CD64 expression by neutrophil granulocytes
Ulrich Sack, Institute of Clinical Immunology, Medical Faculty, Universität Leipzig, Leipzig, Germany
From April 11 to 13 2013, the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) (and ESCCA (European Society for Clinical Cell Analysis) conducted a flow cytometry course in St. Etienne, France, covering a broad range of tests commonly applied in laboratory diagnostics. Detection of CD64 expression by neutrophil granulocytes was one of the topics presented due to its potential clinical impact.
Background Neutrophil granulocytes in the peripheral blood represent the central cell population in innate immunity. Expression of the Fc receptor CD64 on their surface has been shown to correlate with complications in sepsis [1-3], infectious diseases [4;5], and even in solid organ transplanted patients [6]. CD64, also called FcγRI (Fc γ receptor I), is a class of plasma membrane receptors on human myeloid cells. It contains three extracellular immunoglobulin-like domains that represent binding sites for the Fc portion of IgG [3]. Expression level of CD64 on neutrophil granulocytes can be detected easily. It is upregulated in inflammatory and septic complications; upregulation over an index of 1.5 as calculated against bead standard is of diagnostic value for patients with inflammatory response [2]. Physiology In healthy persons, CD64 is constitutively presented by monocytes, macrophages, eosinophils, and neutrophil granulocytes. With less than 1,000 sites per cell the neutrophil granulocyte CD64
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/cyto.b.21216 This article is protected by copyright. All rights reserved.
Cytometry: Part B - Clinical Cytometry
expression is negligible in a state of health and is at the level of detection by flow cytometry [2]. CD64 expression by monocytes has no diagnostic impact. On neutrophil granulocytes it becomes upregulated by various interleukins and depends on the intensity of the stimulation [2;3;7]. It is mainly induced by bacterial infection via CD14 and toll like receptors [1-3;8-13].
Inflammation In situations of increased inflammatory response like sepsis, SIRS (systemic inflammatory response syndrome), local infections, or tissue injuries, the cell surface expression of CD64 by neutrophil granulocytes elevates within four to six hours after stimulation, and the amount of detectable CD64 mRNA increases and can be measured by Northern Blot analysis already within one to three hours [2]. CD64 expression by neutrophil granulocytes also varies by the type of infection (Gramnegative bacteria induce higher expression compared to Gram-positive bacteria [2]). This appears to directly correlate with enhanced bactericidal and antifungal activity [2]. The antibodydependent cross-linking of expressed CD64 [14] contributes to cellular cytotoxicity, phagocytosis, and the clearance of immune complexes [3]. Compared to other haematological indices, expression level of CD64 by neutrophil granulocytes shows a biphasic response to LPS administration in humans. A correlation between pro-inflammatory cytokine serum levels, but not of the anti-inflammatory IL-10, and CD64 expression was demonstrated, illustrating that CD64 expression by neutrophil granulocytes is a measure of innate immunity [15].
Method For clinical application, a CE approved in vitro diagnostic test kit is available (Leuko64, Trillium diagnostics, Brewer, Maine, U.S.A.). This test guarantees a fair internal quality control [16]. Within our course in clinical flow cytometry, we decided to use this this test system. After collecting blood samples (50 µl EDTA-anticoagulated blood) the measurement of
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Page 2 of 7
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Cytometry: Part B - Clinical Cytometry
CD64 expression on neutrophil granulocytes was performed by quantitative flow cytometry on a Gallios (Beckman-Coulter, Krefeld, Germany) by using the Leuko64™ kit, a whole-blood lysed no-wash method containing internal calibration beads for quantification. The proprietary CD64 software was used for data analysis. This kit reports leukocyte expression of CD64 and CD163 as an index (Figure) using fluorescein-labelled calibration beads [2]. Validation of this test kit reveals good sensitivity (90.5 %) and specifity (96.3 %) as well as high positive and negative predictive values for sepsis [2]. In our hands, the inter-assay imprecision is below 15 %, and calibration beads allow day-to-day comparisons of quantitative values.
