# Neutrophil and Monocyte CD64 and CD163 Expression in Critically Ill Neonates and Children with Sepsis: Comparison of Fluorescence Intensities and Calculated Indexes

Authors: Mojca Groselj-Grenc; Alojz Ihan; Metka Derganc
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Objective.  To evaluate the expression of CD64 and CD163  on neutrophils and monocytes in SIRS with/without sepsis and to compare the diagnostic accuracy of CD64 and CD163 molecules expression determined as (1) mean fluorescence intensities (MFI) of CD64 and CD163; and (2) the ratio (index) of linearized MFI to the fluorescence signal of standardized beads. Patients and methods. Fifty-six critically ill neonates and children with systemic inflammatory response syndrome (SIRS) and suspected sepsis, classified into two groups: SIRS with sepsis (n=29) and SIRS without sepsis (n=27). Results. CD64 and CD163 MFI measured on neutrophils and monocytes were elevated in patients with SIRS with sepsis. Diagnostic accuracy of indexes was equal to diagnostic accuracy of MFI for CD64 on neutrophils (0.833 versus 0.854 for day 0 and 0.975 versus 0.983 for day 1) and monocytes (0.811 versus 0.865 for day 0 and 0.825 versus 0.858 for day 1), and CD163 on neutrophils (0.595 versus 0.655 for day 0 and 0.677 versus 0.750 for day 1), but not for CD163 on monocytes.  Conclusion. CD64 MFI, CD163 MFI, CD64 indexes for neutrophils and monocytes, and CD163 index for neutrophils can all be used for discrimination of SIRS and sepsis in critically ill neonates and children. CD64 index for neutrophils, however, is superior to all other markers.


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## Body

## 1. Introduction

CD64 is a high-affinity and
restricted isotype-specificity FcγRI receptor expressed on macrophages,
monocytes, neutrophils, and eosinophils [1, 2]. There are several reports
regarding its potential utility for the diagnostic assessment of sepsis or
infection in adults [3–8] and neonates [9–12], but only a few in children [13, 14]. In an adult study, a higher intensity of CD64 expression has been found on
neutrophils from patients with systemic inflammatory response syndrome (SIRS)
and sepsis than on neutrophils from patients with SIRS only [3]. High CD64
expression on monocytes accompanying high CD64 expression on neutrophils has
been reported in some adult studies as well [5, 15, 16].CD163 is a
monocyte/macrophage-associated antigen which has recently been identified as a
haemoglobin scavenger receptor [17]. Apart from clearance of haemoglobin, it
has also anti-inflammatory properties and an immunoregulatory role [18]. It has
been found that CD163 expression on a monocyte surface is inversely related to
the concentration of its soluble form (sCD163) in randomly selected patients
[19]. During human experimental endotoxinemia, a rapid rise in plasma sCD163
has been observed together with reduced surface CD163 expression on isolated
monocytes following lipopolysaccharide (LPS) administration, suggesting that
LPS induces shedding of CD163 from the surface of isolated monocytes.
Twenty-four hours after LPS administration in humans, CD163 surface expression
is consistently increased over the baseline expression [20]. Soluble CD163 has
been found to be elevated in sepsis in adults [21–23], whereas surface
expression of membrane CD163 (mCD163) in sepsis has not yet been evaluated.The first aim of the study was to evaluate the expression
of CD64 and CD163 on neutrophils and monocytes in critically ill neonates and
children with SIRS and sepsis and to find out whether it can discriminate
between infectious and noninfectious SIRS. SIRS is most commonly caused by
infection, but other conditions such as low-cardiac output syndrome,
respiratory distress, and haemorrhage can trigger it in neonates and children.
It has been found that as many as 82% of children admitted to a paediatric
intensive care unit have two or more signs of SIRS [24].The second aim of the study was to compare the
diagnostic accuracy of CD64 mean fluorescence intensity (MFI), CD163 MFI, CD64
index of linearised MFI to standardised beads, and CD163 index of linearised
MFI to standardized beads measured on neutrophils and monocytes. With recently
available software for data analysis and index calculation, sources of
technical errors and subjectivity, which are present in manual methods, are
reduced and a comparison of results between different laboratories is possible
[25].


