Properties of Mononuclear Cells Isolated by the Ficoll-Paque Method

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



Böyum A. 1968. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl,. 9777-89.
Bøyum A. 1968. Isolation of leucocytes from human blood – further observations. (Paper II). Scand. J. Clin. Lab. Invest. 21 Suppl, . 9731-50.
BØYUM A. 1976. Isolation of Lymphocytes, Granulocytes and Macrophages. 59-15.
Almeida AP, A. Beaven M. 1980. Gel formation with leucocytes and heparin. Life Sciences. 26(7):549-555.
Holland NT, Smith MT, Eskenazi B, Bastaki M. 2003. Biological sample collection and processing for molecular epidemiological studies. Mutation Research/Reviews in Mutation Research. 543(3):217-234.
Marteau J, Mohr S, Pfister M, Visvikis-Siest S. 2005. Collection and Storage of Human Blood Cells for mRNA Expression Profiling: A 15-Month Stability Study. 51(7):1250-1252.
Kaplan J, Nolan D, Reed A. 1982. Altered lymphocyte markers and blastogenic responses associated with 24 hour delay in processing of blood samples. Journal of Immunological Methods. 50(2):187-191.
Imeri F, Herklotz R, Risch L, Arbetsleitner C, Zerlauth M, Risch GM, Huber AR. 2008. Stability of hematological analytes depends on the hematology analyser used: A stability study with Bayer Advia 120, Beckman Coulter LH 750 and Sysmex XE 2100. Clinica Chimica Acta. 397(1-2):68-71.
Paietta E. 2003. How to optimize multiparameter flow cytometry for leukaemia/lymphoma diagnosis. Best Practice & Research Clinical Haematology. 16(4):671-683.
Dolan BP, Gibbs KD, Ostrand-Rosenberg S. 2006. Dendritic Cells Cross-Dressed with Peptide MHC Class I Complexes Prime CD8+ T Cells. J Immunol. 177(9):6018-6024.
Perper RJ, Zee TW, Mickelson MM. 1968. Purification of lymphocytes and platelets by gradient centrifugation. The Journal of Laboratory and Clinical Medicine. 72(5):842-848.
VIVES J, PARRA M, CASTILLO R. Platelet Aggregation Technique Used in the Preparation of Lymphocyte Suspensions. 1(6):276-278.
Alexander EL, Titus JA, Segal DM. 1978. Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using plasmagel and Ficoll-Hypaque. Journal of Immunological Methods. 22(3-4):263-272.
Hokland P, Heron I. 1980. Analysis of the lymphocyte distribution during isopaque-ficoll isolation of mononuclear cells from human peropheral blood. Journal of Immunological Methods. 32(1):31-39.
HOKLAND P, HERON I. 1980. The Isopaque-Ficoll Method Re-evaluated: Selective Loss of Autologous Rosette-forming Lymphocytes during Isolation of Mononuclear Cells from Human Peripheral Blood. Scand J Immunol. 11(3):353-356.
AssumpcióRomeu M, Mestre M, González L, Valls A, Verdaguer J, Corominas M, Bas J, Massip E, Buendia E. 1992. Lymphocyte immunophenotyping by flow cytometry in normal adults. Journal of Immunological Methods. 154(1):7-10.
Lin S, Chao H, Yan D, Huang Y. 2002. Expression of adhesion molecules on T lymphocytes in young children and infants - a comparative study using whole blood lysis or density gradient separation. Clin Lab Haematol. 24(6):353-359.
Bain B, Pshyk K. 1973. Reactivity in mixed cultures of mononuclear leucocytes separated on Ficoll-Hypaque. Proceedings 7th Leucocyte Culture Conference, (Ed. Daguillard, F.), Academic Press; New York p. 29–37.
Wu Y, Lu H, Cai J, He X, Hu Y, Zhao H, Wang X. 2009. Membrane Surface Nanostructures and Adhesion Property of T Lymphocytes Exploited by AFM. Nanoscale Res Lett. 4(8):942-947.
Guia S, Cognet C, de Beaucoudrey L, Tessmer MS, Jouanguy E, Berger C, Filipe-Santos O, Feinberg J, Camcioglu Y, Levy J, et al. 2008. A role for interleukin-12/23 in the maturation of human natural killer and CD56+ T cells in vivo. 111(10):5008-5016.
