Journal of Immunological Methods 206 Ž1997. 61–71
Changes in the polymorphonuclear leukocyte function of blood
samples induced by storage time, temperature and agitation
Gerd Egger
a,)
a
, Elisabeth M. Kukovetz a , Marianne Hayn b, Judith S. Fabjan
b
Institute of General and Experimental Pathology, UniÕersity of Graz, Graz, Austria
b
Institute of Biochemistry, UniÕersity of Graz, Graz, Austria
Received 21 February 1997; revised 3 April 1997; accepted 16 May 1997
Abstract
We have investigated changes in polymorphonuclear leukocyte ŽPMN. functions of blood samples caused by such typical
elements of laboratory handling as storage time, temperature and agitation. The blood of five healthy subjects was stored
upright in test tubes at 4, 22 and 378C over periods of 20 min, one, two, six and 24 h. Controlled agitation was performed on
a shaker. The following PMN functional parameters were measured: the white blood cell count ŽWBC., migration, elastase
ŽEL. release, reactive oxygen species ŽROS. production and lipid peroxidation. Migration was determined in a whole-blood
membrane filter assay; ROS production by latex-stimulated, luminol-enhanced chemiluminescence ŽCL. in a whole-blood
assay; EL as EL a 1-antitrypsin complex; and lipid peroxidation by malondialdehyde ŽMDA. generation. The reactions after
handling were compared with the values measured immediately after blood withdrawal which served as reference values of
‘genuine’ PMN reactivity. The outstanding result was the marked scatter between the individual reactions. Overall, the
proportion of migrating PMNs in the blood total decreased, while CL, correlating positively with MDA, increased with the
time of storage. EL increased considerably in some of the samples. Agitation raised CL and MDA. The effect of temperature
was apparent only after 24 h at 378C. There was evidence that inhomogeneities in the blood samples were another interfering
factor, since resuspension of sedimented blood after storage can be incomplete. In order to obtain reliable results from PMN
functional tests, whole-blood assays and processing of blood samples within 20 min after blood withdrawal are recommended. q 1997 Elsevier Science B.V.
Keywords: Polymorphonuclear leukocyte functions; Blood samples; Storage; Handling; Thermal stress
1. Introduction
Abbreviations: ANOVA, analysis of variance; CL, chemiluminescence; DC, distribution characteristic; EL, elastase; FMLP,
N-formyl–methionyl–leucyl–phenylalanine; HBSS, Hanks balanced salt solution; HPLC, high performance liquid chromatography; MDA, malondialdehyde; MPO, myeloperoxidase; PMN,
polymorphonuclear leukocyte; PUFA, polyunsaturated fatty acid;
ROS, reactive oxygen species; RT, room temperature; TBA,
thiobarbituric acid; TMI, total migration index; WBC, white blood
cell count
)
Corresponding author. Tel.: q43-316-3804292; Fax: q43316-3809640.
Measuring various functions of blood polymorphonuclear leukocytes ŽPMN. is becoming increasingly important in medical diagnosis and prognosis.
Deficiencies in the first-line defence system create a
high risk for infections that may even include septic
complications. In contrast, overactivated phagocytes
may lead to autoaggressive damage of tissues at the
local level Že.g. gout, rheumatoid arthritis and emphysema; reviewed by Gordon et al., 1988; Harris,
0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 0 8 5 - 9
62
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
1988; Janoff, 1988., or at the systemic level to
multiple organ failure, systemic inflammatory response syndrome and adult respiratory distress syndrome Žreviewed by Simon and Ward, 1988; Goris,
1993; Redl et al., 1993.. PMNs circulate in a ‘priming’ state, which is a ‘pre-tuned for future tasks’
state reflecting the organism’s readiness for defence
and, therefore, is of a high predictive value. However, this extremely sensitive priming state can be
substantially disturbed by cell isolation procedures
usually preceding functional tests. Therefore,
whole-blood techniques avoiding damage by cell
separation are increasingly preferred ŽA-Hadithy et
al., 1981; Selvaray et al., 1982; Krause et al., 1983;
Bruchelt and Schmidt, 1984; Rice and Bignold, 1992;
Slater, 1992; Egger et al., 1994; Stevens et al., 1994;
Van Antwerpen et al., 1995; Miesel et al., 1995;
Kukovetz et al., 1995.. Our group measures routinely in fresh whole blood four PMN functional
parameters as follows: migration, reactive oxygen
species ŽROS. release, and the levels of elastase ŽEL.
and of malondialdehyde ŽMDA., a product of lipid
peroxidation. Migration and ROS release are measured in vitro by a standardized challenge of PMN
reactivity that discloses the priming state and, hence,
the prospective potency of these cells, while EL and
MDA blood levels reflect PMN activities that have
already taken place in the organism. In order to
optimize these techniques, one must establish whether
alterations in laboratory handling such as storage
time, temperature and agitation of blood samples
exert any effect on the sensitive PMNs and their
products. The final aim of this study was to define
relevant methodical limits for these PMN parameters.
