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Alex Quach [1; 2] and Antonio Ferrante [1; 2; 3]

Academic Editor: Nejat K. Egilmez

1, Department of Immunopathology, SA Pathology at the Women’s and Children’s Hospital, North Adelaide, SA, Australia, sapathology.sa.gov.au

2, School of Medicine, Robinson Research Institute and School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia, adelaide.edu.au

3, School of Pharmacy and Medical Sciences, Sansom Institute, University of South Australia, Adelaide, SA, Australia, unisa.edu.au

Received Jun 18, 2017; Revised Sep 8, 2017; Accepted Sep 18, 2017

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Some form of blood manipulation is required to achieve isolation of neutrophils from other blood constituents, and the challenge remains to minimise artefactual neutrophil activation. This is important as it may alter the responsiveness of neutrophils to agonists and hence undermine the experimental findings or testing for neutrophil function in diagnostic and research laboratories. There are several different methods for isolating neutrophils from human blood, including the rapid “1-step” method of centrifugation on high-density Ficoll-Hypaque (1.114 g/mL) medium [1, 2], “2-step” erythrocyte sedimentation on polysucrose-based media coupled with centrifugation on low-density Ficoll-Hypaque (1.077 g/mL) media [3, 4], and discontinuous Percoll density gradient centrifugation [5], as well as flow cytometric cell sorting [6] and immunomagnetic bead separation [7]. There are advantages and disadvantages between the uses of these techniques, but centrifugation on Ficoll-Hypaque medium and erythrocyte sedimentation techniques are the most commonly used methods.

There are variations in the separation media used but dextran-based sedimentation is typically used in conjunction with a Ficoll-Hypaque density gradient centrifugation step. In the method originally devised by Boyum [3], centrifugation on a density gradient, a concoction of a polysucrose (Ficoll) to aggregate erythrocytes and sodium diatrizoate (Hypaque) to adjust density, acts as the first step to separate out the peripheral blood mononuclear cells (PBMC), followed by dextran sedimentation to purify the neutrophils from the erythrocytes. Interestingly, many publications since also show a variation in the order of these steps, with sedimentation preceding density gradient centrifugation [5, 8–11]. This raises the possibility that monocytes under the action of dextran release mediators which activate neutrophils [12], making the separation of neutrophils by...

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