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Comparison of Neutrophil CD64 Expression,

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Laboratory Hematology 11:137-147
© 2005 Carden Jennings Publishing Co., Ltd.
doi: 10.1532/LH96.04077
Comparison of Neutrophil CD64 Expression, Manual
Myeloid Immaturity Counts, and Automated Hematology
Analyzer Flags as Indicators of Infection or Sepsis
BRUCE H. DAVIS, NANCY C. BIGELOW
Trillium Diagnostics, LLC, and Maine Medical Center Research Institute, Scarborough, Maine, USA
Received December 23, 2004; received in revised form January 30, 2005; accepted February 10, 2005
ABSTRACT
There is a clear need for improved indicators of infec-
tion or sepsis to increase the sensitivity and specificity of
both diagnosis and therapeutic monitoring. One of the
effects of inflammatory cytokines on the innate immune
response is the rapid up-regulation of CD64 expression on
the neutrophil membrane. We and others have hypothe-
sized that the measurement of neutrophil CD64 expression
might represent an improved diagnostic indicator of infec-
tion and sepsis. In this study we assessed the relative ability
of flow cytometric neutrophil CD64 measurements, neu-
trophil counts, myeloid immaturity differential counts, and
flagging on an automated hematology analyzer to correlate
with the presence of infection, as determined by a retro-
spective clinical scoring system of infection or sepsis. A
total of 160 blood samples were randomly selected to
derive equal proportions of the 3 categories of flags on a
Coulter STKS blood counter that indicate the presence of a
myeloid left shift. The patients for these samples were
scored by retrospective chart review and placed into 4
groups on the basis of likelihood of infection, sepsis, or
severe tissue injury. Neutrophil CD64 expression demon-
strated a superior sensitivity (94.1%), specificity (84.9%),
and positive predictive likelihood ratio (6.24), compared
with neutrophil counts (sensitivity, 79.4%; specificity,
46.8%; positive predictive likelihood ratio, 1.49), band
counts (sensitivity, 87.5%; specificity, 43.5%; positive pre-
dictive likelihood ratio, 1.55), myeloid immaturity fraction
(sensitivity, 94.6%; specificity, 84.5%; positive predictive
likelihood ratio, 2.12), and flagging on an automated
hematology analyzer (sensitivity, 94.1%; specificity, 40.5%;
Correspondence and reprint requests: Bruce H. Davis, MD, Maine
Medical Center Research Institute, 81 Research Dr, Scarborough, ME
04074, USA (e-mail: davisb@mmc.org).
137
ISLH
Official Publication
positive predictive likelihood ratio, 1.58). Relative to the
other laboratory parameters, the neutrophil CD64 parame-
ter also provided the best separation of the 4 clinical
groups. The findings indicate that neutrophil CD64
expression as determined by quantitative flow cytometry is
an improved diagnostic indicator of infection/sepsis relative
to current laboratory indicators of relative or absolute
myeloid cell counts or hematology analyzer flagging algo-
rithms. Lab Hematol. 2005;11:137-147.
KEY WORDS: Inflammation · Sepsis · Infection ·
Leukocyte differential · Laboratory
automation · Fc receptor · Sepsis
assay · Sepsis test · Neutrophil
activation · Immature granulocytes
INTRODUCTION
Diagnostic laboratory indicators of a systemic acute
inflammation response to infection or sepsis have shown lit-
tle evolution in the past few decades despite a marked
advance in the understanding of the molecular and cell biol-
ogy of the effector myeloid cells and cytokines. The labora-
tory evaluation of patients with a suspected infection or an
acute inflammatory disease process has remained a battery of
tests including a complete blood count with leukocyte differ-
ential counting, an erythrocyte sedimentation rate determi-
nation, a C-reactive protein level measurement, and microbi-
ologic cultures. These laboratory tools have been available
with minimal modification for more than the last 2 decades.
Additional laboratory tests with less specificity that show