2. Materials and methods
The methodical approach of this study was to
simulate everyday laboratory conditions including
agitation by transport and storage influences such as
time and temperature on blood samples and to measure the effects of these conditions on blood PMN
functional parameters. Values obtained within a period not longer than 5 min after blood withdrawal
were considered to reflect the true situation in the
circulating blood and hence were taken as the refer-
ence basis classed as ‘100%’. The changes caused by
the laboratory conditions on sample aliquots were
individually expressed as a percentage in relation to
the 100% value.
2.1. Test subjects
Five healthy test persons without clinical symptoms of inflammation, three females, aged 25, 29
and 30 years, and two males, aged 34 and 56 years,
were checked. Two of them were frequent cigarette
smokers typically exhibiting increased PMN reactions. Blood was taken from the antecubital vein
with informed consent. The PMN functional parameters tested were: Chemiluminescence ŽCL., migration, and the white blood cell count ŽWBC. with
blood withdrawn into SARSTEDT Monovette NH4
heparin syringes; PMN elastase ŽEL. and malondialdehyde ŽMDA. in blood withdrawn into SARSTEDT Monovette EDTA K syringes. The blood samples were divided into 1.5 ml aliquots in SARSTEDT plastic test tubes Nr. 72690 and stored upright
in a rack before further treatment.
2.2. Simulation of laboratory handling conditions
Ž1. The 100% reference: All measurements or
procedures were performed within a maximum of 5
min after blood withdrawal. Ž2. Influence of agitation: Within this 5 min limit, a 1 min agitation on an
IKA test tube shaker ŽJanke and Kuner, Germany. at
medium speed was performed. Ž3. Influence of time
and temperature: The samples were kept for 20, 60
min, 2, 6 and 24 h at 48C, at a room temperature of
20–228C ŽRT. and at 378C, respectively, before the
measurementsrprocedures were performed. For the
20 and 60 min intervals the 48C step was omitted
because cooling samples for such short periods is not
typical for routine laboratories.
2.3. PMN functional tests
CL, migration and the WBC were made from
blood in the same test tube. Before processing, the
samples were drawn up 5 times in a 1 ml automatic
pipette without bubbling in order to mix the sedimented blood.
CL was measured according to Kukovetz et al.
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
Ž1995. in a BIO ORBIT 1251 luminometer with
automated dispenser ŽTurku, Finland.. The tests were
performed in duplicate using 1 m l of whole blood for
each. The peak height and the peak time of light
emission were evaluated.
The migration test was described by Egger et al.
Ž1994.. 350 m l blood was pipetted into another
plastic test tube, diluted with 1050 m l Hanks balanced salt solution ŽHBSS. and mixed again by
drawing it up 5 times in a 1 ml automatic pipette. A
migration chamber was filled with 200 m l of this
mixture. A test unit included the chemoattractant
peptide N-formyl–methionyl–leucyl–phenylalanine
ŽFMLP. and blank chambers, each in triplicate. The
following parameters were evaluated: Ža. the percentage of PMNs migrating from the blood sample
into the migration filter Žthe ‘total migration index’,
TMI.; Žb. the penetration depth at which half of the
migrating PMN bulk had covered the filter Žthe
‘distribution characteristic’, DC. and Žc. the individual FMLPrblank quotient of TMI and DC.
2.3.1. WBC
A 40 m l sample was taken and the leukocytes
were counted in a COULTER COUNTER ZM. Differential counts were made visually from smears.
2.3.2. EL
The 1.5 ml blood samples were inverted three
times, then spun for 10 min at 600 g at room temperature, and 600 m l of the plasma supernatant were
kept frozen at y808C for several weeks until measurement by the IMAC test kit ŽMERCK, Germany.
in an automated analyzer ŽGREINER G-450..
2.3.3. MDA
After centrifugation as above, 200 m l of the
plasma were stored at y808C for several weeks.
MDA was measured by the thiobarbituric acid ŽTBA.
reaction using the method described by Wong et al.
Ž1987. and Rabl et al. Ž1992., further slightly modified by one of us ŽM.H... The plasma samples were
thawed immediately before the assay and a volume
of 100 m l was mixed with 100 m l distilled water,
300 m l 0.15 M phosphoric acid, 10 m l butylated
hydroxytoluene Ž0.2% methanolic solution. and 100
m l 0.6% TBA. The mixtures were incubated at 958C
for 60 min. The chromogen was extracted with 1.25
63
ml butanol-1 and analyzed by HPLC with fluorometric detection Žexcitation wavelength 525 nm, emission wavelength 550 nm.. The MDA-TBA adduct
was calibrated with tetramethoxypropane standard
solutions processed in the same manner as the plasma
samples. Because of the constant nature of multiple
measurements, MDA was only measured once.
