JIM-11904; No of Pages 4 Journal of Immunological Methods xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Letter to the editor
Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients Mariela Granero Farias a,⁎, Natália Pieruccini de Lucena b, Suzane Dal Bó a, Simone Martins de Castro c a b c
Unit of Hematology, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil Specialization in Clinical Analysis, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 2 February 2014 Received in revised form 8 May 2014 Accepted 28 July 2014 Available online xxxx Keywords: CD64 Infection Flow cytometry Diagnosis
a b s t r a c t Introduction: Infection and sepsis are major health problems. Therefore, the need for improved diagnostic 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 minimal changes in the last two decades. Objective: The aim of this study was to establish the correlation of neutrophil CD64 with indicators of infection and sepsis. Methods: We established the correlation of the neutrophil CD64 expression with the following variables: complete white blood count, band count, neutrophils, C-reactive protein (CRP), cultures, flags released by automated hematology analyzers and clinical groups. Accordingly clinical groups were divided into two: patients “without clinical or laboratory evidence of infection or inflammatory process” and “clinical or laboratory evidence of a systemic inflammatory response (SIRS) and systemic sepsis” based upon identification of organisms by culture. We analyzed 93 whole blood samples anticoagulated with K3EDTA of patients admitted in the Intensive Care Unit (ICU) of a community hospital. Results: The expression CD64 was statistically significant with clinical groups, flags, and neutrophils and was not significantly correlated with total count of white blood cells and cultures. Conclusion: Our results indicate that high expression of CD64 is an indicator important in the diagnosis of infection and sepsis. © 2014 Elsevier B.V. All rights reserved.
Despite being one of the most frequent causes of mortality and morbidity in the world, infection and sepsis indicators for diagnoses have not been improved in recent decades. White blood cell (WBC) count, neutrophils, presence of immature myeloid forms in the peripheral blood, erythrocyte sedimentation rate and C-reactive protein (CRP) levels (Davis et al., 2006; Calandra and Cohen, 2005), which are available procedures since the 1970s and earlier, are still being used for
⁎ Corresponding author at: Unit of Hematology, Hospital de Clinicas de Porto Alegre, Ramiro Barcelos, 2350, 90035-903 Porto Alegre/RS, Brazil. Tel.: +55 51 3359.7647. E-mail address: [email protected] (M.G. Farias).
laboratory diagnoses. More recently, the new generation of improved diagnostics tests for infection is based on soluble biomarkers 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 the differential analysis are nonspecific markers (Davis et al., 2006). Observers' discrepancies and non-inflammatory causes, which may increase the band count, are jeopardizing the usage of these markers. Moreover, CRP level, recognized as objective diagnostic assay, is an acute-phase protein, and its concentration increases as a response to non-specific inflammatory process (Cid et al., 2010). Since the gold standard for bacteremia diagnosis is fraught with difficulties, bacterial infection diagnoses are sometimes challenging. It is known
http://dx.doi.org/10.1016/j.jim.2014.07.011 0022-1759/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
2
M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
that the incubation of bacteria may take 2–4 days; genuine bacteremia may remain undetected in significant amount of infected cases because of the small volume of blood collected (Simon et al., 2004). Thus, improved indicators of infection and/or sepsis are needed in order to increase the specificity of both diagnoses and therapeutic monitoring. Recent findings in immunology have indicated new potential markers for sepsis diagnosis as cell surface antigens, cytokines and acute phase reactants (Davis et al., 2006). Surface receptors of neutrophils recognize bacterial antigens and this interaction activates the neutrophils to phagocyte. Phagocytosis is eased by receptors for immunoglobulin-G (IgG) in neutrophils (Cid et al., 2011). CD64, a leukocyte surface antigen, is a high affinity Fc receptor (FcγRI), which binds to monomeric IgG. The Fc receptors are involved with the innate and adaptive immune response, stimulating either phagocytosis or antibody-mediated cytotoxicity (Qureshi et al., 2001). Several studies have pointed neutrophil CD64 expression as a good candidate for a more specific laboratory indicator for detection of sepsis/infection (Calandra and Cohen, 2005; Fjaertoft et al., 2007). The high expression of CD64 in immature myeloid cells tends to decrease during their maturation until the stage of segmented neutrophil (Davis et al., 2006). So, healthy patients have non-remarkable expression of CD64 on the surface of neutrophils; however, it is stored in intracellular fluid and can be activated by exogenous or endogenous stimuli. Their expression in neutrophils can be induced by certain inflammatory cytokines and the presence of bacterial products in the cell wall (Fjaertoft et al., 2007). The preset study aims at establishing the association of