Journal of Immunological Methods xxx (2014) xxx–xxx

JIM-11904; No of Pages 4

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Journal of Immunological Methods

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Letter to the editor

Neutrophil CD64 expression as an important diagnostic markerof infection and sepsis in hospital patients

Mariela Granero Farias a,⁎, Natália Pieruccini de Lucena b, SuzaneDal Bó a, SimoneMartins de Castro c

a Unit of Hematology, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazilb Specialization in Clinical Analysis, Federal University of Rio Grande do Sul, Porto Alegre, Brazilc Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, Brazil

a r t i c l e i n f o

⁎ Corresponding author at: Unit of Hematology, HospiAlegre, Ramiro Barcelos, 2350, 90035-903 Porto Alegre/3359.7647.

E-mail address: mg.farias@yahoo.com.br (M.G. Faria

Please cite this article as: Farias, M.G., et al.,in hospital patients, J. Immunol. Methods (

http://dx.doi.org/10.1016/j.jim.2014.07.0110022-1759/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

Article history:Received 2 February 2014Received in revised form 8 May 2014Accepted 28 July 2014Available online xxxx

Introduction: Infection and sepsis are major health problems. Therefore, the need for improveddiagnostic indicators, as well as for better therapeutic monitors in the treatment of infection,remains, since the current diagnostic tools have low specificity and passed through minimalchanges in the last two decades.Objective: The aim of this studywas to establish the correlation of neutrophil CD64with indicatorsof infection and sepsis.Methods: We established the correlation of the neutrophil CD64 expression with the followingvariables: complete white blood count, band count, neutrophils, C-reactive protein (CRP),cultures, flags released by automated hematology analyzers and clinical groups. Accordinglyclinical groups were divided into two: patients “without clinical or laboratory evidence ofinfection or inflammatory process” and “clinical or laboratory evidence of a systemicinflammatory response (SIRS) and systemic sepsis” based upon identification of organisms byculture.We analyzed93whole blood samples anticoagulatedwith K3EDTA of patients admitted inthe Intensive Care Unit (ICU) of a community hospital.Results: The expression CD64 was statistically significant with clinical groups, flags, andneutrophils andwas not significantly correlatedwith total count of white blood cells and cultures.Conclusion: Our results indicate that high expression of CD64 is an indicator important in thediagnosis of infection and sepsis.

© 2014 Elsevier B.V. All rights reserved.

Keywords:CD64InfectionFlow cytometryDiagnosis

Despite being one of the most frequent causes of mortalityand morbidity in the world, infection and sepsis indicators fordiagnoses have not been improved in recent decades. Whiteblood cell (WBC) count, neutrophils, presence of immaturemyeloid forms in the peripheral blood, erythrocyte sedimen-tation rate and C-reactive protein (CRP) levels (Davis et al.,2006; Calandra and Cohen, 2005), which are availableprocedures since the 1970s and earlier, are still being used for

tal de Clinicas de PortoRS, Brazil. Tel.: +55 51

s).

Neutrophil CD64 expres2014), http://dx.doi.org/

laboratory diagnoses. More recently, the new generation ofimproved diagnostics tests for infection is based on solublebiomarkers in the serum or plasma, such as TNFa, IL-6, IL-1a,IL-10, and procalcitonin (Cid et al., 2011).

Current tests have shown that the WBC count and thedifferential analysis are nonspecific markers (Davis et al.,2006). Observers' discrepancies and non-inflammatory causes,whichmay increase the band count, are jeopardizing the usageof these markers. Moreover, CRP level, recognized as objectivediagnostic assay, is an acute-phase protein, and its concen-tration increases as a response to non-specific inflammatoryprocess (Cid et al., 2010). Since the gold standard forbacteremia diagnosis is fraught with difficulties, bacterialinfection diagnoses are sometimes challenging. It is known

sion as an important diagnostic marker of infection and sepsis10.1016/j.jim.2014.07.011

Fig. 1. Dot-plot CD163 × CD64. Population yellow: neutrophil (CD163−/CD64+); population blue: monocyte (CD163+/CD64+); population green:beads. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

Table 1Correlation between CD64 and quantitative and qualitative variables.