## 2. Patients and Methods

### 2.1. Patients and Setting

This prospective observational study
was conducted in the level III multidisciplinary neonatal and paediatric
intensive care unit between January 2006 and September 2006. Fifty-six
consecutive patients with SIRS and clinically suspected infections were
eligible for enrolment. SIRS was defined according to the international pediatric
sepsis consensus conference definitions [26]. Clinically suspected infection
was defined as an explicit statement by the physician in the records,
indicating the suspicion of an infection, combined with the initiation of
diagnostic workup to rule out infection and the prescription of empirical
antibiotic therapy. Patients were not included if they had received antibiotic
therapy for more than 24 hours prior to admission, if they had undergone
surgery in the previous week, or if they had a proven viral infection. The
diagnosis of sepsis was confirmed by positive blood cultures or tracheal
aspirates when chest radiographs showed signs of pneumonia. There were no other
cultures (urine, cerebrospinal, or a puncture of normally sterile body fluid)
positive in our patients. The diagnosis of clinical sepsis was established in
patients with negative cultures, but with a strong suspicion of sepsis, who
received a full course of antibiotic therapy. The group without sepsis included
patients with a suspected infection in whom the subsequent clinical course,
laboratory data, and microbiological tests excluded infection, and in whom
antibiotic therapy was discontinued after a few days. Patients were classified
into two groups: SIRS with sepsis and SIRS without sepsis. The classification
was carried out by the attending physician unaware of the results of flow
cytometry. We analyzed data separately for two age groups: neonates aged less
than 28 days, and children older than 28 days, and for gram-positive and gram-negative
sepsis. Patients’ characteristics are summarized in Table 1. Pathogens isolated
from blood cultures were Staphylococcus epidermidis(n=3), Streptococcus
agalactiae(n=2), Neisseria meningitidis(n=1), Streptococcus
pneumoniae(n=1) and Streptococcus mitis(n=1), while pathogens
isolated from tracheal aspirates were Haemophilus influenzae(n=5), Streptococcus
pneumoniae(n=3), Moraxella catarrhalis(n=2), Escherichia
coli(n=1), Staphylococcus aureus(n=1), and Pseudomonas
aeruginosa(n=1).Table 1
Characteristics of the study population.
SIRS with sepsisSIRS without sepsisNumber2927Male/female12/1716/11Median PRISM
III (range)11 (4–26)12 (2–23)Number  of deaths03 (11%)Number of mechanically
ventilated patients27 (93%)27 (100%)Number of patients
with severe sepsis16 (55%)/Number  of patients with septic shock12 (41%)/Number of patients with inotropic
drugs12 (41%)9 (33%)Number of neonates <28 days1522Number of newborns (0–7 days)1320Median age (range); days1 (0–18)0 (0–13)Median gestational age (range); weeks38.2 (32.4–41.0)37.9 (32.4–41.4)Median birth weight (range); g2945 (1400–3850)3025 (1350–3920)Number of children >28 days145Median age (range); months9.3 (1.9–33.0)4.2 (1.4–65.9)Number of gram-positive sepsis11/Number of gram-negative sepsis10/Number  of culture-negative sepsis8 (28%)/Number of positive blood cultures8 (28%)/Number of positive tracheal
aspirates13 (44%)/The two groups were similar regarding gender (χ2 test, P=0.288), Pediatric Risk of Mortality Score (PRISM) III score (ANOVA, P=0.835), age (ANOVA, P=0.212
and P=0.278 for neonates and children, resp.), gestational age
(ANOVA, P=0.839) and birth weight (ANOVA, P=0.720).The study was approved by the
National Medical Ethics Committee of the Ministry of Health, Republic of Slovenia,
and written consent was obtained from parents before blood sampling.


