Oral Presentation Lorne Infection and Immunity 2018

Relative and absolute levels of CD64 and neutrophil elastase have potential for the diagnosis of sepsis (#113)

Riya Palchaudhuri 1 , Suzanne M Crowe 1 2 3 , Clovis S Palmer 1 2 , Mary Garcia 1 , Steve McGloughlin 4 , Shirley Vallance 4 , Emma L Martin 4 , David A Anderson 1 5
  1. Burnet Institute, Melbourne, VIC, Australia
  2. Department of Medicine, Monash University, Melbourne, Vic, Australia
  3. Infectious Diseases Unit, The Alfred, Melbourne, Vic, Australia
  4. ICU, The Alfred Hospital, Melbourne, Victoria, Australia
  5. Immunology, University of Melbourne, Melbourne, Victoria, Australia

Introduction

Improved biomarkers are required for the detection of sepsis, both in laboratory settings and at the point-of-care (POC). Upregulation of surface-CD64 expression (neutrophil CD64 index, nCD64i) has been extensively studied as a sepsis biomarker with ~80% sensitivity, but is not amenable to use at POC. We hypothesised that simultaneous measurement of neutrophil-CD64 and a neutrophil-specific protein in whole blood could yield a surrogate of the nCD64i that might be feasible for development of a POC test. 

Methods

We are recruiting adult ICU patients (n=50) with clinically-suspected sepsis (Alfred ICU) and healthy individuals (n=50). Levels of a selected neutrophil-specific marker, neutrophil elastase (NE), and neutrophil-activation marker (CD64) are measured by commercial sandwich ELISAs. Samples were also tested using the Leuko64TM assay (Trillium Diagnostics) (surface-CD64), and total (surface and intracellular) expression of CD64 was confirmed by flow-cytometry staining, and microscopy.

 Results

A strong correlation between total (whole blood) CD64 and NE was observed in healthy individuals (n=30, R2=0.7, p<0.0001), even without depletion of monocytes. We established assay cutoffs (mean+ 2SD) for CD64 and NE as well as a “Gating” strategy for healthy levels of CD64/NE for the samples with very low neutrophil count. The commercial Leuko64 kit (surface staining by flow) was positive in 20/25 patients. Conversely, total CD64 was highly elevated in most sepsis patients (23/25 positive, p<0.0001 compared to healthy controls), and was elevated compared to the healthy CD64/NE ratio in the remaining two patients (total 25/25 positive, p=0.052 compared to Leuko64). Flow-cytometry using intracellular staining showed that a significant proportion of neutrophil-CD64 was intracellular in patients negative by Leuko64TM.

 Conclusion

We observed high correlation of CD64 and NE in controls, compared with the presence of elevated amounts of total CD64 (including intracellular CD64) and/or NE in whole blood of sepsis patients. Our results suggest that measurement of both total CD64 and NE levels in whole blood has promise as an improved candidate biomarker for diagnosis of sepsis.