Heparinized whole blood in sterile injector must remain in a sterile incubator with 5%CO2 for two hours, vertically. Injector needle must be bent at 90 degrees. After two hours, top level must be discharged gently through the injector needle on top of the Ficoll. After centrifuge, buffy coat will be discharged and the pellet will be taken and washed three times. Pure neutrophils without erythrocytes will be maintained.
Here in Brazil (Instituto Alergoimuno de Americana) we used this method to perform the Leukocyte Adherence Inhibition Test. Actually we realized that for this specific test there is no need to separate the neutrophils by ficol, so we use the whole leukocyte layer. We also realized that the centrigugation is not necessaire, and that one hour is enough to expontaneous sedimentation. We used to wash too, but we realized that the washing compromised the leukocyte vitality, so we stop washed and we succeed to get 100% adherence in the control group.
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