In press, Archives in Pathology and Laboratory Medicine
Neutrophil CD64 is an Improved Indicator of Infection or Sepsis in
Emergency Room Patients
Bruce H. Davis*
Stephen H. Olsen^
Nancy C. Bigelow*
*Trillium Diagnostics, LLC and
Maine Medical Center Research Institute
Scarborough, Maine 04074
^Department of Clinical Pathology
William Beaumont Hospital
Royal Oak, Michigan 48073
Address correspondence to: Bruce H. Davis, M.D.
Maine Medical Center Research Institute
81 Research Drive
Scarborough, Maine 04074
Inflammation, sepsis, infection, leukocyte differential, laboratory automation, Fc
receptor, sepsis assay, sepsis test, neutrophil activation, immature granulocytes, near
patient testing, medical triage
Sepsis is a significant health problem in the United States with an estimated
750,000 new cases annually [1, 2]. Sepsis is among the ten leading causes of death and
the leading cause of mortality in the non-coronary intensive care unit with a mortality rate
of 30-50% [1-4]. It is estimated that the yearly economic burden to the U.S. alone is
nearly $17 billion . The frequency of sepsis has increased over 135% in a ten-year
interval in the most recent decade and is predicted to continue to rise due in part to the
continued increase in antibiotic resistance [1, 2, 6]. In addition to the fully developed
cases of sepsis, there are millions of patients on an annual basis in the U.S. alone that
suffer severe infection or clinical symptoms similar to sepsis that require diagnostic
evaluation. Ironically, although treatment of sepsis has evolved in the last decades with
newer therapeutic options of improved antibiotics and more novel approaches, such as
inhibitors of the inflammatory and coagulation response, little has changed to improve
diagnosis or therapeutic monitoring [1, 3, 4, 6-11]. There has been some consensus put
forth to standardize the clinical signs and symptoms of sepsis, such as the PIRO staging
system . However despite the fact that in appropriate treatment and failure to
diagnosis sepsis represent the leading causes of mortality in sepsis [13, 14], diagnostic
advances in the detection and monitoring of sepsis have not entered clinical practice.
Thus, an improved diagnostic test for infection and sepsis would have both economic
and therapeutic healthcare benefits.
Diagnostic indicators of the systemic acute inflammation response to infection or sepsis
have shown few innovations in the past few decades, despite a marked advance in the
understanding of the molecular and cell biology of the effector myeloid cells and
cytokines involved in the innate immune response. The laboratory evaluation of patients
with suspected infection or sepsis has remained a battery of tests including the complete
blood count (CBC), leukocyte differential counting for evidence of a myeloid left shift,
erythrocyte sedimentation rates (ESR), C-reactive protein (CRP), and microbiologic
cultures. Only the introduction of procalcitonin (PCT) levels, as another form of acute
phase reactant with purported greater specificity to bacterial compared to viral infection,
can be touted as a new diagnostic for the evaluation of the patient with suspected
infection [15-23], but as of this writing PCT is not cleared for in vitro diagnostic use in the
United States nor universally accepted as an improved diagnostic assay of infection [24-
26]. The CBC and leukocyte differential with associated band count or myeloid left shift
of immaturity receive unjustified clinical utilization, particularly the over reliance on the
nonspecific, insensitive band counts [27-33]. Hence a persistent need exists for
improved diagnostic indictors of infection or sepsis, as well as better therapeutic
monitors in the treatment of infection so that antibiotic therapy might be less empiric.