To check whether dilution of blood with buffer
solution prior to storage might help to preserve PMN
priming, aliquots of a blood sample were diluted
with HBSS in the ratio of 1:3 as above and stored for
30 and 60 min at RT and 48C together with undiluted
blood at RT. The prediluted aliquots were mixed
again by drawing them up 5 times in a 1 ml pipette
before introducing them into the migration chambers.
After preparing the aliquots as above, migration tests
were performed and the results compared with the
relevant 5 min reference value.
2.4. Statistics
Changes over time were evaluated using the
Spearman rank correlations. In the case of a lack in
significance, a Kruskal–Wallis analysis of variance
ŽANOVA. was performed. The values after agitation
were not considered in these calculations. Correlations between the functional parameters were calculated with the Spearman rank correlation using a
significance threshold of p - 0.05. Because of the
poor qualities of the data, the 48C values were
exempted from statistical evaluations.
3. Results
3.1. Elastase (Fig. 1, Table 1)
Absolute ‘genuine’ values: 14, 28, 32, 39, 63
m grl.
Agitation considerably activated elastase release
in one blood sample.
Room temperature ŽRT.: In general, there was no
increase over time, whereas the differences between
the reactions of the individual subjects rose after the
60 min interval, causing an increasing scatter of the
over-all reaction.
378C: A significant increase occurred with time,
64
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
Table 1
Spearman rank correlations between PMN functional parameters
and time
Fig. 1. Measurement of polymorphonuclear leukocyte elastase
blood levels illustrating changes caused by storage influences such
as agitation ŽAG, l., temperature of 48C Ž4, '., 20 to 228C
Žroom temperature, RT, B., and body temperature Ž37, v . at
different time intervals Žabscissa. expressed as percentage of the
genuine values Ž100% line. on the ordinate.
and the scatter between the individuals was more
marked than at RT.
3.2. Chemiluminescence, peak height (Fig. 2, Table
1)
Genuine values: 11.7, 19.6, 26.0, 32.6, 34.3 units
of light emission.
Agitation uniformly increased the values.
RT: CL was already clearly enhanced after 20
min of storage and increased with length of storage.
Fig. 2. Polymorphonuclear leukocyte reactive oxygen species
production measured by chemiluminescence, peak height of light
emission, illustrating changes caused by storage influences such as
agitation ŽAG, l., temperature of 48C Ž4, '., 20 to 228C Žroom
temperature, RT, B., and body temperature Ž37, v . at different
time intervals Žabscissa. expressed as percentage of the genuine
values Ž100% line. on the ordinate. Asterisk: difference in
ANOVA, ps 0.0018.
EL
CLrpeak heigh
CLrpeak time
MDA
TMIrFMLP
TMIrblanks
TMIrQ
DCrFMLP
DCrblanks
DCrQ
RT
378C
n.s.
- 0.0001
- 0.0001
n.s.
0.0025
0.0002
n.s.
n.s.
n.s.
n.s.
0.012
n.s.
0.0354
0.0053
0.001
0.0001
n.s.
n.s.
n.s.
n.s.
EL: elastase, CL: chemiluminescence, TMI: migration, total migration index, DC: migration, distribution characteristic, Q: the
individual FMLPrblank quotient of migration. RT: room temperature of 20–228C. p values are indicated. n.s.: not significant
Ž p) 0.05.. The TMIs correlate negatively, the other parameters
positively.
378C: An increase comparable with room temperature occurred until the sixth hour, while there was a
marked drop by 24 h.
3.3. Chemiluminescence, peak time (Fig. 3, Table 1)
Genuine values: 510, 510, 570, 660, 690 s.
The reaction patterns were inversely related to the
peak heights: agitation and time lowered the values.
Fig. 3. Polymorphonuclear leukocyte reactive oxygen species
production measured by chemiluminescence, time of peak light
emission, illustrating changes caused by storage influences such as
agitation ŽAG, l., temperature of 48C Ž4, '., 20 to 228C Žroom
temperature, RT, B., and body temperature Ž37, v . at different
time intervals Žabscissa. expressed as percentage of the genuine
values Ž100% line. on the ordinate.
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
65
Agitation generally caused an increase together
with considerable individual scatter.
RT: There was no demonstrable significant increase over time. At the first 20 min interval, the
scatter was already high.
378C: A significant increase over time was observed.
3.5. Migration, total migration index, TMI (Fig. 5,
Table 1)
Fig. 4. Measurement of malondialdehyde blood levels illustrating
changes caused by storage influences such as agitation ŽAG, l.,
temperature of 48C Ž4, '., 20 to 228C Žroom temperature, RT,
B., and body temperature Ž37, v . at different time intervals
Žabscissa. expressed as percentage of the genuine values Ž100%
line. on the ordinate.