changes during an active acute inflammatory response
include immune complex measurements, leukocyte alkaline
phosphatase enzyme cytochemical studies on neutrophils in
blood smears, and the microscopical differential counting of
138 B. H. Davis and N. C. Bigelow
immature or “left-shifted” myeloid forms. Only the intro-
duction of the measurement of procalcitonin levels as
another form of acute-phase reactant test with purported
greater specificity for bacterial infection than for viral infec-
tion can be touted as a new diagnostic test for the evaluation
of the patient with a suspected infection [1-9]. As of this
writing, however, this test has not been cleared for in vitro
diagnostic use in the United States, and it has not been uni-
versally accepted as an improved diagnostic assay of infection
[10-12]. The complete blood count and leukocyte differen-
tial with the associated band count or the myeloid left shift
of immaturity receive unjustified clinical use, particularly the
overreliance on the nonspecific, insensitive band counts [13-
19]. Hence, there remains a persistent need for an improved
diagnostic indicator of infection or sepsis, as well as for better
therapeutic monitors in the treatment of infection so that
antibiotic therapy might be less empiric.
Our studies have indicated that quantitative measure-
ment of neutrophil CD64 (high-affinity Fc receptor)
expression is a worthwhile candidate for evaluation as a
more sensitive and specific laboratory indicator of sepsis or
the presence of a systemic acute inflammatory response
[20-25]. Other groups have similarly reported on the asso-
ciation between an elevation in neutrophil CD64 expres-
sion and the presence of infection [26-36]. A change in
neutrophil CD64 expression is one of many activation-
related antigenic changes manifested by neutrophils during
the normal pathophysiologic acute inflammatory response.
Neutrophil expression of CD64 is up-regulated under the
influence of inflammation-related cytokines such as inter-
leukin 12, interferon γ, and granulocyte colony-stimulating
factor [22,37-50]. Up-regulation of CD64 in the presence of
such cytokines or in response to infection is but one of
many changes that occur as part of neutrophil activation
that have been elucidated in the last few decades. However,
a change in neutrophil CD64 expression differs from the
parallel changes of increased CD45RA expression [51],
increased expression of CD11b/18 [52-57], and loss of
expression of CD16 [55,58-61], CD62L [53,62], and
CD66b [57] in that the normal baseline expression of
neutrophil CD64 is negligible. Thus, CD64 appears
uniquely ideal as a surrogate marker of neutrophil activa-
tion or a systemic acute inflammatory response because
neutrophil CD64 expression goes from our normal refer-
ence range of less than 2000 sites per cell and becomes
up-regulated in a graded fashion, depending on the
intensity of cytokine stimulation [22,44,46,50]. Further-
more, the up-regulation of neutrophil CD64 expression
appears to be of pathophysiologic significance because
this form of Fc receptor has been shown to function in
eliciting all of the neutrophil functional responses used in
antibacterial responses [22,45,46].
This study was designed to examine the correlation of
neutrophil CD64 expression, as measured by quantitative
flow cytometry, with the current laboratory evaluation of
neutrophil counts and the presence of immature myeloid
forms in the blood in the context of a retrospective clinical
scoring system of infection or sepsis. A determination of the
relationship between quantitative neutrophil CD64 measure-
ments and current laboratory hematology testing method-
ologies is felt to be a prerequisite for prospective clinical
studies into the diagnostic utility of neutrophil CD64 meas-
urement as a new diagnostic approach for inflammation in
infection/sepsis. We report that although neutrophil CD64
expression is correlated with flagging algorithms on a Coulter
STKS (Beckman Coulter, Hialeah, FL, USA) and with the