Our previous studies have indicated that quantitative 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 [34-39]. Other groups have reported a similar association between elevation
in neutrophil (PMN) CD64 expression and the presence of infection [40-50]. Neutrophil
CD64 is one of many activation-related antigenic changes manifested by neutrophils
during the normal pathophysiologic acute inflammatory response. PMN expression of
CD64 is up-regulated under the influence of inflammatory related cytokines such as
interleukin 12 (IL-12), interferon gamma (IFN-γ) and granulocyte colony stimulating factor
(G-CSF) [36, 51-61]. Up-regulation of CD64 in the presence of such cytokines or in
response to infection is but one of many changes elucidated in the last few decades as
part of neutrophil activation. However, PMN CD64 expression differs from the parallel
changes of increased CD45RA  and CD11b/18 [63-68] expression, as well as loss of
expression of CD16 [66, 69-72], CD62L [64, 73] and CD66b , in that the normal
baseline PMN CD64 expression is negligible. Additionally CD64 expression is stable at
room temperature for over 30 hours, in contrast to the labile expression of CD11b and
other PMN antigens [38, 74, 75]. Thus CD64 appears uniquely ideal as a surrogate
marker of neutrophil activation or a systemic acute inflammatory response as its
expression goes from our normal reference range of less than 2,000 sites per cell and
becomes up-regulated in a graded fashion depending upon the intensity of cytokines
stimulation [36, 55, 57, 61]. Furthermore the up-regulation of PMN CD64 expression
appears to be of pathophysiologic significance, as this form of Fc receptor has been
shown to functional in eliciting all the PMN functional responses utilized in anti-bacterial
responses [36, 56, 57].
In order to better assess the potential diagnostic utility of PMN CD64, we evaluated
specimens coming from patients being assessed in the emergency department of a
large tertiary care hospital. The study was also designed to examine the correlation of
PMN CD64 expression, as measured by quantitative flow cytometry, to those laboratory
tests used in the standard of care for the evaluation of the presence of infection or
sepsis. Thus, PMN CD64 was compared to PMN absolute counts, differential cell
counts for the presence of a myeloid left shift in the blood, C-reactive protein (CRP),
erythrocyte sedimentation rate (ESR), and microbiologic culture results. Determination
of the relationship between quantitative PMN CD64 measurements and current
laboratory hematology testing methodologies is a necessary prerequisite to more
focused clinical studies into the diagnostic utility of PMN CD64 as new diagnostic
approach for inflammation. We judged the efficacy of the various laboratory tests in the
diagnosis of infection/sepsis by using a clinical scoring system placing patients in groups
with varying levels of certainty for infection/sepsis. We report that PMN CD64
expression provides superior diagnostic information, relative to the standard laboratory
Methods and Materials
Sample selection. A total of 100 blood samples were randomly selected from the
routine clinical hematology laboratory at a large tertiary care hospital (William Beaumont
Hospital, Royal Oak, Michigan) using the criteria as being collected from patients being
evaluated in the hospital emergency department. Samples were selected for subsequent
flow cytometric measurement of leukocyte CD64 and other laboratory testing, which
performed within 4 hours of the patient draw time. The study protocol was reviewed and
approved by institutional human subjects review board.
Clinical Infection/Sepsis Score. A retrospective chart review on the patients was
performed at the conclusion of the study with the reviewer (EA) blinded to the neutrophil
CD64 results during assessment of the hospital course. Patients were categorized into
4 groups based upon the medical history chart review and the degree or likelihood of a
systemic acute inflammatory 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 systemic
inflammatory response; group 2, clinical or laboratory evidence of infection or
inflammatory process of a extensive localized nature with an undocumented probability
of eliciting a systemic inflammatory response; group 3, unequivocal clinical or laboratory
evidence of a systemic sepsis, infection or inflammatory process based upon
identification of organisms by culture or smear visualization. Group 0 had no laboratory
or clinical signs or symptoms indicative of an infectious process, nor evidence of an
inflammatory medicated disease process. Group 1 also had no evidence of a systemic
process with blood cultures being negative and if infection or tissue injury was found, it
was localized such as a laceration, cystitis, otitis, myocardial infarct, or fractured ankle.
The group 1 was quite heterogeneous, but the clinical and laboratory findings did not
indicate any reason to conclude the presence of a systemic inflammatory process.