### 2.2. Sample Collection and Flow Cytometry

Blood
samples (0.5 ml) were obtained at the time of suspected sepsis (day 0) and 24
hours later (day 1) together with samples for routine laboratory tests. Whole
blood EDTA-anticoagulated samples were immediately transported to the flow
cytometry laboratory during working hours or stored refrigerated (4˚C) during
the night or at weekends (up to 36 hours). Expressions of CD64 and CD163 on
neutrophils, monocytes, and lymphocytes were measured by quantitative flow
cytometry with a FACSCalibur flow cytometer (Becton Dickinson, NY, USA) using the Leuko64 assay (Trillium
Diagnostics, LLC, Me, USA).
The assay is for research use only and is composed of three antibodies with
specificities to CD64 (clones 22 and 32.2, both fluorescein isothiocyanate
(FITC) conjugated) and to CD163 (clone Mac2-148, phycoeritrin (PE) conjugated),
and a fluorescence bead suspension with three fluorescence signals (green
fluorescence due to FITC, orange fluorescence similar to PE, and red
fluorescence of starfire red) for unique identification of beads, and used for
instrument calibration and standardization of leukocyte CD64 and CD163
expression in human blood. The sample preparation and flow cytometer setup were
based on the manufacturer instructions. Briefly, 50 μL of whole blood, or
diluted whole blood to adjust leukocyte concentration to less than 25×109/L, was incubated for 15 minutes in the dark at room temperature with a
mixture of murine monoclonal antibodies followed by red cell lysis with an
ammonium-chloride-based red cell lysis solution (Trillium Lyse). Fluorescence
beads were then added and flow cytometer analysis was performed on a minimum of
50 000 leukocytes. Data analysis for fluorescence intensity was performed by
CellQuest software (Becton Dickinson,
Calif, USA)
(Table 2). MFI was measured as a linearized value of log scale on lymphocytes (red,
negative control, measuring CD64 expression), monocytes (green, positive
control, measuring CD64, and CD163 expression), neutrophils (blue, measuring
CD64 expression), and beads (aqua blue, measuring FITC, and PE expression) (Figure 1). Index calculation was performed by Leuko64 QuantiCalc software (Trillium
Diagnostics, Me, USA).
Index measurements were derived by the ratio of linearized MFI of the cell
population to the FITC signal from the beads. An internal negative control of
the assay was provided by the automated measurement of the lymphocyte CD64
index, which had to be less than 1.0, and an internal positive control of the
assay was provided by automated measurement of the monocyte CD64 index, which
had to be more than 3.0. Flow cytometry was performed up to 36 hours after blood sampling. Before the beginning
of the study the influence of delayed sample analysis was done and no
significant difference in levels of CD64 and CD163 expression was detected in
the first 36 hours after blood sampling. Isotype-control antibodies were
routinely used in each experiment to detect nonspecific staining; however the
calculation of CD64 and CD163 MFI was done without substracting isotype-control
MFI  in order to accurately compare the
ratio (index) of linearized MFI to MFI alone.Table 2
Median CD64 and CD163 MFI and indexes (i) for neutrophils and monocytes with 95%
confidence interval (CI) in critically ill neonates and children at the time of
suspected sepsis (day 0) and 24 hours later (day 1).
Day 0Day 1SIRS with sepsisSIRSPSIRS with sepsisSIRSPNeutrophilsCD64fMFI83
(66–108)44
(36–56).0000*100
(75–162)47
(32–54).0000*CD64i2.65 (2.02–4.03)1.3
(1.06–1.78).0000*3.36
(2.27–4.68)1.47
(1.07–1.78).0000*CD163MFI65
(50–85)47
(44–64).0463*80.5
(57–115)50.5
(40.2–84.8).0056*CD163i456
(423–548)447
(381–491).2250527
(486–621)462
(426–528).0495*MonocytesCD64MFI251
(231–294)168
(149–212).0000*307
(257–354)195
(159–243).0001*CD64i9.36 (7.94–11.2)6.69
(5.6–7.28).0001*11
(8.39–13.4)7.41
(6.43–8.22)CD163MFI2350
(1674–3942)1451
(1234–1637).0063*3281
(2369–4093)2483
(1927–2937).0439*CD163i20080
(9330–27352)9460
(7708–14893).083621136
(13636–32551)20142
(15926–25366).9598*statistically significant differences, Unpaired Mann-Whitney test.Gating of neutrophils, monocytes, and lymphocytes in FSC/SSC
representative FACS diagrams. CD64 and
CD163 histograms (colored) versus CD64 isotype controls (lined) are presented for
granulocytes (red), lymphocytes (green), and monocytes (blue).
(a)(b)(c)(d)(e)(f)(g)(h)


### 2.3. Statistical Analysis

Data were presented
as the median and 95% confidence interval for the median. Comparison between
groups was made using the unpaired Mann-Whitney test and analysis of variance
(ANOVA). Proportions of patients were compared by theχ2 test.
Receiver-operating characteristic (ROC) curves were drawn to define the optimal
sensitivity, specificity, cutoff value, and diagnostic accuracy, determined by
the area under the ROC curve (AUC) of the studied surface antigens [27, 28].
The cutoff values at which the greatest sum of sensitivity and specificity was
obtained were determined by the statistical program. Hanely and McNeil’s
comparison of diagnostic accuracies was performed. The CD64
score point (combination of two variables: CD64 MFI on neutrophils and
monocytes or index CD64 for neutrophils and monocytes) was calculated for CD64
MFI and CD64 index, separately as described elsewhere [29].
When CD64 fluorescence intensity or CD64 index on both neutrophils and
monocytes was under the cutoff level, the score point of 0 was assigned. The
score point of 1 was assigned when the marker on one type of cells was under
and marker on the other type of cells was over the cutoff level and the score
point of 2 was assigned when markers on both types of cells were over the
cutoff levels. The ROC curve analysis of CD64 score point was then performed. The differences were considered to be statistically
significant at the level of P<.05. The statistical analysis was
performed using Medcalc for Windows, version 5.0 (MedCalc Software, Mariakerke,
Belgium) and Statistical Package for the Social Sciences for Windows, version
12.0 (SPSS Inc., Chicago, ILL, USA).