presence of increased proportions of bands and more imma-
ture myeloid forms in blood as determined by manual differ-
ential counts, this correlation is imperfect, and we have
found neutrophil CD64 expression to be a superior diagnos-
tic indicator of infection or sepsis.
METHODS AND MATERIALS
Sample Selection
Laboratory personnel selected 160 blood samples from
the routine clinical hematology laboratory at a 650-bed hos-
pital (Harris Methodist Hospital, Fort Worth, Texas, USA)
to achieve an equal distribution between the groups defined
by the immature granulocyte/band (IG/band) flags on a
Coulter STKS hematology blood counter using software ver-
sion 1H (Beckman Coulter). The 3 categories were no flag
(n = 50), the IG/band 1 flag (n = 55), and the IG/band 2
flag (n = 55). Sample selection was based purely on flagging
algorithms with no regard for absolute neutrophil counts,
band percentages, or knowledge of individual clinical histo-
ries. Samples were selected for subsequent flow cytometric
measurement of CD64, and measurements were performed
within 4 hours of the patient draw time. The study protocol
was reviewed and approved by the institutional human sub-
jects review board.
Clinical-Infection/Sepsis Score
A retrospective chart review of the patients was per-
formed at the conclusion of the study, with the reviewer
(B.H.D.) blinded to the neutrophil CD64 results during the
assessment of the hospital course. Patients were divided into
4 groups on the basis of the medical history chart review
and the degree or likelihood of a systemic acute inflamma-
tory response as follows: group 0, no clinical or laboratory
evidence of infection or inflammatory process; group 1,
clinical or laboratory evidence of localized infection or
inflammatory process with no or low probability of a sys-
temic inflammatory response; group 2, clinical or laboratory
evidence of infection or inflammatory process of a extensive
localized nature with an undocumented probability of elicit-
ing a systemic inflammatory response; group 3, unequivocal
clinical or laboratory evidence of systemic sepsis, infection,
 Neutrophil CD64, Manual Myeloid Immaturity Counts, and Hematology Analyzer Flags
or inflammatory process based on the identification of
organisms by culture or smear visualization. The patients in
group 0 had no laboratory or clinical signs or symptoms
indicative of an infectious process or any evidence of an
inflammatory medicated disease process. The patients in
group 1 also had no evidence of a systemic process. Blood
culture results were negative, and if infection or tissue injury
was found, it was a local infection, such as a laceration, cys-
titis, otitis, myocardial infarct, or fractured ankle. Group 1
was quite heterogeneous, but the clinical and laboratory
findings did not indicate any reason to conclude the pres-
ence of a systemic inflammatory process. The patients in
group 2 had clinical findings to indicate a systemic response
to infection or tissue injury but, unlike those in group 3,
had no laboratory evidence of infection. Group 2 included
patients such as those with suspected ruptured diverticulitis
or radiologic evidence of pneumonia, with cultures not
reported as positive for infection, yet with a clinical decision
to treat for infection having been made. The patients in
group 3 had both clinical evidence of a systemic process of
infection or sepsis and laboratory documentation of an
infectious etiology. This group was the most definitive for
the presence of infection, sepsis, or severe tissue injury.
Quantitation of CD64
Leukocyte CD64 expression was measured as previously
described [20,23]. Briefly, 50 µL of whole blood or whole
blood diluted with phosphate-buffered saline (PBS) to adjust
the leukocyte concentration to less than 2.5 × 109 cells/L was
incubated for 10 minutes at room temperature with saturat-
ing amounts of fluorescein isothiocyanate (FITC)-conjugated
anti-CD64 murine monoclonal antibody (Medarex, Annan-