Clinical indication Given that the neutrophil is the major cellular component of acute response to infection, the detection of molecular changes of neutrophil granulocytes’ activation could provide a rapid and sensitive indicator of a systemic inflammatory response [1;2;9-11;14]. Diagnostic use of a quantitative flow cytometric assay for expression strength of CD64 by neutrophil granulocytes as an in vitro indicator of neutrophil granulocytes’ activation seems to be a valuable marker as an additional utility in the evaluation of patients with suspect acute inflammation or infection [1;3]. CD64 expression by neutrophil granulocytes has also been shown to be a useful parameter for diagnostic evaluation of suspect postoperative complications in transplanted patients [6]; the high diagnostic value for infectious complications shown in other studies is not influenced by immunosuppression in this population. The use of CD64 expression by neutrophil granulocytes as an infection marker also in the postoperative course of transplanted patients should allow early determination of further treatment already within 24 hours without waiting for the definitive microbiologic culture results [9]. Besides the fast performance of the CD64-kit, an anticipated benefit of measuring CD64 expression by neutrophil granulocytes is that it will make the need for performing subjective and laborious manual microscopic differential counts to determine band counts and immature-to-
3 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Cytometry: Part B - Clinical Cytometry
total myeloid ratios superfluous [2], in particular if it is integrated into routine laboratory procedure. In comparison to various other parameters including CRP, CD64 expression by neutrophil granulocytes has a very good performance as summarized in [6] and it is shown there for transplanted patients in detail. Regarding procalcitonin, at least in preterm children, CD64 is even better than PCT [17]. Various studies show superior diagnostic performance in sepsis as compared to other inflammatory parameters [2;7]. Nevertheless, it must be noted that the test is implemented as a diagnostic test in only very few laboratories. Although the potential diagnostic value of CD64 has been known for some time, most laboratories analyze CD64 expression by neutrophil granulocytes for clinical research only. A major barrier could be the nature of the indication (infection/sepsis), which requires a fast turn-around time, which is usually not possible if the test is not yet available on automated instruments or if the necessary specialized 24-7-laboratory is not available on-site.
Legend to figure Expression of CD64 by neutrophil granulocytes is calculated by comparing median of CD64 expression to FITC labeled beads as internal standard.
Conflict of interests This course has been supported by Beckman Coulter.
References 1. Davis BH et al. Neutrophil CD64 expression: Potential diagnostic indicator of acute inflammation and therapeutic monitor of interferon-gamma therapy. Lab Hematol 1995;1:312. 2. Davis BH et al. Improved diagnostic approaches to infection/sepsis detection. Expert Rev
4 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Page 4 of 7
Page 5 of 7
Cytometry: Part B - Clinical Cytometry
Mol Diagn 2005;5:193–207. 3. Qureshi SS et al. Increased distribution and expression of CD64 on blood polymorphnuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol 2001;125:258–265. 4. Cid J et al. Neutrophil CD64 expression as marker of bacterial infection: a systematic review and meta-analysis. J Infect 2010;60:313-9. 5. Nuutila J et al. Simultaneous quantitative analysis of FcgammaRI (CD64) expression on neutrophils and monocytes: a new, improved way to detect infections. J Immunol Methods 2007;328:189-200. 6. Grey D et al. Increased CD64 Expression on Polymorphonuclear Neutrophils Indicates Infectious Complications Following Solid Organ Transplantation. Cytometry Part A 2011;79A:446-460. 7. Davis BH et al. Neutrophil CD64 Is an Improved Indicator of Infection or Sepsis in Emergency Department Patients. Arch Pathol Lab Med 2006;130:654–661. 8. Ng PC et al. Neutrophil CD64 Is a Sensitive Diagnostic Marker for Early-Onset Neonatal Infection. Pediatr Res 2004;56:796-803. 9. Ng PC et al. Neutrophil CD64 Expression: A Sensitive Diagnostic Marker for Late-Onset Nosocomial Infection in Very Low Birthweight Infants. Pediatr Res 2002;51:296–303. 10. Layseca-Espinosa E et al. Expression of CD64 as a potential marker of neonatal sepsis. Pediatr Allergy Immunol 2002;13:319–327. 11. Davis BH et al. Quantitative Neutrophil CD64 Expression: Promissing Diagnostic Indicator of Infection or Systemic Acute Inflammatory Response. Clinical Immunological Newsletter 1996;16:121–130. 12. Fjaertoft G et al. Neutrophils from term and preterm newborn infants express the high affinity Fcgamma-receptor I (CD64) during bacterial infections. Pediatr Res 1999;45:871-876. 13. Fischer G et al. CD64 surface expression on neutrophils is transiently upregulated in patients with septic shock. Intensive Care Med 2001;27:1848-1852.