the neutrophil CD64 expression of adult patients of our hospital emergency department, with the following variables: WBC count, band count, neutrophils, CRP, microbiological cultures and flags released by automated hematology analyzers, laboratory indicators of infection/sepsis and clinical groups. Patients admitted in the Intensive Care Unit (ICU) of Hospital de Clínicas de Porto Alegre were prospectively evaluated and blood samples were randomly selected from routine hematology laboratory. Patients receiving interferon gamma or G-CSF were excluded from the sample to avoid falsepositive results. Patients were organized into two clinical groups based on medical history and degree or likelihood of systemic acute inflammatory response according the criterions of the “Society of Critical Care Medicine” and “American College of Chest Physicians”: group 1: no clinical or laboratory evidence of infection or inflammatory process and group 2: clinical or laboratory evidence of a systemic inflammatory response (SIRS-inflammatory reaction in which two or more of the following conditions must be changed: temperature, heart rate, respiratory rate, WBC count or the presence of bands) and unequivocal clinical or laboratory evidence of a systemic sepsis, infection or inflammatory process based on identification of organisms by culture (Bone et al., 1992). Venous blood samples were collected with K3EDTA and analyzed in the Sysmex XE 2100 (Sysmex Corporation, Kobe, Japan). The equipment performance was monitored using commercial quality controls. Flags were defined as Left Shift (LS) and Immature Granulocytes (IG). Observation of blood smear stained with Wright–Giemsa, was performed in all cases. The CRP was determined quantitatively by BNII Behring
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, the reader is referred to the web version of this article.)
Table 1 Correlation between CD64 and quantitative and qualitative variables. Variable
Group exposed
P
rs
N = 93 Age (years) Gender F M Clinical groups No infection SIRS + sepsis Flag groups No flags LS and/or IG Microbiological culture Negative Positive White blood cells (/μL) Group 1 Group 2 Neutrophils (/μL) Group 1 Group 2 Band count (%) Group 1 Group 2 CRP (mg/L) Group 1 Group 2 CD64 (%) Group 1 Group 2
(15–95) 44 (47.3%) 49 (52.7%) 37 (1.19; 0.83–1.80) 56 (1.51; 1.11–2.30)
0.034 b0.001
53 (1.11; 0.82–1.64) 40 (1.82; 1.33–2.99) 0.529 35 (1.45; 1.07–2.00) 28 (1.54; 1.14–2.70) 0.146
0.161
0.047
0.652
0.002
0.322
b0.001
0.743
9790 (8220–11,720) 15,165 (9848–21,598) 7707 (5191–9135) 11,949 (7219–15,537) (0–1.6) 5.6 (0–11) 84.1 (15.2–231) 126.5 (15.8–346) 1.000 1.19 (0.82–1.80) 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); White blood cells, neutrophils, CRP, CD64 and band count as median (minimum and maximum); LS: Left Shift; IG: Immature Granulocytes; CRP: C-Reative Protein; Group 1: no evidence of infection or inflammatory process; Group 2: evidence of a systemic inflammatory response (SIRS) and sepsis. P b 0.05 was statistically significant.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
nephelometry. Patients' blood was cultured by BacT/ALERT 3D Food® (Biomérieux, St. Laurent, Quebec, Canada) and two of these cultures (aerobic bottles), from two different sites, were observed for five days of incubation. VITEK 2 was used or classic methods for identification and sensitivity tests were performed according to the Clinical and Laboratory Standards Institute (SCLI). Neutrophil CD64 expression was 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 kit is composed by two monoclonal antibodies; CD64FITC and CD163PE (clone mac2-158). CD163 is a scavenger receptor rich in cysteine and it is expressed only in cells of monocyte– macrophage lineage. CD163 was included in the kit to differentiate neutrophils from monocytes (Fig. 1). The calculation index of the expression CD64 value was performed by the analysis QuantiCALC software (Trillium Diagnostics, Brewer, Me, USA). The assay internal control was provided by the automated measurement of the lymphocyte CD64 index (b 1.0) as negative control and monocyte CD64 index (N 3.0) as a positive control. The CD64 index is then calculated using the
3
ratio of the mean fluorescent intensity of the cell populations to the FITC signal from the beads. Statistical analysis was performed using the Spearman correlation (rs) for quantitative variables relating one to another and Student t test, Kruskal–Wallis and Mann–Whitney to 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 (average 62 years). Values of WBC count, neutrophils, band count and CRP, and the association between CD64 with quantitative and qualitative variables, are described in Table 1 and the difference between CD64 expression and the clinical groups, microbiological cultures and presence of flags is shown in Fig. 2. As highlighted, there is a variable relationship among neutrophil CD64 expression and all the laboratory parameters. The association between CD64 expression (index N 1) and WBC count and microbiological cultures showed not to be significant, already the association with CRP (N5 mg/L); neutrophilia (N9.0 × 106/L); band count (N10%) and flags (Left Shift and Immature Granulocytes) emitted by the equipment, was statistically significant. The correlation was weak with band count and strong with CRP levels and neutrophilia.