Frelin C. 2005. Targeting NF- B activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells. Blood. 105(2):804-811.
van den Akker ELT, Baan CC, van den Berg B, Russcher H, Joosten K, Hokken-Koelega ACS, Lamberts SWJ, Koper JW. 2008. Ficoll-separated mononuclear cells from sepsis patients are contaminated with granulocytes. Intensive Care Med. 34(5):912-916.
Chan JK, Hamilton CA, Anderson EM, Cheung MK, Baker J, Husain A, Teng NN, Kong CS, Negrin RS. 2007. A novel technique for the enrichment of primary ovarian cancer cells. American Journal of Obstetrics and Gynecology. 197(5):507.e1-507.e5.
Kluin-Nelemans JC, van-Helden HP. 1980. Non-lymphoid cells obtained by the Böyum technique and their significance in cancer patients. Journal of clinical & laboratory immunology. 4(2):99.
Minami R, Yokota S, Teplitz RL. 1978. Gradient separation of normal and malignant cells. II. Application to in vivo tumor diagnosis. Acta cytologica. 22(6):584-588.
Chang H, Jones OW, Bradshaw C, Sarkar S, Porreco RP. 1981. Enhancement of human amniotic cell growth by ficoll-paque gradient fractionation. In Vitro. 17(1):81-90.
Kekarainen T, Mannelin S, Laine J, Jaatinen T. 2006. BMC Cell Biol. 7(1):30.
Briquet A, Dubois S, Bekaert S, Dolhet M, Beguin Y, Gothot A. 2010. Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages. Haematologica. 95(1):47-56.
Malanga D, Barba P, Harris PE, Maffei A, Del Pozzo G. 2007. The active translation of MHCII mRNA during dendritic cells maturation supplies new molecules to the cell surface pool. Cellular Immunology. 246(2):75-80.
Ciccocioppo R, Ricci G, Rovati B, Pesce I, Mazzocchi S, Piancatelli D, Cagnoni A, Millimaggi D, Danova M, Corazza GR. Reduced number and function of peripheral dendritic cells in coeliac disease. 149(3):487-496.
Hattar K, van Bürck S, Bickenbach A, Grandel U, Maus U, Lohmeyer J, Csernok E, Hartung T, Seeger W, Grimminger F, et al. 2005. Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation. Journal of Leukocyte Biology. 78(4):992-1000.
Lubin I, Faktorowich Y, Lapidot T, Gan Y, Eshhar Z, Gazit E, Levite M, Reisner Y. 1991. Engraftment and development of human T and B cells in mice after bone marrow transplantation. Science. 252(5004):427-431.
Flaherty MP, Abdel-Latif A, Li Q, Hunt G, Ranjan S, Ou Q, Tang X, Johnson RK, Bolli R, Dawn B. 2008. Noncanonical Wnt11 Signaling Is Sufficient to Induce Cardiomyogenic Differentiation in Unfractionated Bone Marrow Mononuclear Cells. Circulation. 117(17):2241-2252.
Kawka DW, Ouellet M, Hétu P, Singer II, Riendeau D. 2007. Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1771(1):45-54.
Zhang Y, Lin H, Frimberger D, Epstein RB, Kropp BP. 2005. Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. BJU Int. 96(7):1120-1125.
Yang G, Qiu C, Zhao H, Liu Q, Shao Y. 2006. Expression of mRNA for multiple serotonin (5-HT) receptor types/subtypes by the peripheral blood mononuclear cells of rhesus macaques. Journal of Neuroimmunology. 178(1-2):24-29.
Xu R, Jiang X, Guo Z, Chen J, Zou Y, Ke Y, Zhang S, Li Z, Cai Y, Du M, et al. 2008. Functional Analysis of Neuron-like Cells Differentiated from Neural Stem Cells Derived from Bone Marrow Stroma Cells in vitro. Cell Mol Neurobiol. 28(4):545-558.
Pearson TW, Roelants GE, Lundin LB, Mayor-Withey KS. 1979. The bovine lymphoid system: Binding and stimulation of peripheral blood lymphocytes by lectins. Journal of Immunological Methods. 26(3):271-282.
Yang TJ, Jantzen PA, Williams LF. 1979. Acid a-naphthyl acetate asterase: presence of activity in bovine and human T and B lymphocytes. J. Immunol.. 3885–93.
