Clinical Chemistry 45: 1543-1547, 1999;
(Clinical Chemistry. 1999;45:1543-1547.)
© 1999 American Association for Clinical Chemistry, Inc.
Preanalytical Factors (Biological Variation) and the Measurement of Serum Soluble Intercellular Adhesion Molecule-1 in Humans: Influence of the Time of Day, Food Intake, and Physical and Psychological Stress
Benoit Duguéa,
Esa Leppänen and
Ralph Gräsbeck
a Author for correspondence. Fax 358-9-4771025.
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Abstract
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Background: Adhesion proteins such as soluble intercellular
adhesion molecule-1 (sICAM-1) may be important markers for early
atherosclerosis and some other diseases. To devise optimum specimen
collection procedures, we investigated the effects on serum sICAM-1 of
several preanalytical factors (factors that occur before a specimen is
analyzed) such as the time of the day, food ingestion, and physical and
psychological stress.
Methods: Three sets of experiments were conducted:
(a) 30 subjects were investigated during the morning,
after an overnight fast, and then after the usual breakfast and at
1200; (b) 20 subjects were studied before and after
exposure to thermal stress (sauna + swimming in ice-cold water); and
(c) 15 volunteers were investigated after their driving
license examination and during a (stress-free) control session.
Conventional methods and kits were used to determine the blood picture
and serum sICAM-1.
Results: All of these preanalytical factors induced a significant
increase (~10%) in the concentration of sICAM-1.
Conclusion: It is advisable to consider timing, food intake, and
stress when collecting specimens and analyzing data on the
concentration of sICAM-1 in serum.
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Introduction
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Intercellular adhesion molecule-1 (ICAM-1)1
is a
widely distributed adhesion factor present on the surfaces of
endothelial cells and leukocytes. This protein mediates adhesion and
transmigration of leukocytes through the endothelium. Surface expressed
ICAM-1 is apparently shed from the cells and then circulates as
soluble ICAM-1 (sICAM-1). A recent study demonstrated that subjects
with basal plasma concentrations of sICAM-1 in the top quartile of the
health-related reference interval have an 80% higher risk of
developing myocardial infarction than those with concentrations in the
lowest quartile (1). Although exercise has a beneficial
effect on cardiovascular morbidity, it may increase the concentrations
of sICAM-1 (2)(3)(4).
Because this analyte may become of importance in routine laboratory
testing, it is important to collect information about its biological
variation and about the effects of preanalytical factors that may
influence the result of the assay. In the present study, we
investigated the effects of several preanalytical factors: food intake,
time of the day, and physical and psychological stress. In addition, we
calculated different kinds of indices related to biological variation.
Finally, the results were considered against the background of
population-based health-related reference values.
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Subjects and Methods
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subjects and design of the experiment
All specimens were taken from 65 subjectively healthy persons
(ages 18–64 years; mean, 35.8 years) who had given informed consent to
participate in the study. All subjects reported the absence of chronic
and inflammatory illness and the use of any medication (other than oral
contraceptives) during the week before the experiment. All of the
specimens were collected by an experienced laboratory technician
following the Scandinavian recommendations (5), i.e., the
subject sat for at least 15 min before venipuncture. Blood was
collected from the cubital vein using minimum tourniquet,
Venoject® needles, and evacuated blood
collection tubes for serum and EDTA plasma (Terumo).
Serum-separator tubes were not used. These studies were approved by the
Ethics Committee of our Institute.
breakfast and time of day
Thirty volunteers participated, 21 women (ages 18–58 years; mean,
36.8 years) and 9 men (ages 23–47 years; mean, 38.2 years). We
recently described this experiment (6)(7).
Sample collection (repeated punctures) took place at around 0800
immediately after a 15-min rest period; at around 1000, after the
volunteers had eaten; and again at 1200. We provided a selection of
foodstuffs commonly used for breakfast in Finland (coffee or tea, bread
and butter, cheese, juice, milk, porridge). The participants were
instructed to have a breakfast as similar as possible to their usual
meal. The energy intake was <500 kJ for 8 participants, 500-1000 kJ
for 14 participants, and >1000 kJ for 8 participants.
winter swimming test
Our experiments took place at the end of the winter season.
Eleven women (ages 25–52 years; mean, 38.7 years) and 9 men (ages
19–62 years; mean, 44.0 years) participated. Among them, five women
and seven men were regular winter swimmers who bathed in ice-cold water
more than once a week during the winter. The experiments took place in
and near a sauna situated 50 m from a lake. The volunteers first
sat naked in the sauna in 95 °C with a relative humidity of 35–50%
until they felt exhausted (mean time in hot atmosphere, 15 min), after
which they walked to the lake to bathe. When they walked to and from
the lake, they were subjected twice for ~1 min to air with a
temperature between -5 and -15 °C. The subjects entered the water
quickly and were immersed in ice-cold water for a mean of 30 s.