Group 2 had clinical findings to indicate a systemic response to infection or tissue injury,
but unlike group 3 had no laboratory evidence of infection. Group 2 included patients
such as those with suspected ruptured diverticulitis or radiological evidence of
pneumonia, cultures not reported as positive, yet a clinical decision to treat for infection
was made. Group 3 have both clinical evidence of a systemic process of infection or
sepsis and laboratory documentation of an infectious etiology; this being the most
definitive group for the presence of infection, sepsis, or severe tissue injury.
Quantitation of CD64. Leukocyte CD64 expression was measured as previously
described [34, 76]. Briefly, 50 µl of whole blood or phosphate buffered saline (PBS)
diluted whole blood to adjust the leukocyte concentration below 2.5 x 109 cells/L was
incubated for 10 minutes at room temperature with saturating amounts of FITC
conjugated anti-CD64 murine monoclonal antibody (clone 22, Medarex, Inc., Annandale,
NJ, now available from Trillium Diagnostics, LLC, Scarborough, ME) or isotype control
murine antibody followed by red blood cell lysis with an ammonium chloride based red
cell lysis solution (Trillium Lyse, Trillium Diagnostics, LLC, Scarborough, ME). Samples
were then washed once and resuspended with PBS with 0.1% bovine serum albumin
(Sigma Chemicals, St. Louis, MO) pH 7.4 to a volume of 0.5 mL. Flow cytometric
analysis was performed to collect log green fluorescence, forward and log right angle
light scatter signals on a minimum of 20,000 leukocytes using a Cytoron Absolute flow
cytometer (Ortho Diagnostics, Raritan, NJ). Interassay standardization and CD64
quantitation was performed using Quantum 24 FITC calibration beads (Flow Cytometry
Standards Corp., San Juan, PR, now available through Bangs Laboratory, Indianapolis,
IN), which were analyzed at the start and finish of each flow cytometric batch run. Data
analysis was performed using light scatter gating to define the PMN population and
CD64 intensity quantified as FITC mean equivalent soluble fluorescence (MESF) units
using QuickCalTM for Winlist (Verity Software House, Topsham, ME). The PMN CD64
expression in MESF units was corrected for any non-specific antibody binding by the
subtraction of values obtained with isotype control antibody stained and flow
cytometrically analyzed in parallel.
Laboratory Tests. Complete blood counts and leukocyte 5 part differential results were
obtained using a Coulter STKS hematology blood counter using software version 1H
(Beckman Coulter, Hialeah, FL). Instrument reagents and calibration were as
recommended by the manufacturer. Microscopic leukocyte differential counts were
performed by a single observer (NCB) on all specimens derived from two Wright’s
stained blood smears based upon a 400 cell counts as detailed by NCCLS document
H20A (NCCLS, Wayne, PA). Separation of mature neutrophil from band forms was
according to the guidelines recommended by the College of American Pathologists .
The immature myeloid fraction was calculated as the number of myeloid cells at the
metamyelocyte stage or younger (myelocyte or promyelocyte) divided by the total
number of myeloid cells (neutrophils, bands, and immature forms). C-reactive protein
was determined using an immunoturbidimetric assay for the in vitro quantitative
determination of CRP in human serum and plasma on automated clinical chemistry
analyzers (Roche Integra, Roche Diagnostics, Indianapolis, IN). Erythrocyte
sedimentation rates were determined using the Westergren method.
Statistical Analysis. Linear regression analysis was performed using EXCEL ’97
software (Microsoft, Redmond, WA). Box plots and paired and unpaired two-tail t-tests
were performed using Statview 5.0 (SAS Institute, Inc., Cary, NC). Statistically
significant differences were defined a p < 0.05.
