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Clinical and Diagnostic Laboratory Immunology, November 1998, p. 755-761, Vol. 5, No. 6
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Levels of Cytokines and Immune Activation Markers
in Plasma in Human Immunodeficiency Virus Infection: Quality
Control Procedures
Najib
Aziz,1,*
Parunag
Nishanian,1 and
John L.
Fahey1,2
Departments of
Medicine2 and
Microbiology and
Immunology,1 Center for Interdisciplinary
Research in Immunology and Disease, Jonsson Comprehensive Cancer
Center and UCLA AIDS Institute, UCLA School of Medicine, Los
Angeles, California 90095-1747
Received 15 May 1998/Returned for modification 8 July 1998/Accepted 28 July 1998
 |
ABSTRACT |
Procedures for quality control (QC) in a laboratory that
concentrates on cytokine and soluble marker measurements in
biological fluids are outlined. Intra-assay, interassay, and
interlaboratory experiences are presented. Plasma and serum
2-microglobulin (2M) and neopterin test data are presented in
greatest detail, along with substantial tumor necrosis factor alpha
(TNF-
), gamma interferon, soluble interleukin-2 receptor-
(sIL-2R), sTNF-RII, IL-4, and IL-6 data. Recommended QC procedures
for cytokine and soluble-marker testing include replicate testing
of two or more reference samples provided by the kit manufacturer,
replicate testing of in-house frozen reference QC samples that
represent normal and abnormal analyte contents, retesting 15 to 20% of randomly selected samples, and comparing normal reference
ranges each year. Also, eight cytokines and soluble markers were
evaluated in human immunodeficiency virus (HIV)-seronegative and
HIV-seropositive individuals stratified on the basis of CD4 T-cell
numbers. Levels of some but not all cytokines in serum increased in HIV
infection. There was a tendency for cytokines to increase with more
advanced disease, defined by reduced CD4 T-cell numbers. Cytokine
changes did not relate closely to CD4 level, indicating that separate
information was provided by the measurements of TNF-, sTNF-RII,
sIL-2R, 2M, and neopterin. Serum IL-4 and TNF- levels were not
increased. The quality of laboratory data can impact on clinical
relevance. Interlaboratory comparisons revealed substantial
differences at some sites and documented the need for external
proficiency-testing quality assurance programs.
| |
INTRODUCTION |
Cytokine levels and changes in
biological fluids are now recognized as potential and useful markers of
ongoing clinical disorders, indicating their stage and severity and
disease prognosis (1, 10, 13, 42). Initial evidence of
immune activation in human immunodeficiency virus (HIV) infection
included increases in several phenotypic antigens on circulating
lymphocytes as well as increases in levels of soluble products of
cytokine activity in plasma (2, 3, 15, 38, 39, 43, 47).
2-microglobulin (2M) increases were reported early in the
characterization of AIDS (21, 35) but were not perceived as
related to disease course until several years later (11).
2M represents the activity of several cytokines throughout the body
(19, 20) and is a relatively nonspecific marker of immune
activation. Neopterin, on the other hand, which is induced by gamma
interferon (IFN-
) activation of monocytes, was found at elevated
levels in HIV infection and was related to prognosis (11, 23, 28,
48).
The measurement of the levels of cytokines and/or soluble markers of
immune activation can provide reliable information regarding the
disease diagnosis, disease stage, prognosis, and the evaluation of
therapy. However, difficulties and inaccuracy have been reported, and a
number of factors have been shown to affect the validity and the
quality of such measurements (5, 17, 29, 30, 46, 49).
Immunoassays are the most widely used technique for these measurements,
although pitfalls and limitations are known (24, 36, 37).
Differences in levels of measured analytes for identical samples in the
range of 10- to 100-fold have been reported (26, 31,
32). Thus, a number of studies, including international
collaborative studies organized by the World Health Organization for
standardization of cytokine measurements, have been conducted (4,
7, 16, 31-33, 40). Variations in results have been shown to
be due at least in part to differences in the standards used in the
assays (16, 26, 30-33, 40) or in sample collection,
processing, and storage (9, 27, 41, 45).