This experiment was organized on four different occasions during late
winter because it was not possible to accept more than six individuals
per session. The above sequence of events is typical among winter
swimmers. Three blood specimen collections took place: at rest before
the sauna bath after a period of 15 min of sitting, just after the
sauna, and after exposure to cold and the return to the dressing room.
driving license exam
Fifteen volunteers participated, 8 men (ages 18–47 years; mean,
24.3 years) and 7 women (ages 18–50 years; mean, 28.1 years). The
theoretical part of the Finnish driving license is an individual test
delivered through a computer at a public office. Because only three
computers were available, the sessions involved a maximum of three
persons. The exam consisted 60 questions with a 30-min time limit for
answers. There were two types of questions: written questions and
questions about pictures, with time limits for answering of 30 s
and 10 s, respectively. Approximately 75% of all those taking the
test passed. The volunteers were recruited just before the test. After
they finished the exam and learned the result, the volunteers were
invited to estimate their degree of wakefulness, irritability,
annoyance, and good mood, using a scale rated from 1 (low) to 10
(high). Blood specimens were collected after the volunteers finished
the questionnaire. A control session was organized during weekends at
Maria Hospital, Helsinki, at the same hour (mean of 5 weeks between the
sessions). A snack was arranged after the sessions to thank (and
motivate) the volunteers.
sICAM-1 MEASUREMENTS
ELISA kits for the determination of sICAM-1 were purchased from
R&D Systems Europe. The intraassay imprecision
(CVA) was 4.6% and 4.8% for sICAM-1
concentrations of 126 and 476 µg/L, respectively. These results were
obtained from serum samples assayed in replicates of 10.
blood picture and differential counts
Blood smears were drawn immediately after the venipuncture (from
specimens drawn into EDTA tubes). May-Grünwald-Giemsa stain was
used for the differential count. The specimens for the blood picture
determination were analyzed in a Sysmex-1000 machine.
data processing and statistical analysis
The data were analyzed with nonparametric statistical methods: the
Wilcoxon signed-rank test for paired data and the Mann–Whitney test
for group comparisons. When three groups were compared, the Friedman
test was used. The biovariability was analyzed as described by Fraser
and Harris (8). Two CVs were calculated, representing
within- and between-subject variation (CVI and
CVG, respectively). The CVI
included the analytical component. We also calculated the index of
individuality (CVI/CVG);
the index of heterogeneity {i.e., the ratio of
CVI to the theoretical CV, which is [2/(n
- 1)]1/2, where n is the number of specimens
collected per subjects}.
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Results
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The results are summarized in Tables
1 and
2. We observed that all the factors studied (breakfast and
physical and psychological stress) led to a modest but significant
increase of the concentration of sICAM-1 in serum.
A time delay after the fasting state in early morning led to an
increase in the concentration of sICAM-1. When the results were
corrected for hemoconcentration, the increase in sICAM-1 after
breakfast (specimens taken at 1000) was still significant (7.2%;
P <0.05) but did not reach significance at 1200. However, a
significant increase in leukocyte counts occurred.
After winter swimming (sauna and the ice-cold baths), the increase was
rather modest and similar to the rise in hematocrit. Actually, when the
results were corrected for hemoconcentration, no significant change was
detected (P <0.6). However, the subjects who practiced
regular winter swimming exhibited a slightly higher basal concentration
of sICAM-1 compared with occasional winter swimmers (257 ± 54 vs
194 ± 35 µg/L; Mann–Whitney test, P <0.01). The
women who regularly practiced winter swimming had a higher basal
concentration of white blood cells [7.9 (± 0.8) x
109 vs 6.3 (± 0.9) x
109/L; P <0.02] and sICAM-1
(234 ± 30 vs 184 ± 36 µg/L; P <0.05).
The significant increase in sICAM-1 observed after the driving license
session did not correlate with hemoconcentration and changes in the
leukocyte counts. When the results were corrected for
hemoconcentration, the changes were still significant (6%;
P <0.01). The subjective feelings of irritability and
wakefulness were significantly higher after the exam than during the
control session (3.0 of 10 vs 6.2 of 10 and 3.3 of 10 vs 5.1 of 10,
respectively). Surprisingly, after the stress session, there were
significant correlations between the subjective feelings of
irritability and wakefulness and the serum concentration of sICAM-1
(Spearman correlation, P <0.01). In all of these
experiments, no effects attributable to the age and gender of the
volunteers were detected.
The biological short-term variation of sICAM-1 during the morning was
8.6% for the within-subject variability and 37.2% for the
between-subject. The
CVI/CVG was 0.23, and the
index of heterogeneity was 0.82. The data from the winter swimming and
the driving license studies showed similar variability.
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Discussion
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Soluble adhesion proteins such as sICAM-1 may become
important markers for early atherosclerosis (1) and some
other diseases [e.g., see Refs. (9)(10)(11)(12)(13)(14)(15)(16)]. We
present here the influence of several preanalytical factors on the
outcome of this interesting analyte.
The significant increase observed in the sICAM-1 concentration after
breakfast does not appear to be related to changes in plasma volume.