Fig. 2. Difference between neutrophil CD64 expression and (A) clinical groups: group 1 — no evidence of infection or inflammatory process and group 2 — evidence of a systemic inflammatory response (SIRS) and sepsis. P = 0.034; (B) microbiological culture negative and positive. P = 0.529; and (C) groups with and without flags (Left Shift and/or Immature Granulocytes). P b 0.001.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
4
M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
Previous studies have indicated that neutrophil CD64 expression is highly correlated to the presence of infection or inflammatory process (Cid et al., 2010, 2011; Groselj-Grenc et al., 2008). A significant increase in neutrophil CD64 expression is a typical finding in patients with bacterial infections. However, it is still controversial to know if this significant increase is caused by the density of the molecule surface increase or if there are more neutrophils expressing CD64, 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, may explain the lack of significance of our study. It was seen that neutrophil CD64 expression was lower in the patients of clinical group 1 compared to that in clinical group 2 patients. The neutrophil CD64 expression was statistically significant (P = 0.034), agreeing with Davis et al. who showed a high correlation between clinical groups and presence of infectious and inflammatory processes, GroseljGrenc et al. had already shown higher correlation for neutrophils in patients with SIRS and sepsis compared to patients with noninfectious SIRS (Groselj-Grenc et al., 2008). The determination of neutrophil CD64 expression needs standardization, because most of the clinical studies have used manually determined fluorescence intensity (MFI) (Fjaertoft et al., 2007). This method presents variations because of instruments and the comparison between different laboratories cannot be reliable. By using available software for data analysis and index calculation, a novel approach for reducing lot-to-lot variations (Groselj-Grenc et al., 2008) may be possible and the measurement of neutrophil CD64 expression could become an ordinary clinical test with a high compatibility and reproducibility in different laboratories. Neutrophil CD64 expression levels can be used as a barometer of systemic inflammatory responses, having a potential role in therapeutic monitoring of antibiotic use. Some authors have even suggested that, because of its high sensitivity, measurement of neutrophil CD64 expression may allow clinicians to discontinue antimicrobial treatment if there is a negative CD64 expression within 24 h of suspected infection, without having to wait for the definitive microbiological results (Ng P & Lam, 2006). This and other previous studies indicate that high expression of CD64 is an important biomarker for infection or sepsis diagnoses, even better than traditional hematologic parameters
and CRP. It was shown that CD64 assay is relatively simple, more specific and less subjective than other laboratory tests and can easily be implemented in the laboratory practice of a large university hospital. Obviously, more clinical experiences are needed to determine the complete use of neutrophil CD64 expression measurements, as well as studies on the association with other biomolecular markers have to be carried out. The neutrophil CD64 expression would meet the fast turnaround demands of hospital emergency department allowing the fast diagnoses of infections and sepsis making therapeutic 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 of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 101, 1644. Calandra, T., Cohen, J., 2005. The international sepsis forum consensus conference on definitions of infection in the intensive care unit. Crit. Care Med. 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 systematic review 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 patients presenting 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 is an improved indicator of infection or sepsis in emergency department patients. 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 bacterial infection: 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 CD64 and 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 polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin. Exp. Immunol. 125, 258. Simon, L., Gauvin, F., Amre, D.K., Saint-Louis, P., Lacroix, J., 2004. Serum procalcitonin and C-reactive protein levels as markers of bacterial: a systematic review and meta-analysis. Clin. Infect. Dis. 39, 206.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Letter to the editor
Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients Mariela Granero Farias a,⁎, Natália Pieruccini de Lucena b, Suzane Dal Bó a, Simone Martins de Castro c a b c
Unit of Hematology, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil Specialization in Clinical Analysis, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 2 February 2014 Received in revised form 8 May 2014 Accepted 28 July 2014 Available online xxxx Keywords: CD64 Infection Flow cytometry Diagnosis
