Martinez Alfredo, Prizont Robert, Anadon Rosario, Klosowski Federico
Clin Lab. 2017 Jul 1;63(7):1279-1282. doi: 10.7754/Clin.Lab.2017.161108.
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.
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.
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.
The combined dextran plus Ficoll-Hypaque method changes offer a fast and simple method to obtain an adequate purity of granulocytes for PCR.
本研究的目的是确定一种快速、简单的从人血中分离多形核（PMN）粒细胞的方法，该方法适用于对提取的DNA进行聚合酶链反应（PCR）。
对葡聚糖加聚蔗糖-泛影葡胺联合方法进行改良，该改良方法最终用于41份正常人白细胞样本。这些改变包括降低葡聚糖与血液的比例（2:1）、缩短白细胞离心时间、减少聚蔗糖-泛影葡胺悬浮液的体积以及降低最后一次离心的速度。最后一个关键点是省略红细胞裂解步骤以避免盐溶液的干扰。
这些改良使得PMN粒细胞的平均回收率和纯度超过95%，变异系数非常窄，为2%-8%，并且没有改变DNA提取的纯度。
葡聚糖加聚蔗糖-泛影葡胺联合方法的改变提供了一种快速、简单的方法，可获得用于PCR的足够纯度的粒细胞。