Surprisingly, no effects on the concentration of sICAM-1 were observed
after a 100-g oral glucose tolerance test (17). Because of
the large increases in leukocyte counts in our study, it is possible
that there was an increase in the number of leukocytes expressing
ICAM-1 on their surfaces and also an increased activity of the
secretases (18) responsible for the cleavage of sICAM-1 from
the cells.
Acute physical stress may cause increased concentration of circulating
sICAM-1 (2)(3)(4)(19). However, in the reports of
these studies (2)(3)(4), no comments were made on possible
correlation with the changes in plasma volume. In one study
(3), the specimens were taken from supine subjects. If the
specimens after stress were taken immediately after the volunteers laid
down, the higher hydrostatic pressure produced by the previous upright
posture may have influenced the results. Detailed kinetics of changes
in plasma volumes (maximum change, 14% of the total volume;
t1/2=2.8 min) attributable to changes
in posture have been published recently
(20)(21). Therefore, the clear-cut increase
(24%) in the concentration of sICAM-1 (3) may be only
partly attributable to muscular exercise. Actually, an increase of only
11% in the sICAM-1 concentration in serum was observed after physical
exercise (19) when the changes were corrected for
hemoconcentration.
After the sauna and ice-cold baths, we observed a significant increase
in the serum concentration of sICAM-1. However, these changes seemed
related to hemoconcentration. These stimuli also induced a large
increase in the concentration of cortisol (data not shown). Moreover,
at the end of the winter season, regular winter swimmers had a higher
concentration of sICAM-1 compared with occasional winter swimmers.
Interestingly, exercise training over a period of 2 months leads to an
increase in sICAM-1 (2). However, regular winter swimmers
tend to exhibit a similar enhancement in both the sICAM-1 concentration
and the leukocyte counts compared with occasional winter swimmers.
Therefore, a part of the sICAM increase could be produced by an
increase in the number of leukocytes expressing ICAM on their cell
surfaces. However, the physical stress we studied was of a totally
different kind than those reported previously. Surprisingly, a recent
publication reports increased concentrations of sICAM-1 (22%) after a
4-min cold water immersion of the hands only (22). Our
findings are at variance with those results because our subjects were
totally immersed in cold water, but no such changes were seen.
To our knowledge this is the first report of a significant increase of
sICAM-1 after psychological stress, i.e., the driving license test.
Although it was only moderately stressing (but enough to significantly
increase the concentration of cortisol; data not shown), we detected a
significant increase in the concentration of sICAM-1 not related to
hemoconcentration or leukocyte count. A previous study failed to reveal
any changes in sICAM-1 after psychological stress (23).
Our biological variabilities were larger than those reported previously
(37% vs 20% for CVG; 8.6% vs 6.1% for
CVI) (24). However, this previous
study was performed on a few young males only (n = 18 for
CVG and n = 5 for
CVI). In our study, the low index of
heterogeneity indicated that all subjects presented similar intrinsic
variances (8). When the
CVI/CVG is <1.4,
population-based reference values are useful, but when it is <0.6,
they become unsuitable for assessing whether a change has occurred (the
reference change being 2.77 CVA+I for P
<0.05 when the "pure" CVI is calculated)
(25). The
CVI/CVG for sICAM-1 was
clearly lower than 0.6, indicating marked individuality. Interestingly,
the index calculated with the data just discussed (24) gave
a similar result. However, our CVIs represent
only short-term variation (i.e., during the morning hours). It is
reasonable to assume that if the CVIs were
obtained from specimens collected during a longer period they would be
slightly higher than ours. However, similar results were obtained using
the data from our driving license test, in which the specimens were
collected with a mean interval of 1 month. Therefore, it is likely that
assays of sICAM-1 are of limited usefulness in detecting early
disease-associated changes evaluated using health-related reference
values.
Here, we present the effects of several preanalytical factors on
sICAM-1 in serum. The changes induced were <9% and should be compared
with the ~25% higher concentrations present in atherosclerosis
(26) or diabetes (27). Clinical and statistical
significance should not be confused. However, the existence of
statistically significant changes suggests that it is important to
consider timing, food intake, stress, and similar preanalytical factors
when analyzing data on the concentration of sICAM-1 in serum.
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Acknowledgments
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This research was supported by the Signe and Ane Gyllenberg, the
Ella and Georg Ehrnrooth, the Magnus Ehrnrooth, the Oskar Öflund,
and the Yrjö Jahnsson Foundations. We thank Arja Haapalinna,
Liisa Haapalinna, Jaana Friman, and Vesa Yli-Pelkonen for excellent
technical help. We also thank the volunteers who participated in this
study, the city of Vantaa for allowing us to use the facilities at Lake
Kuusijärvi, and the Driving License Office in Helsinki.
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Footnotes
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Minerva Foundation Institute for Medical Research, Tukholmankatu 2, 00250 Helsinki, Finland.
1 Nonstandard abbreviations: ICAM-1, intercellular adhesion molecule-1; sICAM-1, soluble ICAM-1; CVI, within-subject variability; CVG, between-subject variability; and CVI/CVG, index of individuality. 
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[Abstract]
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Abstract
Introduction