In our early work with the assessment of neopterin and 2M
concentrations in plasma and in subsequent testing of cytokines and the
products of cytokine activity, we have had large numbers of samples
available to test but limited funds. As a consequence, we looked for a
means to conserve costly reagents but, at the same time, to ensure
consistency and accuracy of testing. Initial testing showed good
agreement between duplicate samples. Thus, we chose to do single
determinations rather than duplicates but also to randomly retest
approximately 15% of samples. This approach had the advantage of
providing representative duplicate measurements and a check on
comparability between analytic runs. As an additional quality control
(QC) procedure, we established a method of preparing a large number of
frozen aliquots from sizable pools of plasma or serum, one each
representing normal levels and abnormally elevated levels of the
cytokines and soluble markers of activation. Aliquots of these
reference standards were required to be included in each analysis of
cytokines or activation markers. Upon repeated testing, we were able to
establish the validity of runs and the comparability of reagents and
technical performance. These QC procedures are now routinely used for
testing neopterin, 2M, soluble tumor necrosis factor receptors I and
II (sTNF-RI and -RII), soluble interleukin 2 receptor-alpha
(sIL-2R), soluble CD8 antigen, the cytokines TNF-, IFN-, IL-1,
IL-2, IL-4, IL-6, IL-10, and IL-12, and chemokines.
Manufacturers of commercial kits for the measurement of these analytes
provide data characterizing their performance with samples of their own
selection. However, each laboratory has its own performance
characteristics. In our present report, the same QC samples were used
for all assays. We report here on the procedures used and on the
coefficient of variation (CV) obtained in control populations and at
different stages of HIV infection. In addition, intra-assay,
interassay, and interlaboratory variabilities are reported.
(These data were presented in part at the 3rd International Symposium
on Clinical Immunology in San Francisco, Calif., 20 to 23 July 1995.)
| |
MATERIALS AND METHODS |
Samples.
Serum and plasma samples were obtained from healthy
volunteers from the University of California, Los Angeles (UCLA)
community and from HIV-seronegative and HIV-seropositive subjects
participating in the Multicenter AIDS Cohort Study (MACS) of the
natural history of AIDS, who were recruited and monitored at UCLA at
approximately 6-month intervals from 1984 (25). Blood was
collected by venipuncture into 15-ml sterile Vacutainers (Becton
Dickinson) containing heparin as an anticoagulant for plasma samples
and without anticoagulant for serum samples. Serum and plasma samples
were separated and stored at 
70°C for subsequent batch testing of
cytokine and soluble immune activation markers.
The general reference (normal) samples were obtained from male and
female employees at UCLA with an age range of 24 to 65 years; 32% were
males and 68% were females. Human Subject Protection Committee
approval was obtained for all studies. The study of cytokine and
soluble marker changes in HIV infection was conducted with sera from 15 subjects who were negative for HIV antibodies at the time of selection
and from 56 HIV-seropositive subjects who were separated into four
groups (13 to 15 subjects in each group) according to their absolute
number of CD4 T cells. The defining levels of CD4 T cells were 500 to
700, 350 to 499, 200 to 349, or fewer than 200/mm3.
The in-house QC samples were prepared in our laboratory as two
high-volume pools of serum or plasma (500 ml each). One pool
from
HIV-negative donors had levels of cytokines and soluble markers
within
the normal range. The other pool was prepared from HIV-seropositive
samples and was distinctly abnormal, with elevated levels of many
cytokines and soluble markers. The samples used for QC purposes
were
comparable to the study patient samples. Aliquots of 1 ml
each were
stored in labeled tubes in a
70°C freezer until removed
and thawed
for
assay.
Quantitation of levels of cytokines and soluble activation
markers in plasma.
2M was quantified by microparticle
enzyme immunoassay (Abbott Laboratories, Abbott Park, Ill.) and
reported in milligrams per liter. Neopterin was measured by
competitive radioimmunoassay kit (IMMUtest Neopterin; BRAHMS,
Berlin, Germany) and expressed as nanomoles per liter. sIL-2R was
measured with an enzyme immunoassay (EIA) kit (T Cell Diagnostics,
Cambridge, Mass.; now available from Endogen, Woburn, Mass.) and
reported in units per milliliter. sTNF-RI and sTNF-RII (1:20 dilution)
were quantified in plasma with EIA kits (R&D Systems) and expressed as
picograms and nanograms per milliliter, respectively. TNF- was
measured by Innotest hTNF EIA kits (Innogenetics N.V., Antwerp,
Belgium) and reported in picograms per milliliter. IFN- was
measured with radioimmunoassay kits (Centecor; no longer available) by
a Center for Interdisciplinary Research in Immunology and Disease
modified protocol (increases of incubation time to overnight and of
number of washings between steps to 10 times) and was reported in units
per liter. IL-4 and IL-6 were measured with EIA kits (Genzyme,
Cambridge, Mass.). The measurements of cytokine and activation marker
levels were performed according to the manufacturer's instructions.