dale, NJ, USA; now available from Trillium Diagnostics,
Scarborough, ME, USA) or isotype control murine antibody
followed by red blood cell lysis and fixation with the Coulter
Q-Prep system (Beckman Coulter). Samples then were
washed once and resuspended with PBS containing 1%
bovine serum albumin (Sigma Chemical Company, St.
Louis, MO, USA), pH 7.4, to a volume of 0.5 mL. Flow
cytometric analysis was performed with a Cytoron Absolute
flow cytometer (Ortho Biotech, Raritan, NJ, USA) to collect
logarithmic green fluorescence and forward and logarithmic
right-angle light scatter signals on a minimum of 20,000
leukocytes. Interassay standardization and CD64 quantita-
tion was performed with Quantum 24 FITC calibration
beads (Flow Cytometry Standards Corporation, San Juan,
PR, USA; now available through Bangs Laboratories, Indi-
anapolis, IN, USA), which were analyzed at the start and fin-
ish of each flow cytometric batch run and were used to stan-
dardize and calibrate the software used for the analysis of list
mode files. Data analysis was performed with light scatter
gating to define the major leukocyte populations of lympho-
cytes, monocytes, and neutrophils, with the CD64 intensity
quantified as FITC mean equivalent soluble fluorescence
139
(MESF) units by means of QuickCal for Winlist (Verity
Software House, Topsham, ME, USA). Neutrophil CD64
expression in MESF units was corrected for any nonspecific
antibody binding by subtracting the values for isotype con-
trol antibody obtained by staining and flow cytometric
analysis of the controls in parallel with the test samples.
Leukocyte Differential Analysis
Complete blood counts and leukocyte 5-part differential
results were obtained with a Coulter STKS hematology
blood counter with software version 1H. Instrument
reagents and calibration were as recommended by the man-
ufacturer. Instrument leukocyte flagging by the STKS soft-
ware results in the determination of the myeloid population
as normal, IG/band 1, or IG/band 2. Microscopical leuko-
cyte differential counts were performed by a single observer
(N.C.B.) on all specimens derived from 2 Wright-stained
blood smears and were based on a 400-cell count, as
detailed by CLSI/NCCLS document H20-A [63]. Mature
neutrophils were separated from band forms according to
the guidelines recommended by the College of American
Pathologists [64]. The immature myeloid fraction was cal-
culated as the number of myeloid cells at the metamyelo-
cyte stage or younger (myelocyte or promyelocyte) divided
by the total number of myeloid cells (neutrophils, bands,
and immature forms).
Statistical Analysis
Linear regression analysis was performed with Microsoft
Excel software (Microsoft Corporation, Redmond, WA,
USA). Paired and unpaired 2-tailed Student t tests were per-
formed with Statview 5.0 (SAS Institute, Cary, NC, USA) to
determine correlations between measurements. Statistically
significant differences were defined at a P level of <.05.
RESULTS
As in previous studies, clinical samples were found to have
variable expression of neutrophil CD64 (range, 408-60,452
MESF units), ranging from the normal or healthy low levels
of <2500 MESF units to levels greater than those seen in
monocytes from healthy individuals. This variability in CD64
expression demonstrates the significant in vivo ability of the
neutrophil to modulate expression of this Fc receptor. As was
also observed in previous studies, the neutrophil population
in clinical samples demonstrated a near-Gaussian unimodal
distribution of CD64 expression in flow cytometric analyses,
rather than the appearance of subpopulations of neutrophils
with different surface expression levels of the antigen.
Whether neutrophil CD64 expression was low as in the
healthy state or was markedly increased as seen in individuals
with sepsis or a systemic acute inflammatory response, the cir-
culating population of neutrophils in the blood was observed
as a single cell cluster with regard to neutrophil CD64 expres-
140 B. H. Davis and N. C. Bigelow