## 2.1. Patients and Setting

This prospective observational study
was conducted in the level III multidisciplinary neonatal and paediatric
intensive care unit between January 2006 and September 2006. Fifty-six
consecutive patients with SIRS and clinically suspected infections were
eligible for enrolment. SIRS was defined according to the international pediatric
sepsis consensus conference definitions [26]. Clinically suspected infection
was defined as an explicit statement by the physician in the records,
indicating the suspicion of an infection, combined with the initiation of
diagnostic workup to rule out infection and the prescription of empirical
antibiotic therapy. Patients were not included if they had received antibiotic
therapy for more than 24 hours prior to admission, if they had undergone
surgery in the previous week, or if they had a proven viral infection. The
diagnosis of sepsis was confirmed by positive blood cultures or tracheal
aspirates when chest radiographs showed signs of pneumonia. There were no other
cultures (urine, cerebrospinal, or a puncture of normally sterile body fluid)
positive in our patients. The diagnosis of clinical sepsis was established in
patients with negative cultures, but with a strong suspicion of sepsis, who
received a full course of antibiotic therapy. The group without sepsis included
patients with a suspected infection in whom the subsequent clinical course,
laboratory data, and microbiological tests excluded infection, and in whom
antibiotic therapy was discontinued after a few days. Patients were classified
into two groups: SIRS with sepsis and SIRS without sepsis. The classification
was carried out by the attending physician unaware of the results of flow
cytometry. We analyzed data separately for two age groups: neonates aged less
than 28 days, and children older than 28 days, and for gram-positive and gram-negative
sepsis. Patients’ characteristics are summarized in Table 1. Pathogens isolated
from blood cultures were Staphylococcus epidermidis(n=3), Streptococcus
agalactiae(n=2), Neisseria meningitidis(n=1), Streptococcus
pneumoniae(n=1) and Streptococcus mitis(n=1), while pathogens
isolated from tracheal aspirates were Haemophilus influenzae(n=5), Streptococcus
pneumoniae(n=3), Moraxella catarrhalis(n=2), Escherichia
coli(n=1), Staphylococcus aureus(n=1), and Pseudomonas
aeruginosa(n=1).Table 1
Characteristics of the study population.
SIRS with sepsisSIRS without sepsisNumber2927Male/female12/1716/11Median PRISM
III (range)11 (4–26)12 (2–23)Number  of deaths03 (11%)Number of mechanically
ventilated patients27 (93%)27 (100%)Number of patients
with severe sepsis16 (55%)/Number  of patients with septic shock12 (41%)/Number of patients with inotropic
drugs12 (41%)9 (33%)Number of neonates <28 days1522Number of newborns (0–7 days)1320Median age (range); days1 (0–18)0 (0–13)Median gestational age (range); weeks38.2 (32.4–41.0)37.9 (32.4–41.4)Median birth weight (range); g2945 (1400–3850)3025 (1350–3920)Number of children >28 days145Median age (range); months9.3 (1.9–33.0)4.2 (1.4–65.9)Number of gram-positive sepsis11/Number of gram-negative sepsis10/Number  of culture-negative sepsis8 (28%)/Number of positive blood cultures8 (28%)/Number of positive tracheal
aspirates13 (44%)/The two groups were similar regarding gender (χ2 test, P=0.288), Pediatric Risk of Mortality Score (PRISM) III score (ANOVA, P=0.835), age (ANOVA, P=0.212
and P=0.278 for neonates and children, resp.), gestational age
(ANOVA, P=0.839) and birth weight (ANOVA, P=0.720).The study was approved by the
National Medical Ethics Committee of the Ministry of Health, Republic of Slovenia,
and written consent was obtained from parents before blood sampling.