The reference standards for each test were provided by the
manufacturer. For most of the assays the manufacturers have calibrated
the kit standards to the reference standards of the NIBSC and the World
Health Organization, if already available.
| |
RESULTS |
Intra-assay variation.
Intra-assay variability was evaluated
with 10 replicates of two different QC plasma samples in the same run.
Intra-assay variabilities of 2M, neopterin, sIL-2R, sTNF-RII,
TNF-, and IFN- are presented in Table
1. CVs of soluble markers are under
7.5%, except for TNF- and IFN- (Table 1). Similar results were
found when two or more QC samples were tested in three different wells
during routine assays (data not shown). Common criteria for acceptable performance cited by a clinical laboratory improvement amendment (14) in other quantitative plasma immunology tests are the
target values plus or minus 3 standard deviations, but we use 2 standard deviations in our laboratory.
Interassay variation.
Aliquots of the two in-house QC samples
were included on each assay day. The CV of serial assays for 2M,
neopterin, sIL-2R, sTNF-RII, TNF-, and IFN- are listed in Table
2. The CV was less than 15% for all
markers except TNF- and IFN- in normal control samples. Similar
data are seen in repeat testing of four plasma activation markers in 15 to 20% of patient samples on a subsequent assay day (Table
3). Actual data for serial testing of the
in-house QC samples for neopterin and sTNF-RII tests over an 18-month
period are presented graphically in Fig.
1.
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|
FIG. 1.
Interassay variations in concentrations of neopterin
(NPT) (A) and sTNF-RII (B) in normal (filled circles) and abnormal high
(open circles) in-house QC preparations. Each point represents the mean
value of triplicate testing.
|
|
Normal population values tested in different years.
Volunteer
healthy donors, men and women from 24 to 65 years of age, from the UCLA
community are tested as normal reference controls every year. The
values obtained in three recent years were assembled and compared
(Table 4). Because the reference populations were not identical from year to year, we did not expect to
obtain identical values. These data provide reference ranges with which
to judge the changes in reagents and technical staff performance from
year to year.
Interlaboratory variation: external proficiency testing for quality
assurance.
From 1989 to 1992, four laboratories participated in a
QC program for two soluble activation markers (2M and neopterin). The same kits and analytic reagent lots were used in each laboratory (Pharmacia AB, Uppsala, Sweden, supplied the 2M assay kits used for
this study). The test samples were shipped frozen in dry ice overnight to each participating laboratory. The results are presented in Fig. 2. Agreement was better
after the third sample. There was generally good agreement between
laboratories for 2M measurements with relatively low CVs. The range
of CVs for normal-level neopterin QC samples was 9 to 70% (Fig. 2D),
and the range of CVs for elevated neopterin levels was 3 to 64% (Fig.
2C). Laboratory 2 reported lower levels of neopterin than the other
laboratories on most dates. This laboratory was the only one using
round-bottom tubes and may have been less successful in the washing
step of the neopterin test.
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FIG. 2.
Levels in plasma of elevated (A and C) and normal (B and
D) external QA samples for 2M (A and B) and neopterin (C and D)
assayed in laboratories 1 ( ), 2 ( ), 3 ( ), and 4 ( ),
participating in an external QA program from 1989 to 1992. To avoid a
change in the presentation of the other data, the outlier value of test
11 in panel A is reported without being plotted in the scale.
|
|
Cytokine and immune activation markers in plasma in HIV
infection.
The plasma samples from 56 HIV-seropositive and
15 HIV-seronegative subjects from the MACS population were
measured for levels of 10 cytokines and soluble markers. When the
HIV-positive individuals were stratified by CD4 levels (Fig.
3), progressive increases in the levels
of IFN-, TNF-, IL-6, sIL-2R, sTNF-RII, 2M, and neopterin were
generally seen. IL-4 levels, in contrast, tended to be reduced
(P < 0.05). Preliminary results indicated that
levels of TNF-, IL-1, and sTNF-RI were not significantly
different between HIV-negative and HIV-positive sera. There were
substantial spreads of the cytokine and soluble marker levels in each
CD4 category (Fig. 3). This is consistent with data indicating that the
levels of immune activation markers in plasma provide different information than CD4 T-cell levels on the pathogenic mechanisms in HIV
infection (11, 12).
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FIG. 3.
Cytokine and soluble marker levels in serum (means ± standard error) of 15 HIV-seronegative () and 56 HIV-seropositive
subjects stratified by CD4+-lymphocyte levels: >500 (A),
350 to 499 (B), 200 to 349 (C), and <200 (D) lymphocytes per µl.
|
|
| |
DISCUSSION |
The procedures recommended for intralaboratory quality control of
cytokine and soluble marker testing in biological fluids are outlined
in Table 5.