The reactions at 378C after 24 h were within the
same limits as the others.
3.4. Malondialdehyde (Fig. 4, Table 1)
Genuine values: 0.275, 0.335, 0.383, 0.476, 0.684
m Mrl.
Genuine values: FMLP 21.5, 22.2, 29,7, 38.6,
49.6%, controls 31.1, 31.5, 32.3, 34.1, 51.6%. Agitation did not influence the reactions of the blank
controls, but, dependent on the individual, could
cause a marked increase or decrease of the reactions
toward FMLP.
RT and 378C: Neither a thermal effect nor differences between the FMLP values and the blank controls could be demonstrated in terms of quantity or
individual scatter. Storage conditions had no influence on the FMLPrblank quotients. After just 20
min, the scatter was high and could amount to more
than "50%. There was, however, a significant overall decrease with time under both FMLP and control
conditions.
Fig. 5. Measurement of polymorphonuclear leukocyte migration demonstrated as total migration index ŽTMI. illustrating changes caused by
storage influences such as agitation ŽAG, l., temperature of 48C Ž4, '., 20 to 228C Žroom temperature, RT, B., and body temperature Ž37,
v . at different time intervals Žabscissa. expressed as percentage of the genuine values Ž100% line. on the ordinate. Filled symbols, left row:
Reactions to FMLP. Open symbols, right row: blank reactions.
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
66
Fig. 6. Measurement of polymorphonuclear leukocyte migration demonstrated as distribution characteristic ŽDC. illustrating changes caused
by storage influences such as agitation ŽAG, l., temperature of 48C Ž4, '., 20 to 228C Žroom temperature, RT, B., and body temperature
Ž37, v . at different time intervals Žabscissa. expressed as percentage of the genuine values Ž100% line. on the ordinate. Filled symbols, left
row: Reactions to FMLP. Open symbols, right row: blank reactions. Asterisk: difference in ANOVA, p s 0.0399.
3.6. Migration, distribution characteristic, DC (Fig.
6, Table 1)
FMLP was increased compared with the other time
points.
Genuine values: FMLP 19.9, 23.3, 24.6, 29.0,
34.4 m m, controls 18.7, 20.0, 20.9, 21.3, 36.5 m m.
Agitation induced reactions comparable to TMI: there
was no substantial influence on the reactions to the
blanks, but high individual variabilities of the reactions toward FMLP.
RT: there was no influence of time on the reactions to FMLP and the blanks, or the relationship
between them Žthe FMLPrblank quotient.. The individual scatter tended to increase with time.
378C: findings were as for room temperature and
no continuous increase over time could be demonstrated. After 24 h, however, the reaction toward
3.7. Cooling to 48C
For all inflammation parameters, the values at 48C
were generally similar to those at RT.
3.8. PMN r m l (Fig. 7)
Genuine values: 1878, 1890, 3750, 5729, 7395
PMNrm l.
There were no statistically significant differences
between the 48C, RT and 378C values after 5 min, 2
and 24 h ŽANOVA.. However, the scatter between
the individuals was surprisingly high.
Table 2
Spearman rank correlation between particular PMN functional parameters
DC:TMI FMLP
DC:TMI blanks
CL peak height:peak time
CL peak height:MDA
n.s.
n.s.
n.s.
0.0044
DC: migration, distribution characteristic, TMI: migration, total migration index, CL: chemiluminescence, MDA: malondialdehyde. p
values are indicated. n.s.: not significant Ž p ) 0.05.. CL and MDA correlate positively.
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
Fig. 7. Polymorphonuclear leukocyte counts per 1 m l blood
illustrating changes caused by temperatures of 48C Ž4, '., 20 to
228C Žroom temperature, RT, B., and body temperature Ž37, v .
at different time intervals Žabscissa. expressed as percentage of
the genuine values Ž100% line. on the ordinate. ANOVA: no
significant differences.
There were no significant correlations between
the individual migration parameters DC and TMI,
nor between the CL parameters peak height and peak
time ŽTable 2.. There was a strong positive correlation between the CL peak values and MDA blood
levels.
We investigated the influence of dilution and
cooling of a blood sample on the migration parameters TMI and DC ŽFigs. 8 and 9.. In comparison with
normal resting values Ži.e. the measurement made 3
min after blood withdrawal., the least evidence of
change was found in undiluted blood at room tem-
67
Fig. 9. Influences on the migration parameter ‘distribution characteristic’ ŽDC. caused by dilution Ždi. of a blood sample with
HBSS prior to storage at room temperature ŽRTdi. or 48C Ž48di. in
comparison to undiluted, native blood at room temperature ŽRTna.
for 30 and 60 min. Filled columns: reactions to FMLP. Open
columns: reactions to blanks. Ordinate: Percentage changes in
comparison to the genuine values Žthe 100% line..
perature, and the most change in diluted blood at
48C.