forms (metamyelocyte stage or younger). Eosinophils express

FIGURE 1. Representative histograms of CD64 expression on
lymphocytes (first peak), neutrophils (second peak), and
monocytes (third peak) in blood samples with normal levels
(top histogram) and elevated levels (bottom histogram).
sion (Figure 1). The only circumstances in which myeloid
cells in the blood demonstrated a skewed unimodal or
bimodal distribution were in samples with a relative increase
in eosinophils or with the presence of immature myeloid
low levels of CD64 and higher levels of endogenous autofluo-
rescence that overlap with or are slightly higher than the neu-
trophil expression levels in samples from healthy individuals,
but eosinophils may also become a lower-intensity population
distinct from the neutrophil population exhibiting up-regulation
of CD64. Compared with neutrophils, immature myeloid
cells constitutively express higher levels of CD64. The higher
CD64 expression of immature myeloid cells is down-regulated
or lost with normal maturation to the mature neutrophil
stage. Thus, the presence of immature myeloid forms in the
blood without neutrophil up-regulation may give a left-sided
skewness or tail to the myeloid population distribution of
CD64 expression.
The relationships of neutrophil CD64 expression to neu-
trophil counts, manual band differential counts, the immature
myeloid ratio, and blood counter flagging for immaturity are
summarized in Table 1 and Figures 2-6. Samples with an
abnormal immature myeloid fraction (>0.00) were identified
in all groups defined by the STKS flagging, but only the
IG/band 2 group had significantly more abnormal samples rel-
ative to the no flag group (P = .0167) or the IG/band 1 group
(P = .0279). As shown in Figure 2, there is no significant rela-
tionship between the neutrophil count and neutrophil CD64
expression. Relationships between increasing band counts, cir-
culating immature myeloid cells, and immaturity flagging, and
increased neutrophil CD64 expression were noted, although
the correlations were not strong (Figure 3). Neutrophil CD64
expression had a stronger correlation with the band percentage
(r = 0.539) than with the absolute band count (r = 0.270;
results not shown). Whereas there was less of a correlation
between neutrophil CD64 expression and the presence of cir-
culating immature myeloid forms (r = 0.169; Figure 3), only
28% of the specimens had such cells noted in the microscopi-
cal differential count, and most of those counts had an imma-
ture myeloid fraction of less than 0.02.
Correlation of Laboratory Parameters to Clinical Status
Chart reviews of all 160 patients were performed, and
clinical histories with regard to the episode approximate to
the blood sampling at the time of the laboratory studies were
scored in a blinded fashion with regard to laboratory results
into 1 of 4 groups defined as follows: (1) score 0, no clinical

TABLE 1. Summary Statistics of the Neutrophil Parameters Collected in this Study Separated by the Blood Cell Counter Flags for Left Shift*

Blood Counter Flags Neutrophil Count, ×109/L Bands, % Band Count, ×109/L Neutrophil CD64, MESF Units

Normal (n = 50) 4.95 ± 2.0 (1.52-8.70) 10.46 ± 9.7 (1-55) 0.88 ± 0.88 (0.03-4.23) 2586 ± 1807 (804-10,761)
IG/Band 1 (n = 55) 10.78 ± 3.36 (2.43-20.77) 18.96 ± 14.06 (1-55) 2.58 ± 2.23 (0.7-9.38) 3983 ± 3268 (408-16,024)
IG/Band 2 (n = 55) 12.93 ± 7.2 (1.07-37.15) 2.21 ± 18.83 (3-69) 4.09 ± 3.78 (0.32-16.05) 11,472 ± 12,295 (1126-60,542)

