasma cytokines and soluble activation markers. The comparability between kits for measurements of cytokine levels in samples obtained from a diverse population, including HIV-positive subjects with different stages of HIV disease as reflected by CD4 counts, was generally good. This may relate to the fact that international standards are available for many cytokines (5, 9, 20, 41, 47, 50).
Measurements of the soluble products of immune activation provide
different information than measurements of cytokine levels in plasma.
Cytokine levels reflect the production, removal, and retention of these
molecules. The soluble markers of immune activation, however, indicate
the biological effects of cytokines. Unfortunately, significant
differences in measurements of soluble activation markers were observed
when commercial kits from different manufacturers were used. This was
especially noteworthy for sIL-2R and 2M. The studies reported here
emphasize the differences between reference standards provided by
manufacturers. In some instances they are similar but not in every
case. However, there are not international standards for the soluble
receptors, such as sTNF-RII and sIL-2R, or for other products of
cytokine activity, such as neopterin or 2M.
Another problem has been changes made in reagents by manufacturers, which may give different quantitative results or may change the level of sensitivity of assays. Unfortunately, many workers in this field have discovered these changes when their results change, without having received forewarning by the manufacturers or suppliers.
Other reports have noted that some monoclonal antibodies recognize only monomers rather than naturally occurring polymers, a fact which can account for differences between one kit and another (38, 44). Soluble cytokine receptors in plasma and/or serum samples may affect the recognition of cytokines by immunoassay (3, 52, 58). Cytokines bound to soluble receptors may or may not be detected by any particular immunoassay, and methodologies such as EIA, RIA, and bioassay may provide different results (3, 13, 37).
Collection and storage procedures may influence the detectable levels
of cytokines and soluble markers. To avoid breakdown of some cytokines,
collection of blood into endotoxin-free tubes and prompt processing
before storage as cell-free plasma or serum at 70°C has been
recommended, especially for TNF- (14, 30, 33, 55). By and
large, the cytokines and soluble markers studied here were quite stable
if refrigerated or frozen. Samples kept at room temperature for 20 days, however, had TNF- levels appreciably lower than the mean
values in samples kept at 4 and 70°C. Plasma and serum cytokine and
soluble marker levels were almost the same. In our laboratory the
correlation coefficient for a comparison of serum and plasma neopterin
levels was 0.961 (Table 3). Only the mean level in plasma for IFN-
was higher (77%) than the level in serum.
Several studies have demonstrated substantial differences between
laboratories that were using the same kits for soluble marker measurements (reference 4 and unpublished
observations). Thus, factors relating to assay performance can
contribute to differences in observed values for cytokines and for
immune activation markers. Furthermore, the quality of laboratory data
can be important in clinical evaluations of prognosis or prediction of
disease progression. We have studied a set of 594 plasma samples that
originated at UCLA and were tested for neopterin levels at UCLA and
another site. The correlation between the data was not strong
(r was 0.614). The log-likelihood for the regression model
(17) of AIDS occurrence in these patients within the 3 years
following the sample draw was 248 based on the UCLA neopterin data
and 264 based on the data at the second laboratory. The larger
log-likelihood number in the second set of data indicates poorer
precision of prediction. Thus, it is evident that the quality of
laboratory data can have an impact on clinical relevance. Furthermore,
the difference in the proportion with AIDS at 3 years between subject
groups in the lowest and the highest quartiles of Kaplan-Meyer plots
for neopterin was 0.58 for UCLA and 0.38 for the other laboratory. Similar experiences in the early days of CD4 T-cell measurement (8, 22) indicated that superior laboratory performance
related to clinical outcome, whereas poor relationships were found with data from laboratories that demonstrated more erratic testing. Also,
differences in laboratory performance could have been responsible for
the differences in prognostic value of CD4 levels vis-à-vis HIV
viral load, where the CD4 level was found to have no value (34) and substantial value (35).
Research laboratories and service laboratories may differ significantly in their approaches to and capacities to conduct clinical investigations. Research laboratories may not have the resources or not be attuned to the needs for determination of intraassay, interassay, and intrasubject variation and interlaboratory differences, for CV calculations, for routine quality control procedures, for factors relevant to testing of serially obtained samples, or for measurements in appropriate reference (normal) populations.
Some service laboratories may need to modify preexisting procedures or reagent selection in order to meet the needs for uniform testing at multiple sites, as is required in clinical trials. Frozen storage of samples for long periods and determinations of intrasubject variability are not part of the routine in most service laboratories. Also, established charges for clinical tests may be outside the budget allowances of clinical studies.
Attention to the issues noted in this report is needed to avoid
conclusions that provide misinformation, in terms of both inappropriate
claims and failures to detect significant relationships between
immunological changes and clinical events. Measuring cytokines and
soluble activation markers is not quite as simple as ordering kits out
of the catalogue and following the manufacturer's instructions. Potential problems can be avoided by conducting assays with careful attention to quality control procedures (4) and issues
reported here. Suggested evaluation steps for ELISA kits and comparison of different suppliers of reagents are summarized in Table
5.
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Institution of external proficiency testing and/or quality assurance programs could assist laboratories that are conducting cytokine and soluble activation marker measurements. The field will benefit by the establishment of international reference standards for the soluble cytokine receptors and other immune activation products. The use of resources now available, e.g., the international reference standards for cytokines, can also be recommended.
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ACKNOWLEDGMENTS |
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We express our appreciation of the participants and the staff of the Adult AIDS Clinical Trial Unit and the Advanced Technology Laboratory at UCLA and Jonathan Kagan and Daniella Livnat of the Drug Development and Clinical Sciences Branch, TRP, DAIDS, NIAID, National Institutes of Health, who made these studies possible. Some samples discussed in this manuscript were collected by the Multicenter AIDS Cohort Study with centers (principal investigators) at the following institutions: The Johns Hopkins School of Public Health (Joseph B. Margolick and Alvaro Muñoz); Howard Brown Health Center and Northwestern University Medical School (John Phair); UCLA (Roger Detels and Janis V. Giorgi); and University of Pittsburgh (Charles Rinaldo). We are grateful for the organizational support of Susan Stehn, technical assistance of Hripi Nishanian, statistical efforts of Joanie Chung, and manuscript preparation by Deborah Mathieson.
Support was provided by grants AI-36086, AI-38858, AI-35040, and AI-27660 from the National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90095-1747. Phone: (310) 825-6568. Fax: (310) 206-1318. E-mail: jlfahey{at}ucla.edu.
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Clinical and Diagnostic Laboratory Immunology, January 1999, p. 89-95, Vol. 6, No. 1
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