a b s t r a c t Introduction: Infection and sepsis are major health problems. Therefore, the need for improved diagnostic 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 minimal changes in the last two decades. Objective: The aim of this study was to establish the correlation of neutrophil CD64 with indicators of infection and sepsis. Methods: We established the correlation of the neutrophil CD64 expression with the following variables: complete white blood count, band count, neutrophils, C-reactive protein (CRP), cultures, flags released by automated hematology analyzers and clinical groups. Accordingly clinical groups were divided into two: patients “without clinical or laboratory evidence of infection or inflammatory process” and “clinical or laboratory evidence of a systemic inflammatory response (SIRS) and systemic sepsis” based upon identification of organisms by culture. We analyzed 93 whole blood samples anticoagulated with K3EDTA of patients admitted in the Intensive Care Unit (ICU) of a community hospital. Results: The expression CD64 was statistically significant with clinical groups, flags, and neutrophils and was not significantly correlated with total count of white blood cells and cultures. Conclusion: Our results indicate that high expression of CD64 is an indicator important in the diagnosis of infection and sepsis. © 2014 Elsevier B.V. All rights reserved.
Despite being one of the most frequent causes of mortality and morbidity in the world, infection and sepsis indicators for diagnoses have not been improved in recent decades. White blood cell (WBC) count, neutrophils, presence of immature myeloid forms in the peripheral blood, erythrocyte sedimentation rate and C-reactive protein (CRP) levels (Davis et al., 2006; Calandra and Cohen, 2005), which are available procedures since the 1970s and earlier, are still being used for
⁎ Corresponding author at: Unit of Hematology, Hospital de Clinicas de Porto Alegre, Ramiro Barcelos, 2350, 90035-903 Porto Alegre/RS, Brazil. Tel.: +55 51 3359.7647. E-mail address: [email protected] (M.G. Farias).
laboratory diagnoses. More recently, the new generation of improved diagnostics tests for infection is based on soluble biomarkers 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 the differential analysis are nonspecific markers (Davis et al., 2006). Observers' discrepancies and non-inflammatory causes, which may increase the band count, are jeopardizing the usage of these markers. Moreover, CRP level, recognized as objective diagnostic assay, is an acute-phase protein, and its concentration increases as a response to non-specific inflammatory process (Cid et al., 2010). Since the gold standard for bacteremia diagnosis is fraught with difficulties, bacterial infection diagnoses are sometimes challenging. It is known
http://dx.doi.org/10.1016/j.jim.2014.07.011 0022-1759/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
2
M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
that the incubation of bacteria may take 2–4 days; genuine bacteremia may remain undetected in significant amount of infected cases because of the small volume of blood collected (Simon et al., 2004). Thus, improved indicators of infection and/or sepsis are needed in order to increase the specificity of both diagnoses and therapeutic monitoring. Recent findings in immunology have indicated new potential markers for sepsis diagnosis as cell surface antigens, cytokines and acute phase reactants (Davis et al., 2006). Surface receptors of neutrophils recognize bacterial antigens and this interaction activates the neutrophils to phagocyte. Phagocytosis is eased by receptors for immunoglobulin-G (IgG) in neutrophils (Cid et al., 2011). CD64, a leukocyte surface antigen, is a high affinity Fc receptor (FcγRI), which binds to monomeric IgG. The Fc receptors are involved with the innate and adaptive immune response, stimulating either phagocytosis or antibody-mediated cytotoxicity (Qureshi et al., 2001). Several studies have pointed neutrophil CD64 expression as a good candidate for a more specific laboratory indicator for detection of sepsis/infection (Calandra and Cohen, 2005; Fjaertoft et al., 2007). The high expression of CD64 in immature myeloid cells tends to decrease during their maturation until the stage of segmented neutrophil (Davis et al., 2006). So, healthy patients have non-remarkable expression of CD64 on the surface of neutrophils; however, it is stored in intracellular fluid and can be activated by exogenous or endogenous stimuli. Their expression in neutrophils can be induced by certain inflammatory cytokines and the presence of bacterial products in the cell wall (Fjaertoft et al., 2007). The preset study aims at establishing the association of the neutrophil CD64 expression of adult patients of our hospital emergency department, with the following variables: WBC count, band count, neutrophils, CRP, microbiological cultures and flags released by automated hematology analyzers, laboratory indicators of infection/sepsis and