*Data are expressed as the mean ± SD (range). MESF indicates mean equivalent soluble fluorescence; IG/Band, immature granulocyte/band.
 Neutrophil CD64, Manual Myeloid Immaturity Counts, and Hematology Analyzer Flags
FIGURE 2. The relationship of absolute neutrophil (PMN)
count to neutrophil CD64 expression (top) and to the per-
centage of band forms (bottom) shows no significant corre-
lation or a weak correlation among the 160 blood samples
selected for this study.
indication of acute inflammation; (2) score 1, weak clinical
suspicion of localized infection or acute inflammatory
process; (3) score 2, moderate clinical evidence of infection
and/or acute inflammation; and (4) score 3, definite clinical
indication of infection and/or severe tissue trauma, shock, or
acute inflammatory process. The clinical-sepsis scoring
demonstrated varying degrees of correlation with the labora-
tory indicators of infection, including the instrument results
of immature myeloid cell flagging.
The mean clinical scores of the 3 STKS flagging algo-
rithm groups differed significantly (P < .0001). The 3 flag-
ging groups also showed significant differences in the
percentages of band forms (Figure 4C), neutrophil counts
(Figure 4B), and the quantities of CD64 expression in neu-
trophils (Figure 4A). The presence of circulating immature
myeloid cells differed significantly among the IG/band flag-
ging groups, but to a lesser degree (Figure 4D). The clinical-
141
FIGURE 3. The relationship between neutrophil CD64
expression and neutrophil band forms from a microscopical
leukocyte differential count shows a correlation when band
counts are expressed as a percentage of leukocytes (top), but
less of a correlation was observed with the presence of circu-
lating myeloid forms that were less mature (bottom).
sepsis score groups similarly showed significant differences in
the various neutrophil quantitative and maturity parameters
(Figure 5). Even the blood cell counter flagging categories
showed a significant difference with regard to the clinical-
sepsis scores or the presence of infection/sepsis (Figure 6).
Yet, neutrophil CD64 expression gave the best separation
between the clinical-sepsis score groups, particularly between
the groups with clinical evidence of infection and those with-
out findings of infection/sepsis.
The results for laboratory parameters measured in this study,
when further segregated into normal or abnormal values,
showed varying degrees of diagnostic power in separating the
clinical-sepsis score groups. However, the neutrophil CD64
measurement showed the highest apparent specificity and sensi-
tivity with regard to separating the clinical-sepsis score groups.
142 B. H. Davis and N. C. Bigelow
FIGURE 4. Neutrophil CD64 expression levels (A), band counts (B), neutrophil counts (C), and the immature myeloid fraction (D)
all show significant differences between the various blood cell counter immature granulocyte/band (IG/band) flagging groups for
myeloid immaturity. CBC indicates complete blood count.
The relative diagnostic performances of the laboratory parame-
ters were determined in 2 ways. To determine the relative diag-
nostic utility of each parameter in the most ideal conditions, we
assessed the performance in the patients with clinical-infection/
sepsis scores of 0, 2, and 3 (Table 2) to allow elimination of the
variable clinical group with a score of 1. The second assessment
was done with all samples. Even when the heterogeneous group
with score 1 was included in the diagnostic assessment (Table
3), neutrophil CD64 expression still demonstrated clear superi-
ority with a sensitivity of 94.12%, a specificity of 84.92%, an
efficiency of 86.88%, and a positive likelihood ratio of 6.24.
Other parameters approached the sensitivity of neutrophil
CD64 expression, but all had much lower specificities, efficien-
cies, and likelihood ratios.
DISCUSSION
Neutrophil CD64 expression is regulated in a fashion that
seemingly parallels the degree of the acute inflammatory
response to a significant clinical process of infection or tissue
injury. The expression of CD64 is highly correlated with the
presence of infection [20,21,26-36]. Its regulation is con-
trolled in a myeloid-specific fashion and influenced by the
cytokines involved in the acute inflammatory response, such
as interleukin 12, interferon γ, and granulocyte colony-
stimulating factor [22,37-50]. The results of this study show
good correlation with flagging algorithms present on one
representative blood cell counter, moderate correlation with
the percentage of band and immature myeloid forms, and no
significant correlation with the neutrophil count. The corre-
lation of neutrophil CD64 expression with the flagging algo-
rithm was observed in this study with the use of only 1
model of blood cell counter by a single manufacturer, which
has now been superseded by newer versions of this instru-
ment. However, similar correlations have also been observed
when neutrophil CD64 measurements were correlated with
the algorithm of a more contemporary blood counter instru-
ment made by another manufacturer that used a different
 Neutrophil CD64, Manual Myeloid Immaturity Counts, and Hematology Analyzer Flags 143

FIGURE 5. Clinical-infection/sepsis score groups show the most significant differences in neutrophil CD64 expression (A), but neu-
trophil counts (B) and morphologic assessment of a left shift by band counts (C) or by the immature myeloid fraction (D) also
show distinctions between the clinical groups.

