Role of CD-64 on Neutrophils and HLA-DR on Monocytes as Markers of Neonatal Sepsis: A Cross-sectional Study

Abstract

Introduction: Neonatal sepsis remains a diagnostic burden globally, responsible for about 30-50% of the total neonatal deaths each year in developing countries. Neutrophil CD-64 is found to be a promising marker for the diagnosis of early and late infections in newborns. Human Leukocyte Antigen-DR (HLA-DR) is a glycosylated cell surface transmembrane protein expressed on monocytes, allowing antigen presentation to T-cells and playing a crucial role in initiating the immune cascade during sepsis. Decreased expression of HLA-DR on monocytes has been associated with decreased survival in newborns with sepsis. Aim: To evaluate the role of neutrophil CD-64 and monocyte HLA-DR expression as markers of neonatal sepsis. Materials and Methods: This was a cross-sectional prospective study carried out at the Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India, between July 2016 and June 2017. Total of 70 full-term neonates with clinical suspicion of sepsis were enrolled. A 2 mL peripheral venous blood sample was collected for flow cytometry, blood culture, and sepsis screening in all patients. The expression of cell surface markers (CD-64 on neutrophils and HLA-DR on monocytes) was measured by an eight-color flow cytometer. A composite parameter was derived by dividing the Mean Fluorescence Intensity (MFI) values of nCD-64 and their respective mHLA-DR, multiplying the ratio by 100, and terming it as the sepsis index (Sepsis Index = nCD64/mHLA-DR x 100). A region was drawn on monocytes on an SSC/CD14 plot. Gating was performed on ‘not monocytes’ on the SSC/CD45 bivariate dot plot, and regions were drawn on lymphocytes and neutrophils. Data were entered into a Microsoft Excel spreadsheet and analysed using Statistical Package for Social Sciences (SPSS) version 21.0 statistical software. The Chi-square test was applied for proportions, and the Analysis of Variance (ANOVA) test was applied for normally distributed data. Results: In this study, 70 symptomatic neonates clinically suspected to have sepsis were enrolled and categorised into the sepsis group and the no Sepsis group. The sepsis group was further subgrouped into Definite Sepsis (Blood culture positive) and Probable Sepsis (Symptomatic baby with sepsis screen positive but sterile blood culture). nCD-64 positivity was observed in all cases (n=19) of definite sepsis. nCD-64 revealed 100% sensitivity, 87.5% specificity, 86.36% Positive Predictive Value (PPV), 100% Negative Predictive Value (NPV), and 93.02% diagnostic accuracy in culture-positive sepsis. However, downregulation of mHLA-DR observed in the present study alone showed poor diagnostic utility. The Sepsis index showed sensitivity of 94.73%, specificity of 62.50%, PPV of 66.66%, NPV of 93.75%, and accuracy of 76.74% in the definite sepsis group. Conclusion: Flow cytometric assessment of neutrophil CD-64 may be considered a rapid and reliable marker for the diagnosis of bacterial neonatal sepsis. mHLA-DR may be beneficial for monitoring patients at a later point in time for the identification of delayed immuno-suppression in neonatal sepsis.