The results of the statistical tests are included in
Tables 1 and 2 and in the graphs.
4. Discussion
The main issue underlying this study was a technical one: to what extent does the treatment of blood
samples such as temperature, storage time and mechanical stress influence the functions of the contained PMNs or, in other words, where is the borderline between true results and handling artefacts?
We can identify three outstanding reasons for
deviations of particular findings from the ‘genuine
values’ that we have defined as the values immediately after blood withdrawal: flaws in the measurements, inhomogeneities in the blood samples, and
changes in blood-cell reactivities caused by storage
conditions.
4.1. DeÕiations within measurements
Fig. 8. Influences on the migration parameter ‘total migration
index’ ŽTMI. caused by dilution Ždi. of a blood sample with
HBSS prior to storage at room temperature ŽRTdi. or 48C Ž48di. in
comparison to undiluted, native blood at room temperature ŽRTna.
for 30 and 60 min. Filled columns: reactions to FMLP. Open
columns: reactions to blanks. Ordinate: Percentage changes in
comparison to the genuine values Žthe 100% line..
Measurement errors on identical samples depend
on the accuracy of the methods used and on the
quality of the laboratory. Multiple measurements
minimize the error rate. Outliers obviously originating from errors in measurement were discarded or, if
possible, another sample aliquot was analyzed.
68
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
4.2. Inhomogeneities of blood samples
The blood samples progressively separate during
storage by aggregation and sedimentation of the red
blood cells, while the white cells are left behind
forming a buffy coat. Mixing sedimented blood samples by drawing them up 5 times into a pipette is not
sufficient to reconstitute a homogeneous suspension
ŽFig. 7.. A more thorough mixing is not indicated
because mechanical stress may influence various
PMN functions ŽFigs. 1–6.. It is desirable that blood
sedimentation is prevented by keeping the samples in
gentle motion in a multi-axle rotating mixer.
4.3. Changes in blood PMN reactiÕities caused by
storage conditions
This study has demonstrated the sensitivity of
PMN priming toward storage conditions. Astonishingly high individual diversities were observed with
storage altering the reactions in both a positive as
well as in a negative sense. This capriciousness, the
cause of which is unknown, is the reason for the
considerable scatter between the individual reactions.
For some of the parameters, storage increases PMN
activities as a whole together with the scatter. ŽTable
1, Figs. 1 and 2.. Thermal influences were only
observed after 24 h storage at 378C ŽFigs. 2, 5 and
6.. However, the ways these functional parameters
behave under the given storage conditions should be
discussed individually and in detail.
4.4. Migration
The TMI parameter ŽFig. 5. is extremely sensitive
with respect to time of storage. Test subjects can
respond with an increase as well as with a decrease
in reactivity in comparison to their ‘genuine’ values.
The directions of the changes are, in our experience,
unpredictable and can cause individual deviations
higher than 50%. In general, the TMI decreases over
time ŽTable 1.. The DC parameter ŽFig. 6. shows an
individual scattering tendency that is somewhat lower
than for the TMI but the over-all reaction remains
unchanged over time ŽTable 1.. Both the TMI and
the DC do not react to FMLP in comparison to the
controls. The reactions to agitation within 5 min after
blood withdrawal indicate that the strong individual
scatter is not a flaw in the measurement but a
phenomenon inherent to the PMNs themselves: the
deviations of the controls are small and within the
methodological limits, while the reactions to FMLP
scatter considerably Žcf. Figs. 5 and 6..
4.5. CL and MDA
ŽFigs. 2–4. ROS release by blood phagocytes
following latex stimulation and estimated by the
peaks of light emission increases markedly over time
ŽFig. 2, Table 1.. The course of the peak time is
inversely related to the peak height ŽFig. 3, Table 1..
A high and early peak suggests an elevated priming
level ŽHofer et al., 1994; Kukovetz et al., 1995..
MDA only increases significantly at 378C ŽFig. 4,
Table 1.. MDA determination by HPLC after derivation with TBA measures free MDA and MDA bound
via Schiff bases to free amino groups originating
from oxidation processes of PUFAs abundant in cell
membranes, mainly from arachidonic acid oxidation
ŽHalliwell and Gutteridge, 1989, Halliwell, 1991,
Schaur et al., 1994.. Additionally the test is generally accepted as a measure for lipid hydroperoxides,
because MDA is generated from these compounds
during the derivation procedure ŽJanero, 1990.. Increased MDA concentrations measured after longer
storage may not reflect the genuine situation in the
blood stream, but rather an artefact caused by in
vitro lipid peroxidation by PMNs. The strong correlation between individual CL and MDA values supports this suggestion ŽTable 2.. The marked drop of
CL peak values after 24 h at 378C is unique among
all parameters ŽFig. 2.. Since luminol is mainly
oxidized via the MPO –H 2 O 2 –halide system
ŽDahlgren and Stendhal, 1983; Briheim et al., 1984;
Dahlgren et al., 1989., a temperature dependent
breakdown of this pathway over time is probable.