## 2.2. Sample Collection and Flow Cytometry

Blood
samples (0.5 ml) were obtained at the time of suspected sepsis (day 0) and 24
hours later (day 1) together with samples for routine laboratory tests. Whole
blood EDTA-anticoagulated samples were immediately transported to the flow
cytometry laboratory during working hours or stored refrigerated (4˚C) during
the night or at weekends (up to 36 hours). Expressions of CD64 and CD163 on
neutrophils, monocytes, and lymphocytes were measured by quantitative flow
cytometry with a FACSCalibur flow cytometer (Becton Dickinson, NY, USA) using the Leuko64 assay (Trillium
Diagnostics, LLC, Me, USA).
The assay is for research use only and is composed of three antibodies with
specificities to CD64 (clones 22 and 32.2, both fluorescein isothiocyanate
(FITC) conjugated) and to CD163 (clone Mac2-148, phycoeritrin (PE) conjugated),
and a fluorescence bead suspension with three fluorescence signals (green
fluorescence due to FITC, orange fluorescence similar to PE, and red
fluorescence of starfire red) for unique identification of beads, and used for
instrument calibration and standardization of leukocyte CD64 and CD163
expression in human blood. The sample preparation and flow cytometer setup were
based on the manufacturer instructions. Briefly, 50 μL of whole blood, or
diluted whole blood to adjust leukocyte concentration to less than 25×109/L, was incubated for 15 minutes in the dark at room temperature with a
mixture of murine monoclonal antibodies followed by red cell lysis with an
ammonium-chloride-based red cell lysis solution (Trillium Lyse). Fluorescence
beads were then added and flow cytometer analysis was performed on a minimum of
50 000 leukocytes. Data analysis for fluorescence intensity was performed by
CellQuest software (Becton Dickinson,
Calif, USA)
(Table 2). MFI was measured as a linearized value of log scale on lymphocytes (red,
negative control, measuring CD64 expression), monocytes (green, positive
control, measuring CD64, and CD163 expression), neutrophils (blue, measuring
CD64 expression), and beads (aqua blue, measuring FITC, and PE expression) (Figure 1). Index calculation was performed by Leuko64 QuantiCalc software (Trillium
Diagnostics, Me, USA).
Index measurements were derived by the ratio of linearized MFI of the cell
population to the FITC signal from the beads. An internal negative control of
the assay was provided by the automated measurement of the lymphocyte CD64
index, which had to be less than 1.0, and an internal positive control of the
assay was provided by automated measurement of the monocyte CD64 index, which
had to be more than 3.0. Flow cytometry was performed up to 36 hours after blood sampling. Before the beginning
of the study the influence of delayed sample analysis was done and no
significant difference in levels of CD64 and CD163 expression was detected in
the first 36 hours after blood sampling. Isotype-control antibodies were
routinely used in each experiment to detect nonspecific staining; however the
calculation of CD64 and CD163 MFI was done without substracting isotype-control
MFI  in order to accurately compare the
ratio (index) of linearized MFI to MFI alone.Table 2
Median CD64 and CD163 MFI and indexes (i) for neutrophils and monocytes with 95%
confidence interval (CI) in critically ill neonates and children at the time of
suspected sepsis (day 0) and 24 hours later (day 1).
Day 0Day 1SIRS with sepsisSIRSPSIRS with sepsisSIRSPNeutrophilsCD64fMFI83
(66–108)44
(36–56).0000*100
(75–162)47
(32–54).0000*CD64i2.65 (2.02–4.03)1.3
(1.06–1.78).0000*3.36
(2.27–4.68)1.47
(1.07–1.78).0000*CD163MFI65
(50–85)47
(44–64).0463*80.5
(57–115)50.5
(40.2–84.8).0056*CD163i456
(423–548)447
(381–491).2250527
(486–621)462
(426–528).0495*MonocytesCD64MFI251
(231–294)168
(149–212).0000*307
(257–354)195
(159–243).0001*CD64i9.36 (7.94–11.2)6.69
(5.6–7.28).0001*11
(8.39–13.4)7.41
(6.43–8.22)CD163MFI2350
(1674–3942)1451
(1234–1637).0063*3281
(2369–4093)2483
(1927–2937).0439*CD163i20080
(9330–27352)9460
(7708–14893).083621136
(13636–32551)20142
(15926–25366).9598*statistically significant differences, Unpaired Mann-Whitney test.Gating of neutrophils, monocytes, and lymphocytes in FSC/SSC
representative FACS diagrams. CD64 and
CD163 histograms (colored) versus CD64 isotype controls (lined) are presented for
granulocytes (red), lymphocytes (green), and monocytes (blue).
(a)(b)(c)(d)(e)(f)(g)(h)


## 2.3. Statistical Analysis

Data were presented
as the median and 95% confidence interval for the median. Comparison between
groups was made using the unpaired Mann-Whitney test and analysis of variance
(ANOVA). Proportions of patients were compared by theχ2 test.
Receiver-operating characteristic (ROC) curves were drawn to define the optimal
sensitivity, specificity, cutoff value, and diagnostic accuracy, determined by
the area under the ROC curve (AUC) of the studied surface antigens [27, 28].
The cutoff values at which the greatest sum of sensitivity and specificity was
obtained were determined by the statistical program. Hanely and McNeil’s
comparison of diagnostic accuracies was performed. The CD64
score point (combination of two variables: CD64 MFI on neutrophils and
monocytes or index CD64 for neutrophils and monocytes) was calculated for CD64
MFI and CD64 index, separately as described elsewhere [29].
When CD64 fluorescence intensity or CD64 index on both neutrophils and
monocytes was under the cutoff level, the score point of 0 was assigned. The
score point of 1 was assigned when the marker on one type of cells was under
and marker on the other type of cells was over the cutoff level and the score
point of 2 was assigned when markers on both types of cells were over the
cutoff levels. The ROC curve analysis of CD64 score point was then performed. The differences were considered to be statistically
significant at the level of P<.05. The statistical analysis was
performed using Medcalc for Windows, version 5.0 (MedCalc Software, Mariakerke,
Belgium) and Statistical Package for the Social Sciences for Windows, version
12.0 (SPSS Inc., Chicago, ILL, USA).


