Blood collection technique, anticoagulants and storing temperature have minor effects on the isolation of polymorphonuclear neutrophils

Julia Krabbe,Nadine Ruske,Thomas Kraus,Svenja Böll,Christian Martin,Anke Seifert,Hanif Alamzad-Krabbe,Viktor Beilmann

doi:10.1038/s41598-020-71500-1

Julia Krabbe, Nadine Ruske + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-71500-1

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Abstract

In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils. To elucidate the influence of the blood collection technique, anticoagulants and storing temperature on isolated PMNs healthy volunteers provided blood samples with different needles and collection techniques, anticoagulants (EDTA, heparin, citrate) and storing temperatures (4, 22, 37 °C). From each blood sample PMNs were isolated and compared regarding number of PMNs and oxidative burst. The blood collection technique, anticoagulants and storing temperature had minor impact on isolated PMNs. All three tested cannulas and anticoagulants can be used to obtain blood samples for PMN isolation. For storing temperatures 37 °C should be preferred. Regarding time between the PMN isolation and the actual experiments, a time span of maximum 1 h should be targeted.

Highlights

In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils
To elucidate the influence of the blood collection technique on quality and quantity of isolated PMNs 10 healthy volunteers provided 5 different blood samples (10 ml each): (1) blood obtained with a 21 gauge (G) butterfly vein cannula with free flow into a sample vessel (BF-FF), (2) blood obtained with a 21G butterfly vein cannula drawn with a 10 ml syringe (BF-S), (3) blood obtained with a 18G vein catheter with free flow into a sample vessel (VC-FF) (4) blood obtained with a 18G vein catheter drawn with a 10 ml syringe (VC-S) and (5) blood obtained with a 22G vacuum cannula into an vacuum vessel (V-V)
PMNs were identified by expression of the following markers: cluster of differentiation (CD) 45, CD11b, CD66b and CD1612

Summary

Introduction

In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils. The blood collection technique, anticoagulants and storing temperature had minor impact on isolated PMNs. All three tested cannulas and anticoagulants can be used to obtain blood samples for PMN isolation. The hypothesis of this study was that the blood collection technique regarding collection with negative pressure aspiration versus free flow, as well as regarding the inner diameter of the cannula influences the activation of PMNs and lead to Aachen University, University Hospital Aachen, Pauwelstraße 30, 52074 Aachen, Germany. For comparison of anti-coagulants and storing temperatures, 6 volunteers donated blood which was mixed with ethylenediaminetetraacetic acid (EDTA), heparin or sodium citrate, respectively. From those blood samples PMNs were isolated and either immediately analysed or stored at 4, 22 or 37 °C for 3 h

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