However, a 24 h period of passivity is extremely
unphysiological for a PMN. The average time such
cells reside in the circulation before actively leaving
the blood vessels is 6 to 7 h ŽAthens et al., 1961;
Davis et al., 1991..
4.6. EL
The influence of storage had different effects on
the individual blood samples. In some subjects, stor-
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
age altered the basic values to a lesser extent, while
in others, EL enormously increased with time, especially at 378C ŽFig. 1. indicating an intensive in vitro
release from PMNs. Since it is not EL itself, but the
EL a 1-antitrypsin Ž a 1-AT. complex which is measured ŽNeumann et al., 1984., the results may depend on the individual a 1-AT blood levels that can
be depleted by binding to EL in a one-to-one proportion ŽKoj, 1985.. The EL a 1-AT complex has an in
vivo half-life of approximately 1 h; the complex is
cleared by macrophages in the lymphatic system and
the liver ŽOhlsson and Laurell, 1976.. Since such a
clearing mechanism cannot work in vitro, a longer
life-span and, hence, an accumulation, can be assumed.
4.7. Agitation
This increases the PMN activity ŽFigs. 2–4. or, in
the case of migration, influences individual reactivity
toward FMLP, while the controls are only slightly
affected ŽFigs. 5 and 6.. The activating influence of
agitation on blood PMNs may not only have significance for laboratory handling and transport conditions, but also for in vivo situations. Mechanical
stress on PMNs also occurs in the circulation and
may be considerably increased by turbulence caused
by such intima defects as atherosclerotic plaques and
ulcerations. Thus, mechanical irritation may contribute to the increased activity of blood PMNs
observed during atherosclerotic processes ŽGorog,
¨¨
1992; Mazzone et al., 1993; Van der Wal et al.,
1994; Key et al., 1996..
Explanations of how storage acts on PMNs must
remain speculative. One argument is that activation
of the extremely vulnerable blood platelets increases
with the length of storage and may release such
substances as arachidonic acid metabolites, serotonin
and PAF Žreviewed by Weksler, 1988.. Such an
argument, however, has to be questioned since dilution of a blood sample immediately after venepuncture must also reduce the concentration of these
agents and apparently increased PMN activation
ŽFigs. 8 and 9.. Overall, the release of ROS and, in
some subjects, of EL increased, whereas PMN migration ŽTMI. decreased with storage time. Remarkably, we found comparable alterations in relation to
age. Older test subjects exhibited increased CL and
69
decreased TMI migration values compared with juveniles ŽEgger et al., 1995; Kukovetz et al., 1997.. In
the first paper we discussed the possibility of a
prolonged PMN circulation time in older subjects.
Comparable to in vitro storage, a prolonged residence in the blood vessels may increase the chemical
efficacy of PMNs.
4.8. Conclusions
For investigators using whole-blood methods for
determining PMN functions, we can recommend the
following measures: Blood samples should be processed carefully without mechanical stress and as
fast as possible. The procedure adopted during the 20
min after venepuncture that we have already recommended in a former paper ŽEgger et al., 1994. represents reasonable compromise between maintaining
the stability of PMN reactions and the practicability
of the tests. This narrow window of time excludes
transport of blood samples and presupposes a test
facility in the vicinity of the donor. The withdrawn
blood can be handled at room temperature and cooling the blood samples to 48C is not helpful. Dilution
of blood samples with buffer solution prior to storage is disadvantageous. The period of time available
for harvesting plasma for the determination of EL is
a little greater, about 60 min. To prevent sedimentation and the separation of blood components, gentle
rotation of the samples during the short storage
before measurement may be useful. PMN reactivity
changes with the length of storage but, because of
the unpredictable individual scatter, the degree of the
changes cannot be defined in advance. The isolation
of white blood cells cannot prevent chemical and
osmotic stress as well as mechanical and temporal
influences. Such procedures are therefore useless for
the determination of the resting blood PMN functional state. Users of such PMN functional tests
should always check critically the suitability of their
assays.
Acknowledgements
This work was supported by the Austrian Joint
Research Project SFB007 ‘Biomembranes’, Section
70
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
11: Oxidation of membrane lipids and lipoproteins
by neutrophil granulocytes.
References
A-Hadithy, H., Addison, I.E., Goldstone, A.H., 1981. Use of
whole blood in the measurement of neutrophil migration. J.
Clin. Pathol. 43, 158.
Athens, J.W., Haab, O.P., Raab, S.O., Mauer, A.M., Ashenbrucker, H., Cartwright, G.E., Wintrobe, M.M., 1961.