The procedures described here differ from those outlined by the
manufacturers of reagents, in that duplicate testing was not conducted
after initial experience indicated that a technologist with the
reagents achieved CVs under 10% for intra-assay variability and under
10 to 15% for interassay variability, with the exception of TNF-.
The interassay variability was usually greater in the normal
(lower) range, where the cytokine or soluble-marker concentrations were
near the lower limit of the analyte detectability in plasma or serum
and the precision of reference standard data is lower.
The procedures described here differ in some respects from those
described in methodology manuals and reports. We recommend our
procedures to laboratories which do many tests of this type with some
frequency and with technicians of proven skill in this area. Doing
duplicates (or triplicates) as well as the in-house reference sample
aliquots and the manufacturer reference materials is important if
testing is done infrequently. Graphic methods for assessing QC data are
available (22).
External proficiency testing should facilitate the quality and
comparability of laboratory performance. This proved to be important for measurements of CD4 T-cell levels in HIV-infected patients (6, 18). However, until external quality
assurance (QA) proficiency testing programs are available for cytokine
and soluble marker analyses, testing for clinical studies should be conducted in a single experienced laboratory with strong
established internal QC procedures. It is recommended that each
laboratory establish its own ranges for normal reference populations
and test samples from patients with diseases characterized by abnormal levels of cytokines and/or soluble activation markers in order to be
confident that abnormal levels can be detected in representative body
fluids, usually plasma and serum.
While levels of cytokines in plasma may have some appeal (and are
needed in a few research contexts), it is useful to remember that many
cytokines cannot be accurately quantified in plasma or serum (17,
24, 36, 46, 49). Some of those that can be detected may show
substantial variability because assays are at or near their limits of
precise measurement. A variety of other factors could cause such
effects. In contrast, measurement of the levels of immune activation
markers and soluble products of cytokine activity in plasma may be
preferable because they reflect the sum of lymphoid cells contributed
from the entire body and are generally detectable by more precise
quantitative assays.
Several points can be made about the changes in levels of cytokines and
soluble markers of disease activation in plasma. Serial testing of
individuals has revealed several characteristic and different patterns
of cytokine and soluble marker changes in HIV disease progression
(34, 44). A broad range of levels of cytokines and plasma
markers of activation in plasma, representing cytokine activity
throughout the body, were found in each of four major CD4 T-cell
categories in HIV infection. This emphasizes the difference in disease
course or activity as represented by the soluble products of activation
versus the level of damage to the CD4 maintenance systems, as
represented by the CD4 T-cell levels. Epidemiological studies have
shown that these parameters provide different information and that
combinations of the two types of measurement give more precise
prognostic data than either alone (11, 12). There was no
evidence of a shift from a Th1 to a Th2 pattern of cytokine expression
with disease progression in these data. Viral load measurements in
plasma have been shown to give good prognostic information. However,
the CD4 plus cytokine-soluble-marker combinations may approximate
viral load data in prognostic value (8, 12). Furthermore, in
advanced disease, CD4 or activation marker levels may be prognostically
superior to plasma HIV load measurements (8, 12).
Differences in results between laboratories may occur, and examples are
documented here. Also, laboratories can vary in the quality of their
day-to-day performance. This can be attributable to variations in
reagents, to differences in technical proficiency, and to other
factors. The need for proficiency testing programs is evident.
| |
ACKNOWLEDGMENTS |
We appreciate the support of the entire MACS with centers (and
principal investigators) at The Johns Hopkins School of Public Health
(Joseph B. Margolick and Alvaro Muñoz), Howard Brown Health Center and Northwestern University Medical School (John Phair), University of Pittsburgh (Charles Rinaldo), and UCLA (Roger Detels and
Janis V. Giorgi). In particular, Roger Detels has encouraged excellence in laboratory technology contributing to epidemiological studies of HIV and AIDS since 1984. CD4 measurements were performed in
the laboratory of Janis Giorgi at UCLA. We wish to acknowledge the excellent technical assistance of Hripi Nishanian and Mehran Bozorgmehri, the statistical assistance of Joanie Chung, and
the assistance of Deborah Mathieson in manuscript preparation. The many
professional contributions of Bo Hofmann to specific aspects of this
work were noteworthy.
This work was supported by grants AI-35040, AI38858, and AI 36086.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90095-1747. Phone: (310) 825-0825. Fax: (310) 825-0595. E-mail: naziz{at}ucla.edu.
| |
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Clinical and Diagnostic Laboratory Immunology, November 1998, p. 755-761, Vol. 5, No. 6
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