Variable Group exposed P rs

N = 93

Age (years) (15–95)Gender

F 44 (47.3%)M 49 (52.7%)

Clinical groupsNo infection 37 (1.19; 0.83–1.80) 0.034SIRS + sepsis 56 (1.51; 1.11–2.30)

Flag groups b0.001No flags 53 (1.11; 0.82–1.64)LS and/or IG 40 (1.82; 1.33–2.99)

Microbiological culture 0.529Negative 35 (1.45; 1.07–2.00)Positive 28 (1.54; 1.14–2.70)

White blood cells (/μL) 0.146 0.161Group 1 9790 (8220–11,720)Group 2 15,165 (9848–21,598)

Neutrophils (/μL) 0.047 0.652Group 1 7707 (5191–9135)Group 2 11,949 (7219–15,537)

Band count (%) 0.002 0.322Group 1 (0–1.6)Group 2 5.6 (0–11)

CRP (mg/L) b0.001 0.743Group 1 84.1 (15.2–231)Group 2 126.5 (15.8–346)

CD64 (%) 1.000Group 1 1.19 (0.82–1.80)Group 2 1.51 (1.11–2.30)

Data from gender are represented as number (percentage); clinical groups,flags groups and culture are represented as number (median; P25–P75);Whiteblood cells, neutrophils, CRP, CD64 and band count as median (minimumand maximum); LS: Left Shift; IG: Immature Granulocytes; CRP: C-ReativeProtein; Group 1: no evidence of infection or inflammatory process; Group 2:evidence of a systemic inflammatory response (SIRS) and sepsis. P b 0.05 wasstatistically significant.

2 M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx

that the incubation of bacteria may take 2–4 days; genuinebacteremia may remain undetected in significant amount ofinfected cases because of the small volume of blood collected(Simon et al., 2004).

Thus, improved indicators of infection and/or sepsis areneeded in order to increase the specificity of both diagnosesand therapeutic monitoring. Recent findings in immunologyhave indicated new potential markers for sepsis diagnosisas cell surface antigens, cytokines and acute phase reactants(Davis et al., 2006).

Surface receptors of neutrophils recognize bacterial anti-gens and this interaction activates the neutrophils to phago-cyte. Phagocytosis is eased by receptors for immunoglobulin-G(IgG) in neutrophils (Cid et al., 2011). CD64, a leukocyte surfaceantigen, is a high affinity Fc receptor (FcγRI), which binds tomonomeric IgG. The Fc receptors are involved with the innateand adaptive immune response, stimulating either phagocyto-sis or antibody-mediated cytotoxicity (Qureshi et al., 2001).

Several studies have pointed neutrophil CD64 expressionas a good candidate for a more specific laboratory indicatorfor detection of sepsis/infection (Calandra and Cohen, 2005;Fjaertoft et al., 2007). The high expression of CD64 in immaturemyeloid cells tends to decrease during their maturation untilthe stage of segmented neutrophil (Davis et al., 2006). So,healthy patients have non-remarkable expression of CD64 onthe surface of neutrophils; however, it is stored in intracellularfluid and can be activated by exogenous or endogenous stimuli.Their expression in neutrophils can be induced by certaininflammatory cytokines and the presence of bacterial productsin the cell wall (Fjaertoft et al., 2007).

The preset study aims at establishing the association of theneutrophil CD64 expression of adult patients of our hospitalemergency department, with the following variables: WBCcount, band count, neutrophils, CRP, microbiological culturesand flags released by automated hematology analyzers,laboratory indicators of infection/sepsis and clinical groups.

Patients admitted in the Intensive Care Unit (ICU) ofHospital de Clínicas de Porto Alegre were prospectivelyevaluated and blood samples were randomly selected fromroutine hematology laboratory. Patients receiving interferongammaor G-CSFwere excluded from the sample to avoid false-positive results. Patients were organized into two clinicalgroups based on medical history and degree or likelihood ofsystemic acute inflammatory response according the criterionsof the “Society of Critical CareMedicine” and “American Collegeof Chest Physicians”: group 1: no clinical or laboratory evidenceof infection or inflammatory process and group 2: clinical orlaboratory evidence of a systemic inflammatory response(SIRS-inflammatory reaction in which two or more of thefollowing conditionsmust be changed: temperature, heart rate,respiratory rate, WBC count or the presence of bands) andunequivocal clinical or laboratory evidence of a systemic sepsis,infection or inflammatory process based on identification oforganisms by culture (Bone et al., 1992).