Leukokinetic studies. IV. The total blood, circulating and
marginal granulocyte pools and the granulocyte turnover rate
in normal subjects. J. Clin. Invest. 40, 989.
Briheim, G., Stendhal, O., Dahlgren, C., 1984. Intra- and extracellular events in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun. 45, 1.
Bruchelt, G., Schmidt, K.H., 1984. Comparative studies on the
oxidative processes during phagocytosis measured by
luminol-dependent chemiluminescence. J. Clin. Chem. Clin.
Biochem. 22, 1.
Dahlgren, C., Stendhal, O., 1983. Role of MPO in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun. 39, 736.
Dahlgren, C., Follin, P., Johannson, A., Lock, R., Orselius, K.,
1989. Localization of luminol-dependent CL reaction in human granulocytes. J. Biolumin. Chemilumin. 4, 263.
Davis, J.M., Albert, J.D., Tracy, K.J., Calvano, S.E., Lowry, S.F.,
Shires, G.T., Yurt, R.W., 1991. Increased neutrophil mobilization and decreased chemotaxis during cortisol and epinephrine
infusions. J. Trauma 31, 725.
Egger, G., Klemt, Ch., Spendel, S., Kaulfersch, W., Kenzian, H.,
1994. Migratory activity of blood polymorphonuclear leukocytes during juvenile rheumatoid arthritis, demonstrated with a
new whole-blood membrane filter assay. Inflammation 18,
427.
Egger, G., Aglas, F., Rainer, F., 1995. Blood polymorphonuclear
leukocyte migratory activities during rheumatoid arthritis. Inflammation 19, 651.
Gordon, T.P., Terkeltaub, R., Ginsberg, M.H., 1988. Gout: Crystal-induced inflammation. In: Gallin, J.I., Goldstein, I.M.,
Snyderman, R. ŽEds.., Inflammation. Basic Principles and
Clinical Correlates. Raven Press, New York. p. 775.
Goris, R.J.A., 1993. Shock, sepsis and multiple organ failure: The
result of whole-body inflammation. In: Schlag, G., Redl, H.
ŽEds.., Pathophysiology of Shock, Sepsis, and Organ Failure.
Springer Verlag, Berlin Heidelberg, New York, London, Paris,
Tokyo, Hong Kong, Barcelona, Budapest, p. 7.
Gorog,
¨ ¨ P., 1992. Neutrophil-oxidized low density lipoprotein:
Generation in and clearance from the plasma. Int. J. Exp.
Pathol. 72, 485.
Halliwell, B., 1991. Lipid Peroxidation, Free-Radical Reactions,
and Human Disease. Current Concepts. Upjohn Company,
Kalamazoo, Michigan, p. 1.
Halliwell, B., Gutteridge, J.M., 1989. Free Radicals in Biology
and Medicine. Clarendon Press, Oxford, p. 188.
Harris, E.D., Jr., 1988. Pathogenesis of rheumatoid arthritis: A
disorder associated with dysfunctional immunoregulation. In:
Gallin, J.I., Goldstein, I.M., Snyderman, R. ŽEds.., Inflammation. Basic principles and Clinical Correlates. Raven Press,
New York, p. 751.
Hofer, H.P., Egger, G., Bratschitsch, G., Kukovetz, E.M., Petek,
W., Schaur, R.J., 1994. Phagocyte activation and its implication in wound healing following trauma surgery. Med. Sci.
Res. 22, 533.
Janero, D.R., 1990. Malonaldehyde and thiobarbituric acid –
Reactivity as diagnostic indices of lipid peroxidation and
peroxidative tissue injury. Free Radic. Biol. Med. 9, 515.
Janoff, A., 1988. Emphysema: Proteinase–antiproteinase imbalance. In: Gallin, J.I., Goldstein, I.M., Snyderman, R. ŽEds..,
Inflammation. Basic Principles and Clinical Correlates. Raven
Press, New York, p. 803.
Key, N.S., Platt, J.L., Vercellotti, G.M., 1996. Vascular endothelial cell proteoglycans are susceptible to cleavage by neutrophils. Arterioscler. Thromb. 12, 836.
Koj, A., 1985. Biological functions of acute-phase proteins. In:
Gordon, A.H., Koj, A. ŽEds.., The Acute-Phase Response to
Injury and Infection. Elsevier, Amsterdam, New York, Oxford, p. 145.
Krause, P.J., Pock, R.M., Woronick, C.L., Maderazo, E.G., 1983.
Simplified micropore filter assay of neutrophil migration using
whole blood. J. Infect. Dis. 148, 881.
Kukovetz, E.M., Hofer, H.P., Egger, G., Khoschsorur, G.A.,
Bratschitsch, G., Petek, W., Quehenberger, F., Schaur, R.J.,
1995. Assay of phagocyte activation by means of malondialdehyde and luminol-enhanced chemiluminescence during uneventful wound healing following trauma surgery. Redox Report 1, 247.