As in previous studies clinical samples were found to have variable expression of PMN
CD64 (range 261 – 41,585 MESF units) ranging from the normally low levels of <1,500
MESF units to levels above that seen in monocytes from healthy individuals. This
variability in CD64 expression demonstrates the in vivo ability of the PMN to modulate
expression of this Fc receptor. Also as observed with previous studies, the PMN
population in demonstrated a near Gaussian unimodal distribution of CD64 expression
with flow cytometric analysis, rather than the appearance of subpopulations of PMNs
with different surface expression of the antigen. Whether the PMN CD64 expression
was negligible as in the healthy state or significantly upregulated as in individuals with
sepsis or a systemic acute inflammatory response, the circulating population of PMNs in
the blood was a single cell cluster with regard to PMN CD64 express (Figure 1). Due to
this distribution as a shift of the entire population, it would be inappropriate to quantitate
the increase in CD64 expression in simplistic terms of percent positive. The only
circumstances where myeloid cells in the blood demonstrated a skewed unimodal or
approaching a bimodal appearance were observed was associated with samples having
a relative eosinophilia or an increase in immature myeloid forms (metamyelocyte stage
or younger). Eosinophils can express lower levels of CD64 or higher fluorescence due
to autofluorescence, which may differ than the PMN expression of CD64. Immature
myeloid cell normally express higher levels of CD64 compared to PMNs, where CD64 is
down regulated or lost with normal maturation. Hence, if immature myeloid forms are
present in the blood there maybe a skewness or tail to the myeloid population distribution
of CD64 expression. However, the majority of specimens show a symmetric cluster of
expression of the three major leukocyte populations as shown in figure 3.
The relationship among PMN CD64 expression, absolute neutrophil counts, manual band
counts, ESR rate, and C-reactive protein amount the blood samples in this study was
quite variable (figures 2, 3). As shown, there is variable relationship among all the
laboratory parameters, but PMN count and PMN CD64 expression showed no
significant correlation. A better relationship between increasing ESR rate or higher C-
reactive protein levels and increased PMN CD64 expression was noted, although the
correlation was not a strong one. Although the presence of circulating immature myeloid
cells (metamyelocyte and younger) were evaluated, only six of the 100 patients had
evidence of this degree of a myeloid left shift and as such, could not be evaluated for
any relative diagnostic significance or correlation with the other laboratory parameters.
Correlation of Laboratory Parameters to Clinical Observation. Chart review of all 100
patients was performed and clinical histories with regard to the episode approximate to
the blood sampling at the time of laboratory studies were scored in a blinded fashion
with regard to the clinical status into one of 4 groups defined as follows: (0) No clinical
indication of the acute inflammation, (1) Weak clinical suspicion of a significant infection
or acute inflammatory process, (2) Moderate clinical evidence of infection and/or acute
inflammation that resulted in some treatment plan towards this process, and (3) definite
clinical indication of infection and/or severe tissue trauma, shock, or acute inflammatory
process. Each of the laboratory tests was evaluated for the ability to distinguish these
four clinical groups (figure 4). The PMN CD64 expression was found to be the most
effective for significant separation of the four groups, achieving statistically significant
separation among all four groups. The groups 2 and 3 with the most likelihood of having
systemic infection or sepsis had all but two of the PMN CD64 values elevated above the
established normal range of < 1,500 MESF values.
All of the laboratory test employed in this study, when segregated into normal or
abnormal values, showed varying diagnostic power in separating the clinical sepsis
score groups. However, the neutrophil CD64 measurement showed the best specificity
and sensitivity with regard to separating the clinical sepsis score groups. The relative
diagnostic performance of the laboratory tests, as determined by sensitivity, specificity,
efficiency, positive predictive value, negative predictive value, positive and negative
likelihood ratio was evaluated in two manners. In order to determine the relative
diagnostic utility of each parameter in the most ideal conditions, the performance was
assessed in those patients with clinical infection/sepsis scores of 0, 2, and 3 (table 1).