## 3. Results

CD64 MFIs, CD163 MFIs, and CD64
indexes for both neutrophils and monocytes were significantly higher in
patients with SIRS with sepsis compared with patients with noninfectious SIRS,
while CD163 indexes showed statistical differences only on neutrophils on day 0
(Table2). Statistically significant increase (P<.05) from day 0 to
day 1 was found only for CD163 MFI for neutrophils in children with SIRS with
sepsis and for CD163 MFI and index for monocytes in children with noninfectious
SIRS. Medians of CD64 index for neutrophils were higher in children with sepsis
than in neonates with sepsis, although differences were not significant (P>.05) at the time of suspected sepsis and 24 hours later (Figure 2). We did
not find any statistically significant differences for CD64 and CD163 indexes,
and CD64 and CD163 MFIs for neutrophils or monocytes, between gram-positive and
gram-negative sepsis (P>.05 for all comparisons, data not shown).
Optimum diagnostic cutoff levels, AUCs, sensitivity and specificity of CD64 and
CD163 indexes, CD64 and CD163 MFIs for neutrophils and monocytes, and CD64
score points, for SIRS with sepsis at the time of suspected sepsis and 24 hours
later are presented in Table 3. Setting sensitivity at more than 95% in ROC
analysis of CD64 index for neutrophils displayed cutoff point 1.15 on day 0
(sensitivity 96.6% and specificity 40.7%) and 1.71 (sensitivity 100% and
specificity 75.0%) on day 1. There were no significant differences between
diagnostic accuracies of MFI and the corresponding index for either parameter, CD64 and CD163, on neutrophils and monocytes at the time of
suspected sepsis and 24 hours later (P>.05 for all comparisons),
except for diagnostic accuracy of CD163 MFI for monocytes, which was
significantly higher at 24 hours than diagnostic accuracy of the CD163 index
for monocytes (P=.003). The ROC curves of different indexes at the
time of suspected sepsis and 24 hours later are presented in Figure 3.
Diagnostic accuracies of CD64 indexes for neutrophils and monocytes at the time
of suspected sepsis were both significantly higher compared with diagnostic
accuracies of CD163 index for neutrophils (P=.000 and P=.023,
resp.) and of CD163 index for monocytes (P=.028 and P=.050, resp.). Diagnostic accuracy of CD64 index for neutrophils at 24
hours was significantly higher compared with diagnostic accuracies of CD64
index for monocytes (P=.009), CD163 index for neutrophils (P=.000), and CD163 index for monocytes (P=.000). The combinations of two
variables: CD64 MFI for neutrophils with CD64 MFI for monocytes or CD64 index
for neutrophils with CD64 index for monocytes (CD64 score point) insignificantly
increased diagnostic accuracy of CD64 MFI (P=.559) or CD64 index (P=.534) for neutrophils on day 0, while there was an insignificant fall in
diagnostic accuracy (P=.494 and P=.128, resp.) on day 1
(Table 3).Table 3
Optimum diagnostic cutoff level, diagnostic accuracy with 95% confidence
interval (CI) determined by the area under the ROC curve (AUC), sensitivity,
and specificity for given cutoff levels of CD64 and CD163 MFI and indexes (i)
for neutrophils and monocytes and CD64 score points for sepsis prediction in
critically ill neonates and children at the time of suspected sepsis (day 0)
and 24 hours later (day 1).
Cutoff levelAUC (95% CI)Sensitivity (%)Specificity (%)Day01010101CD64MFI for neutrophils72650.854 (0.734–0.934)0.983 (0.885–0.995)65.595.592.695.0CD64i for neutrophils2.452.190.833 (0.709–0.919)0.975 (0.872–0.996)65.586.488.9100CD64MFI for monocytes2282660.865 (0.747–0.941)0.858 (0.715–0.946)72.472.788.995.0CD64i for monocytes8.709.470.811 (0.684–0.903)0.825 (0.677–0.924)65.563.696.395.0CD163MFI for neutrophils47550.655 (0.516–0.777)0.750 (0.592–0.870)75.986.451.960.0CD163i for neutrophils3914820.595 (0.455–0.724)0.677 (0.515–0.813)86.277.337.065.0CD163MFI for monocytes164130330.713 (0.576–0.826)0.682 (0.520–0.817)75.963.674.185.0CD163i for monocytes19257342820.635 (0.495–0.759)0.500 (0.338–0.654)51.727.388.995.0CD64MFI score point>0 (1 and 2)>0 (1 and 2)0.879 (0.764–0.951)0.961 (0.851–0.995)86.2100.085.290.0CD64i score point>0 (1 and 2)>0 (1 and 2)0.864 (0.746–0.941)0.914 (0.785–0.977)82.890.988.980.0CD64 indexes for neutrophils at the time of suspected sepsis (day 0), and 24
hours later (day 1), for separate groups of neonates and children. Data are
presented as box plots (median value and interquartile range). Outliers and
extreme cases of index CD64 are not shown.
(a)(b)Receiver-operating characteristic (ROC) curves of CD64 and CD163 indexes (i)
for neutrophils and monocytes for critically ill neonates and children with
SIRS with sepsis and SIRS without sepsis at the time of suspected sepsis (day
0) and 24 hours later (day 1).
(a)(b)


