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Abstract

SHORT COMMUNICATIONA Modified Combination of Two Methods for Isolation of Polymorphonuclear Granulocytes from Human Blood for Polymerase Chain Reaction (PCR) by Alfredo Martinez, Robert Prizont, Rosario Anadon, Federico Klosowski

Background: The aim of the study was to determine a rapid and simple method for isolation of polymorphonuclear (PMN) granulocytes from human blood appropriate for PCR of extracted DNA.
Methods: A modification of the combined dextran plus Ficoll-Hypaque methods was the final method used in 41 samples of normal white cells from humans. The changes encompassed a lower dextran-blood ratio (2:1), a shorter centrifugation time of leukocytes, a small volume of Ficoll-Hypaque suspension, and a decreased speed in the last centrifugation. The final key point was skipping the lysis of erythrocytes to avoid interference by salt solutions.
Results: These modifications rendered a mean PMN granulocyte recovery and purity of over 95% with a very narrow 2 - 8% coefficient of variation and did not alter the purity of DNA extraction.
Conclusions: The combined dextran plus Ficoll-Hypaque method changes offer a fast and simple method to obtain an adequate purity of granulocytes for PCR.

DOI: 10.7754/Clin.Lab.2017.161108