Venous blood samples were collected with K3EDTA andanalyzed in the Sysmex XE 2100 (Sysmex Corporation, Kobe,Japan). The equipment performance was monitored usingcommercial quality controls. Flags were defined as Left Shift(LS) and Immature Granulocytes (IG). Observation of bloodsmear stainedwithWright–Giemsa,was performed in all cases.The CRP was determined quantitatively by BNII Behring

Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsisin hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011

3M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx

nephelometry. Patients' blood was cultured by BacT/ALERT 3DFood® (Biomérieux, St. Laurent, Quebec, Canada) and two ofthese cultures (aerobic bottles), from two different sites, wereobserved for five days of incubation. VITEK 2was used or classicmethods for identification and sensitivity testswere performedaccording to the Clinical and Laboratory Standards Institute(SCLI).

Neutrophil CD64 expressionwas set using the kit Leuko64™(Trillium Diagnostics, Brewer, Me, USA), in a FACSCalibur®flow cytometer (BD Biosciences, San Jose, California, USA),according to the manufacturer's instructions. The referred kitis composed by two monoclonal antibodies; CD64FITC andCD163PE (clonemac2-158). CD163 is a scavenger receptor richin cysteine and it is expressed only in cells of monocyte–macrophage lineage. CD163 was included in the kit todifferentiate neutrophils from monocytes (Fig. 1). The calcula-tion index of the expression CD64 value was performed by theanalysis QuantiCALC software (Trillium Diagnostics, Brewer,Me, USA). The assay internal control was provided by theautomatedmeasurement of the lymphocyte CD64 index (b1.0)as negative control and monocyte CD64 index (N3.0) as apositive control. The CD64 index is then calculated using the

Fig. 2.Difference between neutrophil CD64 expression and (A) clinical groups: group 1systemic inflammatory response (SIRS) and sepsis. P= 0.034; (B)microbiological cultuShift and/or Immature Granulocytes). P b 0.001.

Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expresin hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/

ratio of themean fluorescent intensity of the cell populations tothe FITC signal from the beads.

Statistical analysis was performed using the Spearmancorrelation (rs) for quantitative variables relating one toanother and Student t test, Kruskal–Wallis andMann–Whitneyto compare quantitative to categorical variables.

We included 93 patients; 49 (52.7%) males and 44 (47.3%)females in an age range of 15 to 95 years old (average62 years). Values of WBC count, neutrophils, band count andCRP, and the association between CD64 with quantitative andqualitative variables, are described in Table 1 and the differencebetween CD64 expression and the clinical groups, microbio-logical cultures and presence of flags is shown in Fig. 2.

As highlighted, there is a variable relationship amongneutrophil CD64 expression and all the laboratory parameters.The association between CD64 expression (index N 1) andWBCcount and microbiological cultures showed not to be signifi-cant, already the association with CRP (N5 mg/L); neutrophilia(N9.0 × 106/L); band count (N10%) and flags (Left Shift andImmature Granulocytes) emitted by the equipment, wasstatistically significant. The correlation was weak with bandcount and strong with CRP levels and neutrophilia.

— no evidence of infection or inflammatory process and group 2— evidence of are negative and positive. P= 0.529; and (C) groupswith andwithout flags (Left

sion as an important diagnostic marker of infection and sepsis10.1016/j.jim.2014.07.011

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Previous studies have indicated that neutrophil CD64expression is highly correlated to the presence of infection orinflammatory process (Cid et al., 2010, 2011; Groselj-Grencet al., 2008). A significant increase in neutrophil CD64expression is a typical finding in patients with bacterialinfections. However, it is still controversial to know if thissignificant increase is caused by the density of the moleculesurface increase or if there are more neutrophils expressingCD64, or even it is a combination of both events.

The WBC count, normally high in infectious processes,which may be reduced in bone marrow exhaustion, mayexplain the lack of significance of our study.

It was seen that neutrophil CD64 expression was lower inthe patients of clinical group 1 compared to that in clinicalgroup 2 patients. The neutrophil CD64 expression wasstatistically significant (P = 0.034), agreeing with Davis et al.who showed a high correlation between clinical groups andpresence of infectious and inflammatory processes, Groselj-Grenc et al. had already shown higher correlation forneutrophils in patients with SIRS and sepsis compared topatients with noninfectious SIRS (Groselj-Grenc et al., 2008).

