Since its introduction in 1968, the mononuclear cell separation method described by Bøyum has been used in numerous immunological investigations.1, 2 This widespread adoption indicates the superior results obtained with this technique and its freedom from impairment of lymphocyte function. Nevertheless, certain effects of the separation procedure have been seen and these are noted below, since research situations may arise in which they are of significance.
Separation with Ficoll-Paque PLUS has been reported to lead to adsorption of cytophilic IgG to the mononuclear leukocytes, resulting in erroneously high estimates of the number of Ig-bearing lymphocytes and too low estimates of the number of cells bearing Fc receptors. 13 This interference can be avoided by washing the blood cells with a balanced salt solution before isolation, thus removing the IgG present in the plasma that gives rise to these artifacts.
Selective loss of a population of lymphocytes that form rosettes with autologous red blood cells has been reported to occur using the standard procedure and evidence was found that this is the result of a specific lymphocyte-red blood cell interaction, not a non-specific trapping.14, 15 This population was found to account for about 6% of the lymphocytes initially present in the blood sample and could be recovered almost quantitatively by resuspending the red cell pellet in medium and recentrifuging over a gradient of slightly higher density than normal (i.e. 1.083 g/mL).15
Slight differences in phenotype compositions have been seen between fresh samples of whole blood and samples purified using Ficoll-Paque PLUS.16, 17 Fresh samples of whole blood show lower percentages of CD4+, CD19+, and CD4+CD45RA+ cells, and higher percentages of CD8+ and CD4+CD29+ subsets than fresh lymphocyte purified on a Ficoll-Paque PLUS gradient.15
Ficoll-Paque density gradient separation (FDS) was associated with significantly higher percentages of CD3+/CD62L+ and CD3+/CD11b+ lymphocytes in young children and adults alike, while the percentages of CD3+/CD54+ cells from adults was not affected by FDS. The percent expression of CD54, CD62L, and CD11b on T cells from both children and adults were significantly higher following FDS, with a greater increase in CD11b expression on T cells from young children, reaching adult levels.16
Lymphocytes separated by the Bøyum procedure have been reported to show enhanced stimulation in mixed lymphocyte cultures as compared with lymphocytes in “leukocyte-rich plasma” (not exposed to Ficoll-Paque PLUS). This enhanced reaction was postulated to depend at least partially on the removal in the Ficoll-Paque PLUS method of neutrophils that appear otherwise to have a suppressive effect on the mixed lymphocyte reaction.18