5 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Cytometry: Part B - Clinical Cytometry
14. Akerley WL 3rd et al. Neutrophil activation through high-affinity Fc gamma receptor using a monomeric antibody with unique properties. Blood 1991;77:607–615. 15. van der Meer et al. Hematological indices, inflammatory markers and neutrophil CD64 expression: comparative trends during experimental human endotoxemia. J Endotoxine Research 2007;13:94-100. 16. Davis BH et al. Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: proposed practical method. Cytometry B Clin Cytom 2013;84:329-37. 17. Berrington JE et al. Proportionate reduction in uncertainty of late onset infection in pre-term infants by neutrophil CD64 measurement. Fetal Pediatr Pathol 2014;33:16-22.
6 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
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Cytometry: Part B - Clinical Cytometry
Expression of CD64 by neutrophil granulocytes is calculated by comparing median of CD64 expression to FITC labeled beads as internal standard.
John Wiley and Sons, Inc. This article is protected by copyright. All rights reserved.
Ulrich Sack, Institute of Clinical Immunology, Medical Faculty, Universität Leipzig, Leipzig, Germany
From April 11 to 13 2013, the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) (and ESCCA (European Society for Clinical Cell Analysis) conducted a flow cytometry course in St. Etienne, France, covering a broad range of tests commonly applied in laboratory diagnostics. Detection of CD64 expression by neutrophil granulocytes was one of the topics presented due to its potential clinical impact.
Background Neutrophil granulocytes in the peripheral blood represent the central cell population in innate immunity. Expression of the Fc receptor CD64 on their surface has been shown to correlate with complications in sepsis [1-3], infectious diseases [4;5], and even in solid organ transplanted patients [6]. CD64, also called FcγRI (Fc γ receptor I), is a class of plasma membrane receptors on human myeloid cells. It contains three extracellular immunoglobulin-like domains that represent binding sites for the Fc portion of IgG [3]. Expression level of CD64 on neutrophil granulocytes can be detected easily. It is upregulated in inflammatory and septic complications; upregulation over an index of 1.5 as calculated against bead standard is of diagnostic value for patients with inflammatory response [2]. Physiology In healthy persons, CD64 is constitutively presented by monocytes, macrophages, eosinophils, and neutrophil granulocytes. With less than 1,000 sites per cell the neutrophil granulocyte CD64
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/cyto.b.21216 This article is protected by copyright. All rights reserved.
Cytometry: Part B - Clinical Cytometry
expression is negligible in a state of health and is at the level of detection by flow cytometry [2]. CD64 expression by monocytes has no diagnostic impact. On neutrophil granulocytes it becomes upregulated by various interleukins and depends on the intensity of the stimulation [2;3;7]. It is mainly induced by bacterial infection via CD14 and toll like receptors [1-3;8-13].