The determination of neutrophil CD64 expression needsstandardization, because most of the clinical studies have usedmanually determined fluorescence intensity (MFI) (Fjaertoft etal., 2007). This method presents variations because of instru-ments and the comparison between different laboratoriescannot be reliable. By using available software for data analysisand index calculation, a novel approach for reducing lot-to-lotvariations (Groselj-Grenc et al., 2008) may be possible and themeasurement of neutrophil CD64 expression could become anordinary clinical test with a high compatibility and reproduc-ibility in different laboratories.

Neutrophil CD64 expression levels can be used as abarometer of systemic inflammatory responses, having apotential role in therapeutic monitoring of antibiotic use.Some authors have even suggested that, because of its highsensitivity, measurement of neutrophil CD64 expression mayallow clinicians to discontinue antimicrobial treatment if thereis a negative CD64 expression within 24 h of suspectedinfection, without having to wait for the definitive microbio-logical results (Ng P & Lam, 2006).

This and other previous studies indicate that high expres-sion of CD64 is an important biomarker for infection or sepsisdiagnoses, even better than traditional hematologic parameters

Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expresin hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/

and CRP. It was shown that CD64 assay is relatively simple,more specific and less subjective than other laboratory testsand can easily be implemented in the laboratory practice of alarge university hospital. Obviously, more clinical experiencesare needed to determine the complete use of neutrophil CD64expressionmeasurements, as well as studies on the associationwith other biomolecular markers have to be carried out.

The neutrophil CD64 expression would meet the fastturnaround demands of hospital emergency departmentallowing the fast diagnoses of infections and sepsis makingtherapeutic decision more effective.

References

Bone, R.C., Balk, R.A., Cerra, R.B., Dellinger, R.P., Fein, A.M., Knaus, W.A., et al.,1992. Definition for sepsis and organ failure and guidelines for the use ofinnovative therapies in sepsis. The ACCP/SCCM Consensus ConferenceCommittee. American College of Chest Physicians/Society of Critical CareMedicine. Chest 101, 1644.

Calandra, T., Cohen, J., 2005. The international sepsis forum consensusconference on definitions of infection in the intensive care unit. Crit. CareMed. 33, 1538.

Cid, J., Aguinaco, R., Sánchez, R., García-Pardo, G., Llorente, A., et al., 2010.Neutrophil CD64 expression as marker of bacterial infection: a systematicreview and meta-analysis. J. Infect. 60, 313.

Cid, J., García-Pardo, G., Reyes, R., Sánchez, R., Llorente, A., et al., 2011.Neutrophil CD64: diagnostic accuracy and prognostic value in patientspresenting to the emergency department. Eur. J. Clin. Microbiol. Infect. Dis.30, 845.

Davis, B.H., Olsen, S.H., Ahmad, E., Bigelow, N.C., et al., 2006. Neutrophil CD64 isan improved indicator of infection or sepsis in emergency departmentpatients. Arch. Pathol. Lab. Med. 130, 654.

Fjaertoft, G., Hakansson, L.D., Pauksens, K., Sisask, G., Venge, P., et al., 2007.Neutrophil CD64 (FcγRI) expression is a specific marker of bacterialinfection: a study on the kinetics and the impact of major surgery. Scand. J.Infect. Dis. 39, 525.

Groselj-Grenc, M., Ihan, A., Derganc, M., 2008. Neutrophil and monocyte CD64and CD163 expression in critically ill neonates and children with sepsis:comparison of fluorescence intensities and calculated indexes. Mediat.Inflamm. 2008, 202646.

Ng, P.C., Lam, H.S., 2006. Diagnostic markers for neonatal sepsis. Curr. Opin.Pediatr. 18, 125.

Qureshi, S.S., Lewis, S.M., Gant, V.A., Treacher, D., Davis, B.H., Brown, K.A., 2001.Increased distribution and expression of CD64 on blood polymorphonu-clear cells from patients with the systemic inflammatory responsesyndrome (SIRS). Clin. Exp. Immunol. 125, 258.

Simon, L., Gauvin, F., Amre, D.K., Saint-Louis, P., Lacroix, J., 2004. Serumprocalcitonin and C-reactive protein levels as markers of bacterial: asystematic review and meta-analysis. Clin. Infect. Dis. 39, 206.

sion as an important diagnostic marker of infection and sepsis10.1016/j.jim.2014.07.011