## 4. Discussion

To our knowledge, this is the first study which
has evaluated expression of leukocyte antigens CD64 and CD163 in critically ill
neonates and children with SIRS with sepsis. In this study, we have shown that
expression of CD64 and CD163 on neutrophils and monocytes is elevated in
patients with sepsis compared with patients with noninfectious SIRS. We have
also shown that diagnostic accuracy of computer-calculated indexes for CD64 on
neutrophils and monocytes and CD163 on neutrophils based on the ratio (index)
of linearized MFI to the fluorescence signal of standardised beads, is equal to the diagnostic
accuracy of manually determined MFI,
if proceeded by the same flow cytometer. Our results therefore suggest that the
cost-effectiveness of tests, based on multiple antibodies (CD64 and CD163) and
standardized beads should be reevaluated.CD64 is expressed at low concentration on the
surface of nonactivated neutrophils [30]. CD64 surface upregulation is induced
by granulocyte colony-stimulating factor (G-CSF) and interferon-γ (INF-γ)
within 4–6 hours of stimulation [25]. Our study confirmed the results from
studies of the adult population that the level of CD64 expression is
significantly higher in patients with SIRS with sepsis compared with patients
with noninfectious SIRS [3]. In our study, the expression of CD64 on
neutrophils was significantly higher in critically ill neonates and children
with SIRS with sepsis compared with those with noninfectious SIRS. So far, CD64
expression on leukocytes has been studied mostly in the neonatal population.
These studies showed that CD64 expression on neutrophils is upregulated in
early-neonatal infection [9, 10], late-neonatal infection [11, 12], and in
preterm neonates with infection [11, 13]. Some authors have even suggested that,
because of its high sensitivity, measurement of neutrophil CD64 expression may
allow clinicians to discontinue antimicrobial treatment if negative within 24
hours of suspected infection, without waiting for the definitive
microbiological results [9]. The results of our research are in accordance with
the results of this other study [9] only to some extent. The AUCs in both
studies were similar, but in the other study [9], higher sensitivity was
achieved, probably because the cutoff values were determined in advance to
enable better sensitivity with the aim to identify all infected cases. Due to
the different method of flow cytometry used for the measurement of CD64
expression in the other study [9], the cutoff values cannot be compared. In our study the cutoff values were determined
by the statistical program to obtain the greatest sum of sensitivity and
specificity for comparisons of different markers. In order to increase the
sensitivity we determined the cutoff values at which the sensitivity was 96.6%
and 100%, respectively, on day 0 and 1. The specificity was then slightly lower
on day 1 than that in the previously mentioned study [9]. Three cases of sepsis
caused by coagulase-negative staphylococci could have lowered the sensitivity
of CD64 expression on neutrophils in our study, whereas in the other study [9]
there were no such cases.
In children, there are only two reports of 14 infants and children, and 8
children, respectively, hospitalized because of different bacterial infections
(mostly pneumonias and pyelonephritides) in whom CD64 expression on neutrophils
was significantly higher compared with noninfected controls [13, 14]. So far,
CD64 expression has not been studied in septic critically ill children. In
adult patients, higher expression of CD64 on neutrophils was found in gram-negative
sepsis compared with gram-positive sepsis [15]. In the recent study in neonates
[31], as in our study, this difference was not confirmed. The cause for this
difference between adults and neonates could be a less expressed neutrophil
response to infection with gram-negative bacteria in neonates. Indeed, we found
lower CD64 index in neonates with sepsis than in children, although the
difference was not statistically significant. A novel marker of infection, the
CD64 score point, which incorporates the quantitative analysis of CD64
expression on both neutrophils and monocytes has
recently been
introduced as a marker in adults which could distinguish between infection and
healthy states [29]. In our study, the CD64 score point was able to discriminate
between SIRS with sepsis and noninfectious SIRS as well, although its
diagnostic accuracy was not higher than that of CD64 expression on neutrophils
themselves.To our knowledge, CD64 expression on
monocytes in neonates and children has not previously been studied. There are
some reports of elevated CD64 expression on monocytes in adult patients with
sepsis [5, 15, 16]. In our study, the results of CD64 on monocytes and
neutrophils were similar at the time of suspected sepsis, but 24 hours later
the diagnostic accuracy of CD64 on monocytes was significantly lower than on
neutrophils. This data could indicate that activation in monocytes is faster or
more rapidly completed than in neutrophils [32].The increased expression of CD163 is
a part of the maturation of a monocyte to a phagocytic macrophage [18]. The
expression of CD163 is upregulated by interleukin-6 and glucocorticoids
together with interleukin-10, and downregulated by LPS and interferon-γ [18].
Although the absolute value of median expression of CD163 was much lower on
neutrophils than on monocytes in our patients with sepsis, the expression of
CD163 on both types of cells could differentiate between patients with SIRS
with sepsis and noninfectious SIRS in our study. As dynamics of fluorescence
intensity for CD64 and CD163 on neutrophils was diagnostically very significant,