technology to identify immature myeloid cells [65]. Hence,
an increase in neutrophil CD64 expression appears to paral-
lel the appearance of immature myeloid forms or the “left
shift” in the blood. Yet, the imperfect and variable correla-
tions to the results of standard laboratory studies by them-
selves would seem to indicate that the measurement of
CD64 expression is not simply a surrogate for existing labo-
ratory tests for infection or sepsis currently available in most
hospital laboratories.
The results of this study seemingly suggest that all labora-
tory parameters are correlated with the presence of acute
inflammation or infection but that they are not reporting the
same information. The correlation of laboratory parameters
with the clinical chart review results and the resulting sepsis
scores indicates that neutrophil counts and immaturity indi-
cators, such as band counts or flagging algorithms, do corre-
late with the clinical condition, albeit imperfectly and with
good sensitivity but with relatively low specificity. The CD64
measurements in this study show a better correlation with
clinical scoring for the presence of an infectious or inflamma-
tory process and manifest a better sensitivity and specificity
with regard to the detection of clinically meaningful acute
inflammation. Our findings on band counts and neutrophil
counts as indicators of infection do not differ significantly
from the findings reported by many previous studies [13-
15,17-19], namely, that band counts, although showing some
correlation with the presence of infection, are an imperfect
laboratory diagnostic parameter. This conclusion suggests that
the lack of correlation between CD64 expression and band
and absolute neutrophil counts is simply the difference in
specificity between CD64 measurements and the other labo-
ratory parameters with regard to the correlation with an acute
inflammatory response. Our interpretation is that these
results indicate that CD64 expression should be a more
promising and meaningful parameter of the acute inflamma-
tory response found in infection and sepsis.
The lack of a single highly specific and sensitive labora-
tory indicator of acute inflammation has resulted in the clini-
cal use of several concurrent laboratory tests in routine prac-
tice to rule in or rule out the presence of an infection or
144 B. H. Davis and N. C. Bigelow

as steroids and adrenergic agents) or the presence of myelo-

FIGURE 6. The relationship between the blood cell counter
immature granulocyte/band (IG/band) flagging and the
clinical-sepsis score showed significant differences between
the 3 flagging levels and the presence of infection/sepsis.
significant acute inflammatory response. Culture results are
often viewed as confirmatory but in practice are often not
used in treatment decisions because of their relatively slow
turnaround times of up to 72 hours or more. Several investi-
gators have proposed varying algorithms to approach the pre-
dictive value of the relatively insensitive tests of sedimenta-
tion rate, C-reactive protein level, and the presence of imma-
ture leukocyte populations, but there is a clear need for
improved diagnostics in this area [66]. Perhaps the relative
specificity of these tests is not unexpected, given the number
of additional factors, such as concurrent drug therapy (such
proliferative disorders, that can result in changes in these
laboratory parameters independent of the presence of infection
or acute inflammation. Myeloid cell CD64 up-regulation
appears to be a more specific and perhaps a more sensitive
avenue of laboratory quantitation, given that this up-regulation
appears to reflect a functional change in neutrophil activa-
tion under the influence of inflammation-related cytokines
and to result in an incremental improvement in neutrophil
cell function. For example, the use of interferon γ has shown
efficacy in clinical trials in patients with chronic granuloma-
tous disease despite little or no evidence of a correction of
the oxidative burst defect in most patients [67]. Interferon γ
administration in the treatment of chronic granulomatous
disease and in healthy individuals results in a detectable
increase in CD64 expression 4 to 6 hours following adminis-
tration, which leads to a demonstrable increase in cellular
functions, such as phagocytosis, that are mitigated through
Fc receptor mechanisms [20,22,45].
Other antigenic changes occurring with neutrophil acti-
vation, such as modulations in CD11b, CD18, CD16,
CD62L, and CD45RA expression, may also occur, but these
changes have relative disadvantages as useful laboratory
parameters compared with neutrophil CD64 expression.
That all of these other antigenic expressions show significant
expression in healthy individuals, often with a moderate
degree of heterogeneity, leads to questions of sensitivity.
Additionally, the expression of some of these antigens, such
as CD11b, show changes that depend on specimen handling
factors that are independent of any disease state [53,68]. Fac-
tors that modulate such expression include specimen storage,
temperature, and centrifugation. Our studies to date have
indicated that neutrophil CD64 expression is stable for at
least 36 hours or longer in anticoagulated blood samples

TABLE 2. Performance Statistics of Diagnostic Parameters in Recognizing Infection/Sepsis with Disease Presence Defined by Infection/Sepsis
Scores 2 and 3 and Disease Absence Defined by Infection/Sepsis Score 0*