Kukovetz, E.M., Bratschitsch, G., Hofer, H.P., Egger, G., Schaur,
R.J., 1997. Influence of age on the release of reactive oxygen
species by phagocytes as measured by a whole blood chemiluminescence assay. Free Radic. Biol. Med. 22, 433.
Mazzone, A., De Servi, S., Ricevutti, G., 1993. Increased expression of neutrophil and monocyte adhesion molecules in unstable coronary artery disease. Circulation 88, 358.
Miesel, R., Zuber, M., Hartung, R., Haas, R., Kroger,
H., 1995.
¨
Total radical-trapping antioxidative capacity of plasma and
whole blood chemiluminescence in patients with inflammatory
and autoimmune rheumatic diseases. Redox Report 1, 323.
Neumann, S., Gunzer, G., Hennrich, N., Lang, H., 1984. ‘PMNelastase assay’: Enzyme immunoassay for human polymorphonuclear elastase complexed with a 1-proteinase inhibitor. J.
Clin. Chem. Biochem. 22, 693.
Ohlsson, K., Laurell, C.B., 1976. The disappearance of enzyme–
inhibitor complexes from the circulation of man. Clin. Sci.
Mol. Med. 51, 87.
Rabl, H., Khoschsorur, G., Colombo, T., Tatzber, F., Esterbauer,
H., 1992. Human plasma lipid peroxide levels show a strong
transient increase after successful revascularization operations.
Free Radic. Biol. Med. 13, 281.
Redl, H., Schlag, G., Kneidinger, R., Dinges, H.P., Davies, J.,
1993. Activationradherence phenomena of leukocytes and
endothelial cells in trauma and sepsis. In: Schlag, G., Redl, H.
G. Egger et al.r Journal of Immunological Methods 206 (1997) 61–71
ŽEds.., Pathophysiology of Shock, Sepsis, and Organ Failure.
Springer Verlag, Berlin, Heidelberg, New York, London, Paris,
Tokyo, Hong Kong, Barcelona, Budapest, p. 549.
Rice, J.E., Bignold, L.P., 1992. Chemotaxis of polymorphonuclear
leukocytes in whole blood in the ‘sparse-pore’ polycarbonate
ŽNucleopore. membranerBoyden chamber assay. J. Immunol.
Methods 149, 121.
Schaur, R.J., Dussing, G., Kink, E., Schauenstein, E., Posch, W.,
Kukovetz, E.M., Egger, G., 1994. The lipid peroxidation
product 4-hydroxynonenal is formed by — and is able to
attract — rat neutrophils in vivo. Free Radic. Res. 20, 365.
Selvaray, R.J., Sbarra, A.J., Thomas, G.B., Cetrulo, C.L., Mitchell,
G.W. Jr., 1982. A microtechnique for studying chemiluminescence response of phagocytes using whole blood and its
application to the evaluation of phagocytes in pregnancy. J.
Retic. Soc. 31, 3.
Simon, R.H., Ward, P.A., 1988. Adult respiratory distress syndrome. In: Gallin, J.I., Goldstein, I.M., Snyderman, R. ŽEds..,
Inflammation. Basic Principles and Clinical Correlates. Raven
Press, New York, p. 215.
Slater, K., 1992. Phagocyte Chemiluminescence in Health and
Disease. Int. Biotech. Lab., May, 12.
71
Stevens, D.L., Bryant, A.E., Huffmann, J., Thompson, K., Allen,
R.C., 1994. Analysis of circulating phagocyte activity measured by whole blood luminescence. Correlations with clinical
status. J. Infect. Dis. 170, 1463.
Van Antwerpen, V.L., Theron, A.L., Richards, G.A., Van der
Merwe, C.A., Viljoen, E., Van der Walt, R., Anderson, R.,
1995. Plasma levels of beta-carotene are inversely correlated
with circulating neutrophil counts in young male cigarette
smokers. Inflammation 19, 405.
Van der Wal, A.-C., Becker, A.E., Van der Loos, C.M., Das,
P.K., 1994. The site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an
inflammatory process irrespective of the dominant plaque
morphology. Circulation 89, 36.
Weksler, B.B., 1988. Platelets. In: Gallin, J.I., Goldstein, I.M.,
Snyderman, R. ŽEds.., Inflammation. Basic Principles and
Clinical Correlates. Raven Press, New York, p. 543.
Wong, S.H.Y., Knight, J.A., Hopfer, S.M., Zaharia, O., Leach,
Ch.N. Jr., Sunderman, F.W. Jr., 1987. Lipoperoxides in plasma
as measured by liquid-chromatographic separation of malondialdehyde–thio-barbituric acid adduct. Clin. Chem. 33, 214.