A Modified Combination of Two Methods for Isolation of Polymorphonuclear Granulocytes from Human Blood for Polymerase Chain Reaction (PCR).

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.