Neutrophil CD64 Immature

Expression Neutrophil Count CBC Analyzer Flag Band Percentage Myeloid Fraction
(>5000 MESF Units) (>7500 × 106/L) (IG/Band 1 or 2) (>10%) (>0.00)

Sensitivity 94.12% 79.41% 94.12% 87.50% 46.88%

Specificity 100.00% 94.59% 97.30% 89.19% 82.86%
PPV 100.00% 93.10% 96.97% 87.50% 71.43%
NPV 94.87% 83.33% 94.74% 89.19% 63.04%
Efficiency 97.18% 87.32% 95.77% 88.41% 65.67%
LR+ >100 14.69 34.82 8.09 2.73
LR– 0.06 0.22 0.06 0.14 0.64

*The positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR–) show the neu-
trophil CD64 parameter to have the best diagnostic performance. CBC indicates complete blood count; IG/Band, immature granulocyte/band.
 Neutrophil CD64, Manual Myeloid Immaturity Counts, and Hematology Analyzer Flags 145

TABLE 3. Performance Statistics of Diagnostic Parameters in Recognizing Infection/Sepsis with Disease Presence Defined by Infection/Sepsis
Scores 2 and 3 and Disease Absence Defined by Infection/Sepsis Scores 0 and 1*

Neutrophil
CD64 Expression Neutrophil Count CBC Analyzer Flag Band Percentage Immature Myeloid
(>5000 MESF Units) (>7500 × 106/L) (IG/Band 1 or 2) (>10%) Fraction (>0.00)

Sensitivity 94.12% 79.41% 94.12% 87.50% 46.88%

Specificity 84.92% 46.83% 40.48% 43.48% 77.88%
PPV 62.75% 28.72% 29.91% 30.11% 37.50%
NPV 98.17% 89.39% 96.23% 92.59% 83.81%
Efficiency 86.88% 53.75% 51.88% 53.06% 71.03%
LR+ 6.24 1.49 1.58 1.55 2.12
LR– 0.07 0.44 0.15 0.29 0.68

*The positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), and the negative likelihood ratio (LR–) show the
neutrophil CD64 parameter to have the best diagnostic performance. CBC indicates complete blood count; IG/Band, immature granulocyte/band.

stored at room temperature; thus, this parameter is quite
amenable to clinical laboratory testing. Additional studies are
required to comprehend more fully the utility of neutrophil
CD64 expression as a potential diagnostic indicator of infec-
tion or sepsis.
The results of this and other studies indicate a potential
for neutrophil CD64 expression as a more sensitive and spe-
cific indicator of clinical infectious conditions requiring ther-
apeutic decisions. This study employed a method of quanti-
tative cytometry using reference bead standards with FITC
MESF units, which are now traceable to new National Insti-
tute of Standards and Technology (NIST) reference materials
for fluorescein, so-called RM 8640 and SRM 1932 [69,70].
This approach, although suitable for a clinical research study,
is likely too costly and complex for a routine clinical labora-
tory test. In fact, our current studies are being performed
with a modification of the assay described here, in which a
single microbead standard in a whole-blood lysate, no-wash
assay is used with dedicated software to derive a neutrophil
CD64 index. This assay, the Leuko64 assay, is much simpler
and more amenable to a clinical laboratory setting. The
Leuko64 assay quantitates CD64 expression as a CD64
index referenced to the microbead standards. This index is
highly correlated with MESF units and can be transformed
by the NIST reference material to molecular equivalents of
CD64 expression. This study and previous studies indicate
that neutrophil CD64 expression is worthy of a more thor-
ough clinical evaluation as an improved diagnostic indicator
of infection and sepsis, particularly in an assay format suit-
able for use in a clinical laboratory.
ACKNOWLEDGMENTS
This work was presented in part at meetings of the
International Society of Laboratory Hematology in Jack-
son Hole, Wyoming, in May 1995, and the Clinical
Cytometry Society in Charleston, South Carolina, in
August 1995. We gratefully acknowledge the skilled tech-
nical assistance of John Smith and Chris Black of Harris
Methodist Laboratories, Fort Worth, Texas, and the secre-
tarial assistance of Janet Kumbier.
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