Di Cesare S, Maloney S, Fernandes BF, Martins C, Marshall J, Antecka E, Odashiro AN, Dawson WW, Burnier MN. 2009. The effect of blue light exposure in an ocular melanoma animal model. J Exp Clin Cancer Res. 28(1):
Hueso P, Rocha M. 1978. Comparative study of six methods for lymphocyte isolation from several mammalian sources and determination of their carbohydrate composition (author's transl). Revista espanola de fisiologia. 34(3):339-344.
Li X, Zhong Z, Liang S, Wang X, Zhong F. 2009. Effect of cryopreservation on IL-4, IFN? and IL-6 production of porcine peripheral blood lymphocytes. Cryobiology. 59(3):322-326.
Blaxhall PC. 1981. A comparison of methods used for the separation of fish lymphocytes. J Fish Biology. 18(2):177-181.
Brandslund I, Rasmussen JM, Fisker D, Svehag S. 1982. Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll®). Journal of Immunological Methods. 48(2):199-211.
Feucht H, Hadam M, Frank F, Riethmüller G. 1980. Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (Percoll). Journal of Immunological Methods. 38(1-2):43-51.
Mizobe F, Martial E, Colby-Germinario S, Livett BG. 1982. An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood. Journal of Immunological Methods. 48(3):269-279.
Sato J, Kawano Y, Takaue Y, Hirao A, Makimoto A, Okamoto Y, Abe T, Kuroda Y, Shimokawa T, Iwai A. 1995. Quantitative and qualitative comparative analysis of gradient‐separated hematopoietic cells from cord blood and chemotherapy‐mobilized peripheral blood. Stem Cells. 13(5):548-555.
EC Guide to GMP (Good Manufacturing Practice), annex 1 “Manufacture of Sterile Medicinal Products”.
United States Pharmacopeia. Recommendations for ancillary materials, chapter <1043>.
Leene W, Roholl PJ, De Groot C. 1976. Lymphocyte differentiation in the rabbit thymus. In Annales d'immunologie. 127911.
BOYUM A, LOVHAUG D, TRESLAND L, NORDLIE EM. 1991. Separation of Leucocytes: Improved Cell Purity by Fine Adjustments of Gradient Medium Density and Osmolality. Scand J Immunol. 34(6):697-712.
Archambault D, Morin G, Elazhary M. 1988. Isolation of bovine colostral lymphocytes: in vitro blastogenic responsiveness to concanavalin A and bovine rotavirus. Ann. Rech. Vet.. 19169-174.
Van Riper G, Siciliano S, Fischer PA, Meurer R, Springer MS, Rosen H. 1993. Characterization and species distribution of high affinity GTP-coupled receptors for human rantes and monocyte chemoattractant protein 1.. 177(3):851-856.
Chin S, Poey AC, Wong C, Chang S, Teh W, Mohr TJ, Cheong S. 2010. Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy. Cytotherapy. 12(1):31-37.
Garayoa M, Garcia JL, Santamaria C, Garcia-Gomez A, Blanco JF, Pandiella A, Hernández JM, Sanchez-Guijo FM, del Cañizo M, Gutiérrez NC, et al. 2009. Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors. Leukemia. 23(8):1515-1527.
Grisendi G, Annerén C, Cafarelli L, Sternieri R, Veronesi E, Cervo GL, Luminari S, Maur M, Frassoldati A, Palazzi G, et al. 2010. GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion. Cytotherapy. 12(4):466-477.
Brooke G, Rossetti T, Pelekanos R, Ilic N, Murray P, Hancock S, Antonenas V, Huang G, Gottlieb D, Bradstock K, et al. 2009. Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials. 144(4):571-579.
Miltenyi S, Müller W, Weichel W, Radbruch A. 1990. High gradient magnetic cell separation with MACS. Cytometry. 11(2):231-238.
Mauldin JP, Nagelin MH, Wojcik AJ, Srinivasan S, Skaflen MD, Ayers CR, McNamara CA, Hedrick CC. 2008. Reduced Expression of ATP-Binding Cassette Transporter G1 Increases Cholesterol Accumulation in Macrophages of Patients With Type 2 Diabetes Mellitus. Circulation. 117(21):2785-2792.
Ali H, Jurga M, Kurgonaite K, Forraz N, McGuckin C. 2009. Defined serum-free culturing conditions for neural tissue engineering of human cord blood stem cells. Acta neurobiologiae experimentalis. 69(1):12-23.
Figueroa-Tentori D, Querol S, Dodi IA, Madrigal A, Duggleby R. 2008. High purity and yield of natural Tregs from cord blood using a single step selection method. Journal of Immunological Methods. 339(2):228-235.

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