clinical groups. Patients admitted in the Intensive Care Unit (ICU) of Hospital de Clínicas de Porto Alegre were prospectively evaluated and blood samples were randomly selected from routine hematology laboratory. Patients receiving interferon gamma or G-CSF were excluded from the sample to avoid falsepositive results. Patients were organized into two clinical groups based on medical history and degree or likelihood of systemic acute inflammatory response according the criterions of the “Society of Critical Care Medicine” and “American College of Chest Physicians”: group 1: no clinical or laboratory evidence of infection or inflammatory process and group 2: clinical or laboratory evidence of a systemic inflammatory response (SIRS-inflammatory reaction in which two or more of the following conditions must be changed: temperature, heart rate, respiratory rate, WBC count or the presence of bands) and unequivocal clinical or laboratory evidence of a systemic sepsis, infection or inflammatory process based on identification of organisms by culture (Bone et al., 1992). Venous blood samples were collected with K3EDTA and analyzed in the Sysmex XE 2100 (Sysmex Corporation, Kobe, Japan). The equipment performance was monitored using commercial quality controls. Flags were defined as Left Shift (LS) and Immature Granulocytes (IG). Observation of blood smear stained with Wright–Giemsa, was performed in all cases. The CRP was determined quantitatively by BNII Behring
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, the reader is referred to the web version of this article.)
Table 1 Correlation between CD64 and quantitative and qualitative variables. Variable
Group exposed
P
rs
N = 93 Age (years) Gender F M Clinical groups No infection SIRS + sepsis Flag groups No flags LS and/or IG Microbiological culture Negative Positive White blood cells (/μL) Group 1 Group 2 Neutrophils (/μL) Group 1 Group 2 Band count (%) Group 1 Group 2 CRP (mg/L) Group 1 Group 2 CD64 (%) Group 1 Group 2
(15–95) 44 (47.3%) 49 (52.7%) 37 (1.19; 0.83–1.80) 56 (1.51; 1.11–2.30)
0.034 b0.001
53 (1.11; 0.82–1.64) 40 (1.82; 1.33–2.99) 0.529 35 (1.45; 1.07–2.00) 28 (1.54; 1.14–2.70) 0.146
0.161
0.047
0.652
0.002
0.322
b0.001
0.743
9790 (8220–11,720) 15,165 (9848–21,598) 7707 (5191–9135) 11,949 (7219–15,537) (0–1.6) 5.6 (0–11) 84.1 (15.2–231) 126.5 (15.8–346) 1.000 1.19 (0.82–1.80) 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); White blood cells, neutrophils, CRP, CD64 and band count as median (minimum and maximum); LS: Left Shift; IG: Immature Granulocytes; CRP: C-Reative Protein; Group 1: no evidence of infection or inflammatory process; Group 2: evidence of a systemic inflammatory response (SIRS) and sepsis. P b 0.05 was statistically significant.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
nephelometry. Patients' blood was cultured by BacT/ALERT 3D Food® (Biomérieux, St. Laurent, Quebec, Canada) and two of these cultures (aerobic bottles), from two different sites, were observed for five days of incubation. VITEK 2 was used or classic methods for identification and sensitivity tests were performed according to the Clinical and Laboratory Standards Institute (SCLI). Neutrophil CD64 expression was 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 kit is composed by two monoclonal antibodies; CD64FITC and CD163PE (clone mac2-158). CD163 is a scavenger receptor rich in cysteine and it is expressed only in cells of monocyte– macrophage lineage. CD163 was included in the kit to differentiate neutrophils from monocytes (Fig. 1). The calculation index of the expression CD64 value was performed by the analysis QuantiCALC software (Trillium Diagnostics, Brewer, Me, USA). The assay internal control was provided by the automated measurement of the lymphocyte CD64 index (b 1.0) as negative control and monocyte CD64 index (N 3.0) as a positive control. The CD64 index is then calculated using the
3
ratio of the mean fluorescent intensity of the cell populations to the FITC signal from the beads. Statistical analysis was performed using the Spearman correlation (rs) for quantitative variables relating one to another and Student t test, Kruskal–Wallis and Mann–Whitney to 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 (average 62 years). Values of WBC count, neutrophils, band count and CRP, and the association between CD64 with quantitative and qualitative variables, are described in Table 1 and the difference between CD64 expression and the clinical groups, microbiological cultures and presence of flags is shown in Fig. 2. As highlighted, there is a variable relationship among neutrophil CD64 expression and all the laboratory parameters. The association between CD64 expression (index N 1) and WBC count and microbiological cultures showed not to be significant, already the association with CRP (N5 mg/L); neutrophilia (N9.0 × 106/L); band count (N10%) and flags (Left Shift and Immature Granulocytes) emitted by the equipment, was statistically significant. The correlation was weak with band count and strong with CRP levels and neutrophilia.