it is highly unlikely that a nonspecific staining would have been the cause. Also,
the representative FACS diagrams made possible quite a sharp distinction
between cell populations without causing serious problems in cell gating. Our
results therefore indicate that, like monocytes, neutrophils are able to some
extent to express CD163.Membrane CD163 expression has not
yet been clinically evaluated. Only one author found increased bone marrow infiltration
with CD163-positive macrophages in postmortem analysis of samples from patients
who died from severe sepsis or septic shock compared with controls [33]. In
contrast, high concentrations of soluble CD163 (sCD163) have been described in
adult septic patients in several studies [3, 21]. It has been shown
experimentally with cultured monocytes that CD163 can be shed from the cell
membrane after LPS stimulus [20], cross-linking of the Fc receptor for
immunoglobulin G [34] or oxidative stress [35]. The shedding of CD163 from
monocyte membrane correlates with decreased expression of membrane CD163 on
cultured monocytes [20, 34]. The decreased expression of membrane CD163 is
probably partly due to shedding of CD163 [20] and partly due to decreased CD163
mRNA synthesis [34, 36]. In healthy adult volunteers, CD163 surface expression
was increased again 24 hours after LPS stimulation [20]. The precise time point
of increased expression is not yet exactly known, but it was estimated to be
0–24 hours after LPS stimulation in adult volunteers [20] or even later in
cultured monocytes [34]. It was shown that stimulation with LPS induced
short-term suppression of CD163 mRNA expression in cultured monocytes, while
long-term cultures of monocytes treated with LPS showed intereleukin-10
dependent recovery of surface CD163 expression [34]. In our clinical
study, we did not observe the transitional fall of membrane CD163 expression on
monocytes. The expression of CD163 on both monocytes and neutrophils was higher
in septic patients than in patients with noninfectious SIRS both at the time of
suspected sepsis and 24 hours later. The reason may be that clinical signs are
observed with significant delay after endotoxinemia.Most clinical studies in the past
have used manually determined fluorescence intensity to describe CD64
expression on neutrophils. This method, however, is subject to day-to-day
operational and instrumental fluctuations and cannot be used for comparison
between different laboratories. The newly developed method for
computer-calculated indexes uses calibration beads which serve as an internal
fluorescence intensity standard and, together with fully automated software for
index calculation, which removes the subjectivity in data analysis by the end
users, provide a novel approach in minimizing lot-to-lot variation, and in
removing the subjectivity due to different range of experience and skills of
operators [25]. In our study, comparison of manually determined fluorescence
intensity and computer-calculated index revealed that diagnostic accuracy of
both parameters was equal for CD64 on neutrophils and monocytes and CD163 on
neutrophils, but not for CD163 on monocytes. Our explanation is that automated
index calculation was originally developed for evaluation of CD64 index on
neutrophils in different clinical conditions [25] and the method is probably
not universally transmittable to evaluate all other antigens on different cell
types for diagnostic purposes. The calculation of CD64 index for neutrophils,
which achieved the highest diagnostic accuracy in our study, was optimal and we
suggest that this parameter should be used in future evaluations. We found no
studies, except for a poster presentation at Sepsis 2007 in Paris
[37], which have used index calculation
for evaluation of CD64 expression on neutrophils.Some limitations of this study merit
consideration. Firstly, the definition of clinical sepsis with culture-negative
patients, particularly critically ill neonates, is still a matter of
discussion. Secondly, the number of patients is relatively small. A greater
number of neonates and children is needed for a more precise evaluation of diagnostic
accuracies of selected infection markers (CD64 and CD163 index for monocytes
and neutrophils) and for comparison with routinely used infection markers in
the future. The blood samples were processed within 36 hours in our study. For
clinical use, the method should be available every day, particularly because
some authors suggest processing of blood specimens for analysis of cell surface
markers immediately after obtaining them to avoid cell apoptosis [30]. However,
other authors claim that measurements for neutrophil CD64 assay are stable for
up to 30 hours at room temperature and up to 72 hours refrigerated [25]. As we
already mentioned, no significant differences in levels of CD64 and CD163
expressions were obtained in the first 36 hours after blood sampling in our
study.


## 5. Conclusion

The data from the present study show
that CD64 and CD163 expressions on neutrophils and monocytes are elevated in
critically ill neonates and children with SIRS with sepsis. Diagnostic
accuracies of computer-calculated indexes for
CD64 on neutrophils and monocytes and CD163 on neutrophils based on the ratio (index)
of linearised MFI to the fluorescence signal of standardized beads are equal to
diagnostic accuracy of manually determined MFI, if processed by the same flow
cytometer. Therefore, CD64 MFI, CD163 MFI, CD64
indexes for neutrophils and monocytes and CD163 index for neutrophils can all be
used for discrimination of SIRS and sepsis in critically ill neonates and
children. CD64 index for neutrophils, however, is superior to all other
markers.



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*Source: 202646-2008-07-01.xml*