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Clin Biochem. 2008 May;41(7-8):570-5. doi: 10.1016/j.clinbiochem.2007.12.021. Epub 2008 Jan 11.

Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection.

Author information

1
REQUIMTE, Physical-Chemistry Department, Faculty of Pharmacy, University of Porto, Rua AnĂ­bal Cunha 164, 4099-030 Porto, Portugal.

Abstract

OBJECTIVES:

To assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA).

DESIGN AND METHODS:

Isolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca(2+)](i)) was measured using Fluo-3 AM, a calcium-sensitive dye.

RESULTS:

EDTA provided the highest number of isolated neutrophils/mL of blood (1.7x10(6)+/-1.5x10(5)) when compared with citrate (0.46x10(6)+/-0.95x10(5)) and heparin (0.66x10(6)+/-0.15x10(5)). EDTA originated less degree of PMA-induced activation (370+/-30%) relatively to citrate (830+/-98%) and heparin (827+/-77%). [Ca(2+)](i) was lower with EDTA (122+/-11 nM) when compared with citrate and heparin (150+/-13 and 230+/-30 nM).

CONCLUSION:

The anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.

[Indexed for MEDLINE]

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