Absolute counting of neutrophils in whole blood using flow cytometry

Brunck, Marion E. G., Andersen, Stacey B., Timmins, Nicholas E., Osborne, Geoffrey W. and Nielsen, Lars K. (2014) Absolute counting of neutrophils in whole blood using flow cytometry. Cytometry Part A, 85 12: 1057-1064. doi:10.1002/cyto.a.22503


Author Brunck, Marion E. G.
Andersen, Stacey B.
Timmins, Nicholas E.
Osborne, Geoffrey W.
Nielsen, Lars K.
Title Absolute counting of neutrophils in whole blood using flow cytometry
Journal name Cytometry Part A   Check publisher's open access policy
ISSN 1552-4930
1552-4922
Publication date 2014-12
Sub-type Article (original research)
DOI 10.1002/cyto.a.22503
Open Access Status
Volume 85
Issue 12
Start page 1057
End page 1064
Total pages 8
Place of publication Hoboken, NJ, United States
Publisher John Wiley & Sons
Collection year 2015
Language eng
Abstract Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucountâ„¢ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens.
Keyword Absolute count
Neutrophils
ANC
Flow cytometry
Volumetry
Single-platform
Double-platform
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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Created: Wed, 10 Dec 2014, 15:45:54 EST by Susan Day on behalf of Queensland Brain Institute