Inflammation In situations of increased inflammatory response like sepsis, SIRS (systemic inflammatory response syndrome), local infections, or tissue injuries, the cell surface expression of CD64 by neutrophil granulocytes elevates within four to six hours after stimulation, and the amount of detectable CD64 mRNA increases and can be measured by Northern Blot analysis already within one to three hours [2]. CD64 expression by neutrophil granulocytes also varies by the type of infection (Gramnegative bacteria induce higher expression compared to Gram-positive bacteria [2]). This appears to directly correlate with enhanced bactericidal and antifungal activity [2]. The antibodydependent cross-linking of expressed CD64 [14] contributes to cellular cytotoxicity, phagocytosis, and the clearance of immune complexes [3]. Compared to other haematological indices, expression level of CD64 by neutrophil granulocytes shows a biphasic response to LPS administration in humans. A correlation between pro-inflammatory cytokine serum levels, but not of the anti-inflammatory IL-10, and CD64 expression was demonstrated, illustrating that CD64 expression by neutrophil granulocytes is a measure of innate immunity [15].
Method For clinical application, a CE approved in vitro diagnostic test kit is available (Leuko64, Trillium diagnostics, Brewer, Maine, U.S.A.). This test guarantees a fair internal quality control [16]. Within our course in clinical flow cytometry, we decided to use this this test system. After collecting blood samples (50 µl EDTA-anticoagulated blood) the measurement of
2 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Page 2 of 7
Page 3 of 7
Cytometry: Part B - Clinical Cytometry
CD64 expression on neutrophil granulocytes was performed by quantitative flow cytometry on a Gallios (Beckman-Coulter, Krefeld, Germany) by using the Leuko64™ kit, a whole-blood lysed no-wash method containing internal calibration beads for quantification. The proprietary CD64 software was used for data analysis. This kit reports leukocyte expression of CD64 and CD163 as an index (Figure) using fluorescein-labelled calibration beads [2]. Validation of this test kit reveals good sensitivity (90.5 %) and specifity (96.3 %) as well as high positive and negative predictive values for sepsis [2]. In our hands, the inter-assay imprecision is below 15 %, and calibration beads allow day-to-day comparisons of quantitative values.
Clinical indication Given that the neutrophil is the major cellular component of acute response to infection, the detection of molecular changes of neutrophil granulocytes’ activation could provide a rapid and sensitive indicator of a systemic inflammatory response [1;2;9-11;14]. Diagnostic use of a quantitative flow cytometric assay for expression strength of CD64 by neutrophil granulocytes as an in vitro indicator of neutrophil granulocytes’ activation seems to be a valuable marker as an additional utility in the evaluation of patients with suspect acute inflammation or infection [1;3]. CD64 expression by neutrophil granulocytes has also been shown to be a useful parameter for diagnostic evaluation of suspect postoperative complications in transplanted patients [6]; the high diagnostic value for infectious complications shown in other studies is not influenced by immunosuppression in this population. The use of CD64 expression by neutrophil granulocytes as an infection marker also in the postoperative course of transplanted patients should allow early determination of further treatment already within 24 hours without waiting for the definitive microbiologic culture results [9]. Besides the fast performance of the CD64-kit, an anticipated benefit of measuring CD64 expression by neutrophil granulocytes is that it will make the need for performing subjective and laborious manual microscopic differential counts to determine band counts and immature-to-
3 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Cytometry: Part B - Clinical Cytometry
total myeloid ratios superfluous [2], in particular if it is integrated into routine laboratory procedure. In comparison to various other parameters including CRP, CD64 expression by neutrophil granulocytes has a very good performance as summarized in [6] and it is shown there for transplanted patients in detail. Regarding procalcitonin, at least in preterm children, CD64 is even better than PCT [17]. Various studies show superior diagnostic performance in sepsis as compared to other inflammatory parameters [2;7]. Nevertheless, it must be noted that the test is implemented as a diagnostic test in only very few laboratories. Although the potential diagnostic value of CD64 has been known for some time, most laboratories analyze CD64 expression by neutrophil granulocytes for clinical research only. A major barrier could be the nature of the indication (infection/sepsis), which requires a fast turn-around time, which is usually not possible if the test is not yet available on automated instruments or if the necessary specialized 24-7-laboratory is not available on-site.