This was done to allow elimination of the variable clinical group with a score of 1 which
are neither disease free, nor having a documented systemic inflammatory process. The
second evaluation of diagnostic performance was done with all samples, including those
with a clinical sepsis score of 1. Even with the heterogeneous group with the score 1
included in the evaluation (table 2), the neutrophil CD64 expression still demonstrated
clear superiority with a sensitivity of 87.9%, specificity of 71.2%, efficiency of 76.8%,
and a positive likelihood ratio of 3.05. Only the CRP paralleled the sensitivity of
neutrophil CD64 expression with 88.2%, but all but the band count had much lower
specificity, efficiency, and likelihood ratios.
Neutrophil CD64 expression is regulated in a graded fashion that would seemingly
parallel the degree of inflammatory response to a significant clinical process of infection
or tissue injury. Previous studies have indicated that PMN CD64 is highly correlated with
the presence of infection [34, 35, 40-47, 49, 50]. Its upregulation is controlled in a
myeloid specific fashion influenced by the cytokines involved in the acute inflammatory
response, such as IL-12, IFN-γ, and G-CSF, which occurs in a matter of hours [36, 51-
61, 78]. Thus between the rapid upregulation of CD64 expression on the neutrophil and
the normal 6 hour half life of the blood neutrophil, determination of the PMN CD64
expression should provide an indication of the current status of a patient’s systemic
acute inflammatory response to an infection. The results of this study indicate that PMN
CD64 expression is an improved diagnostic indicator of infection/sepsis in an
ambulatory patient population encountered in a large tertiary case emergency
department when compared to C-reactive protein, ESR, band counts, and absolute
neutrophil counts. Additionally the imperfect and variable correlations of PMN CD64 to
the laboratory studies currently used for infection detection indicate that CD64
expression is not simply a surrogate for any of these existing laboratory tests for
infection or sepsis currently available in most hospital laboratories.
This study suggests that all laboratory parameters may be correlated with the presence
of acute inflammation or infection, but that they are not reporting the same information.
The clinical chart review indicates neutrophil counts and immaturity indicators, such as
band counts, correlate, albeit imperfectly and with variable sensitivity and specificity, with
the presence of clinical evidence of infection and/or acute inflammation. The CD64
measurements in this study show a better correlation with and separation by clinical
scoring for the presence of an infectious or inflammatory process, suggesting a better
sensitivity and specificity with regard to detection of clinically meaningful acute
inflammation. Our findings on band counts and neutrophil counts do not differ
significantly from that previously reported [27-29, 31-33]. The weak correlation between
PMN CD64 and band counts and absolute neutrophil counts might be simply the
difference in specificity with regard to detection of an acute inflammatory response or
reflect the differing kinetic of response of these measurement during the dynamic
inflammatory response to infection or tissue injury. Our interpretation is that the study
results indicate PMN CD64 to be a more promising and meaningful diagnostic
parameter of the acute inflammatory response found in infection and sepsis.
The lack of a single highly specific and sensitive laboratory indicator of acute
inflammation has resulted in the clinical utilization of several concurrent laboratory tests
in routine practice to rule in or rule out the presence of infection or significant acute
inflammatory response. Culture results are often viewed as confirmatory but in practice
are often not utilized in treatment decisions due to their relatively slow turn around times
of up to 72 hours or more. Some investigators have proposed varying algorithms to
improve the predictive value of the relative insensitive tests of sedimentation rate, C
reactive protein, and presence of immature leukocyte populations, but even with such
approaches, there remains a need for improved diagnostics of infection . The
relative specificity of these tests is not unexpected given the number of additional
factors that can result in changes in these laboratory parameters, independent of the
presence of infection or acute inflammation, such as concurrent drug therapy, such as
steroids and adrenergic agents or the presence of myeloproliferative disorders. Even
PMN CD64 is not universally applicable, as if a patient is receiving therapeutic
interferon-gamma or G-CSF, circulating neutrophils would have a marked elevation in
CD64 without the presence of infection. Aside from these relative uncommon
treatments, myeloid cell CD64 up-regulation would appear to be a more specific and
perhaps sensitive avenue of laboratory quantitation of infection.