Fig. 2. Difference between neutrophil CD64 expression and (A) clinical groups: group 1 — no evidence of infection or inflammatory process and group 2 — evidence of a systemic inflammatory response (SIRS) and sepsis. P = 0.034; (B) microbiological culture negative and positive. P = 0.529; and (C) groups with and without flags (Left Shift and/or Immature Granulocytes). P b 0.001.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
4
M.G. Farias et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
Previous studies have indicated that neutrophil CD64 expression is highly correlated to the presence of infection or inflammatory process (Cid et al., 2010, 2011; Groselj-Grenc et al., 2008). A significant increase in neutrophil CD64 expression is a typical finding in patients with bacterial infections. However, it is still controversial to know if this significant increase is caused by the density of the molecule surface increase or if there are more neutrophils expressing CD64, 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, may explain the lack of significance of our study. It was seen that neutrophil CD64 expression was lower in the patients of clinical group 1 compared to that in clinical group 2 patients. The neutrophil CD64 expression was statistically significant (P = 0.034), agreeing with Davis et al. who showed a high correlation between clinical groups and presence of infectious and inflammatory processes, GroseljGrenc et al. had already shown higher correlation for neutrophils in patients with SIRS and sepsis compared to patients with noninfectious SIRS (Groselj-Grenc et al., 2008). The determination of neutrophil CD64 expression needs standardization, because most of the clinical studies have used manually determined fluorescence intensity (MFI) (Fjaertoft et al., 2007). This method presents variations because of instruments and the comparison between different laboratories cannot be reliable. By using available software for data analysis and index calculation, a novel approach for reducing lot-to-lot variations (Groselj-Grenc et al., 2008) may be possible and the measurement of neutrophil CD64 expression could become an ordinary clinical test with a high compatibility and reproducibility in different laboratories. Neutrophil CD64 expression levels can be used as a barometer of systemic inflammatory responses, having a potential role in therapeutic monitoring of antibiotic use. Some authors have even suggested that, because of its high sensitivity, measurement of neutrophil CD64 expression may allow clinicians to discontinue antimicrobial treatment if there is a negative CD64 expression within 24 h of suspected infection, without having to wait for the definitive microbiological results (Ng P & Lam, 2006). This and other previous studies indicate that high expression of CD64 is an important biomarker for infection or sepsis diagnoses, even better than traditional hematologic parameters
and CRP. It was shown that CD64 assay is relatively simple, more specific and less subjective than other laboratory tests and can easily be implemented in the laboratory practice of a large university hospital. Obviously, more clinical experiences are needed to determine the complete use of neutrophil CD64 expression measurements, as well as studies on the association with other biomolecular markers have to be carried out. The neutrophil CD64 expression would meet the fast turnaround demands of hospital emergency department allowing the fast diagnoses of infections and sepsis making therapeutic 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 of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 101, 1644. Calandra, T., Cohen, J., 2005. The international sepsis forum consensus conference on definitions of infection in the intensive care unit. Crit. Care Med. 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 systematic review 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 patients presenting 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 is an improved indicator of infection or sepsis in emergency department patients. 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 bacterial infection: 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 CD64 and 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 polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin. Exp. Immunol. 125, 258. Simon, L., Gauvin, F., Amre, D.K., Saint-Louis, P., Lacroix, J., 2004. Serum procalcitonin and C-reactive protein levels as markers of bacterial: a systematic review and meta-analysis. Clin. Infect. Dis. 39, 206.
Please cite this article as: Farias, M.G., et al., Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.07.011