Legend to figure Expression of CD64 by neutrophil granulocytes is calculated by comparing median of CD64 expression to FITC labeled beads as internal standard.
Conflict of interests This course has been supported by Beckman Coulter.
References 1. Davis BH et al. Neutrophil CD64 expression: Potential diagnostic indicator of acute inflammation and therapeutic monitor of interferon-gamma therapy. Lab Hematol 1995;1:312. 2. Davis BH et al. Improved diagnostic approaches to infection/sepsis detection. Expert Rev
4 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Page 4 of 7
Page 5 of 7
Cytometry: Part B - Clinical Cytometry
Mol Diagn 2005;5:193–207. 3. Qureshi SS et al. Increased distribution and expression of CD64 on blood polymorphnuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol 2001;125:258–265. 4. Cid J et al. Neutrophil CD64 expression as marker of bacterial infection: a systematic review and meta-analysis. J Infect 2010;60:313-9. 5. Nuutila J et al. Simultaneous quantitative analysis of FcgammaRI (CD64) expression on neutrophils and monocytes: a new, improved way to detect infections. J Immunol Methods 2007;328:189-200. 6. Grey D et al. Increased CD64 Expression on Polymorphonuclear Neutrophils Indicates Infectious Complications Following Solid Organ Transplantation. Cytometry Part A 2011;79A:446-460. 7. Davis BH et al. Neutrophil CD64 Is an Improved Indicator of Infection or Sepsis in Emergency Department Patients. Arch Pathol Lab Med 2006;130:654–661. 8. Ng PC et al. Neutrophil CD64 Is a Sensitive Diagnostic Marker for Early-Onset Neonatal Infection. Pediatr Res 2004;56:796-803. 9. Ng PC et al. Neutrophil CD64 Expression: A Sensitive Diagnostic Marker for Late-Onset Nosocomial Infection in Very Low Birthweight Infants. Pediatr Res 2002;51:296–303. 10. Layseca-Espinosa E et al. Expression of CD64 as a potential marker of neonatal sepsis. Pediatr Allergy Immunol 2002;13:319–327. 11. Davis BH et al. Quantitative Neutrophil CD64 Expression: Promissing Diagnostic Indicator of Infection or Systemic Acute Inflammatory Response. Clinical Immunological Newsletter 1996;16:121–130. 12. Fjaertoft G et al. Neutrophils from term and preterm newborn infants express the high affinity Fcgamma-receptor I (CD64) during bacterial infections. Pediatr Res 1999;45:871-876. 13. Fischer G et al. CD64 surface expression on neutrophils is transiently upregulated in patients with septic shock. Intensive Care Med 2001;27:1848-1852.
5 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Cytometry: Part B - Clinical Cytometry
14. Akerley WL 3rd et al. Neutrophil activation through high-affinity Fc gamma receptor using a monomeric antibody with unique properties. Blood 1991;77:607–615. 15. van der Meer et al. Hematological indices, inflammatory markers and neutrophil CD64 expression: comparative trends during experimental human endotoxemia. J Endotoxine Research 2007;13:94-100. 16. Davis BH et al. Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: proposed practical method. Cytometry B Clin Cytom 2013;84:329-37. 17. Berrington JE et al. Proportionate reduction in uncertainty of late onset infection in pre-term infants by neutrophil CD64 measurement. Fetal Pediatr Pathol 2014;33:16-22.
6 Sons, Inc. John Wiley and This article is protected by copyright. All rights reserved.
Page 6 of 7
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Cytometry: Part B - Clinical Cytometry
Expression of CD64 by neutrophil granulocytes is calculated by comparing median of CD64 expression to FITC labeled beads as internal standard.
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