Thee are other antigenic changes that occur with neutrophil activation, such as
modulations in CD11b, CD18, CD16, CD62L and CD45RA, but these changes have
several relative disadvantages compared to PMN CD64 expression as a potential
diagnostic laboratory test. These other myeloid antigenic expressions show significant
expression in normal healthy individuals, often with a moderate degree of heterogeneity
in expression which may affect the diagnostic sensitivity. Additionally some of the
antigens, such as CD11b, show lability in expression or changes dependant upon
specimen handling independent of any disease state [64, 75]. The PMN expression of
CD11b can be modulated through specimen storage, temperature fluctuations, and
centrifugation. Our studies to date have indicated that PMN CD64 expression is stable
for at least 36 hours or longer in anticoagulated blood samples stored at room
temperature, thus being quite amenable to clinical laboratory testing . However,
additional prospective studies are required to more fully comprehend the utility of PMN
CD64 expression as a diagnostic indicator of infection or sepsis.
The results of this and other studies indicate a potential for PMN CD64 as a more
sensitive and specific indicator of infection or sepsis, which would facilitate therapeutic
decisions. The current study employed a method of quantitative cytometry using
reference beads standards calibrated in FITC MESF units, which now can be traceable
to a new National Institute of Standards and Technology (NIST) reference materials for
fluorescein, so called RM 8640 and SRM 1932 [79, 80]. This approach is quite suitable
for a clinical research study, but is likely too costly and complex to be performed in a
routine clinical laboratory test. Towards this end our subsequent studies are being
performed using a modification of the assay utilized in this reported study. The
subsequent generation PMN CD64 assay now being employed, called the Leuko64
assay, uses a single microbeads standard in a whole blood lyse no wash assay that
derives a PMN CD64 index utilizing dedicated software. This Leuko64 assay format is
much simpler and amenable to a clinical laboratory setting and can be completed in less
than 30 minutes, which would meet the rapid turnaround demands of an emergency
department setting. This and other previous studies to date indicate that PMN CD64 is
worthy of more thorough clinical evaluation as an improved diagnostic indicator of
infection and sepsis.
Dr Davis is owner of Trillium Diagnostics, LLC, which produces Leuko64, currently a
research use only assay kit for the measurement of leukocyte CD64 expression. This
work was presented in part at the Clinical Cytometry Society meeting in October, 2004 in
Long Beach, CA and the 13th annual Seminar on Molecular Pathology, Wm Beaumont
Hospital, Troy, MI, October 7, 2004.
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Neutrophil Download full-text
(> 9,000 x
87.88% 60.00% 68.18% 50.00% 88.24%
77.27% 57.14% 77.27% 66.67% 57.89%
74.36% 50.00% 75.00% 50.00% 65.22%
89.47% 66.67% 70.83% 66.67% 84.62%
81.82% 58.33% 72.73% 60.00% 72.22%
3.87 1.40 3.00 1.50 2.10
0.16 0.70 0.41 0.75 0.20
Table 1. Performance statistics of diagnostic parameters in recognizing infection/sepsis
using disease presence defined by infection/sepsis scores 2 and 3 and disease absence
defined by infection/sepsis score 0. Positive predictive value (PPV), negative
predictive value (NPV), positive likelihood ratio (LR+), and the negative likelihood ratio
(LR-) show neutrophil CD64 to have the best diagnostic performance.
Table 2. Performance statistics of diagnostic parameters in recognizing infection/sepsis
using disease presence defined by infection/sepsis scores 2 and 3 and disease absence
defined by infection/sepsis scores 0 and 1. Positive predictive value (PPV), negative
predictive value (NPV), positive likelihood ratio (LR+), and the negative likelihood ratio
(LR-) show neutrophil CD64 to have the best diagnostic performance.
(> 9,000 x
68.18% 50.00% 88.24%
76.32% 65.52% 59.38%
62.50% 37.50% 53.57%
80.56% 76.00% 90.48%
73.33% 60.98% 69.39%
2.88 1.45 2.17
0.42 0.76 0.20