Further Applications of Ficoll-Paque PLUS and Ficoll-Paque PREMIUM

Extracted from Isolation of Mononuclear Cells, Methodology and applications (PDF)
GE Healthcare, 2010

A great many modifications and extensions of the method have come into use following the introduction of the technique described by Bøyum in 1968 and its subsequent widespread adoption. For example, monocytes (which are recovered in the mononuclear cell fraction, using the standard procedure described in this booklet) can be removed, if desired, by incubating the blood sample with iron (or iron carbonyl) before separation on Ficoll-Paque PLUS. The monocytes phagocytose the iron particles and become denser, with the result that they sediment through the Ficoll-Paque PLUS layer on centrifugation and collect in the red blood cell pellet at the bottom of the tube3.

An important and widely used extension of the original technique is its application, in combination with selective rosetting (clustering), to the isolation of lymphocyte subclasses. Selective rosetting is presented in RosetteSep™ products marketed by StemCell Technologies Inc 19, 20, 21, 59. These products contain tetrameric antibody complexes (TAC) that crosslink unwanted cells to multiple red blood cells present in the sample, forming immunorosettes. Upon centrifugation over Ficoll-Paque product, the immunorosettes sediment to the bottom of the tube together with the red blood cells. Alternatively, the purified lymphocytes obtained by the standard procedure (with or without monocyte removal) can be incubated with an excess of sheep red blood cells (ratio of red blood cells to lymphocytes at least 50:1), whereupon the T lymphocytes spontaneously form rosettes with the sheep red blood cells. On centrifugation for a second time over Ficoll-Paque PLUS, the T lymphocyte rosettes sediment to the bottom of the tube together with the excess red blood cells, leaving the other (non-rosetting) lymphocytes at the interface3.

Such techniques for the separation of lymphocyte subclasses, as well as the standard method for isolating the entire lymphocyte population, have been widely applied to studies of lymphocyte functions and surface markers in disease states as compared to normal controls. Caution is, however, necessary in applying the Ficoll-Paque PLUS technique to pathological blood specimens, since it has been found that the resulting mononuclear cell layer may be contaminated with immature granulocytes in patients with certain infections 22, and particularly cancer 9, 23. In the latter case, elevated numbers of monocytes may also be present 24.

Ficoll-Paque PLUS has been used with success to separate cells from a variety of sources other than peripheral blood, even though its properties have been optimized specifically for blood mononuclear cell isolation. Thus, separation over Ficoll-Paque PLUS facilitated detection and identification of malignant cells in abdominal and pleural fluids 23,25. Separation on Ficoll-Paque PLUS has also been reported to assist in establishing cultures of amniotic fluid cells and to facilitate their subsequent cytogenic analysis 26. Ficoll-Paque PLUS and Ficoll-Paque PREMIUM have also been used for the isolation of cord blood derived hematopoietic stem cells, cord blood derived pluripotent stem cells for regenerative medicine clinical applications, and bone marrow mesenchymal stem cells 27, 28, 60, 61. Ficoll-Paque products are also suitable for granulocyte isolation 29, 30, 31.

Ficoll-Paque PLUS can also be used to isolate mononuclear cells from species other than man. In some cases (e.g. cow, goat, and rabbit) it may be necessary to alter the standard procedure to achieve good results 3 and it should be remembered that the density of Ficoll-Paque PLUS (1.077 g/mL), although optimized for the isolation of human mononuclear cells, may not give optimal yield and purity of mononuclear cells from other species. However, isolation methods using Ficoll-Paque PLUS for preparation of mononuclear cells from peripheral blood (PB) and bone marrow (BM) have been described for mouse ((32(PB), 33 (BM)), dog (34 (PB), 35(BM)), monkey (36 [PB], 37 [BM]), cow (38 [PB], 39 [PB]), rabbit (36 [BM], 40 [PB]), horse (41 [PB]), pig (42 [PB]), and even fish (43). Where it is desired to work with solutions of densities other than 1.077 g/mL it may be convenient to use Ficoll-Paque PREMIUM 1.084, Ficoll-Paque PREMIUM 1.073, or the alternative centrifugation media Percoll/Percoll PLUS since iso-osmotic solutions of different densities are very easily prepared with these media, facilitating the optimization of a particular separation. In research studies, separation with Percoll has also been reported to give improved lymphocyte yields and purities in some cases 44, 45, 46, 47.

Applications using Ficoll-Paque PREMIUM 1.084

Ficoll-Paque PREMIUM 1.084 can be used for isolating higher density cells. It has for instance been shown that the lymphocytes in rodents have a slightly higher average density than lymphocytes in humans 50, 51 and a fraction of these will therefore be lost into the pellet after a standard 1.077 g/mL density gradient centrifugation, contaminating the granulocyte layer and decreasing the mononuclear cell recovery. Dog, bovine, rabbit, mouse, and rat blood cells have successfully been separated using a 1.084 g/mL density gradient media such as Percoll or Ficoll-diatrizoate solutions 52, 53.

It has been reported that selective loss of lymphocytes that form rosettes with autologous blood cells (as described above) may occur by the standard procedure using a 1.077 g/mL Ficoll-Paque products 14, 15 and these lymphocytes have been shown to 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. Thus, Ficoll-Paque PREMIUM 1.084 could potentially be used for this application, although this remains to be shown.

Applications using Ficoll-Paque PREMIUM 1.073

Ficoll-Paque PREMIUM 1.073 can be used for enriching lower density cells such as monocytes from peripheral blood, or mesenchymal stem cells from bone marrow or placenta 54, 55, 56, 57.

Grisendi et al. directly compared Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073 for isolating and expanding mesenchymal stem cells from human bone marrow aspirate and convincingly demonstrated that density gradient separation using the lower density Ficoll-Paque PREMIUM 1.073 was associated with an enrichment of mesenchymal stem cell (MSC) subtypes characterized by a higher proliferation potential. Thus, Ficoll-Paque PREMIUM 1.073 can ultimately benefit clinical applications based on these cells. Brooke et al. recently described a successful manufacturing process for isolating and expanding placenta-derived human MSC using Ficoll-Paque PREMIUM 1.073 for a clinical trial 57.




  1. Isolation of mononuclear cells and granulocytes from human blood. (Paper IV). Bøyum, A., Scand. J., Clin. Lab. Invest. 21 Suppl, 97, 77–89 (1968).
  2. Isolation of leucocytes from human blood – further observations. (Paper II). Bøyum, A., Scand. J. Clin. Lab. Invest. 21 Suppl, 97, 31–50 (1968).
  3. Isolation of lymphocytes, granulocytes and macrophages. Bøyum, A., Scand. J., Immunol. 5 Suppl, 5, 9–15 (1976).
  4. Gel formation with leucocytes and heparin. Almeida, A.P., Beaven, M.A., Life Sci, 26, 549–555 (1980).
  5. Biological sample collection and processing for molecular epidemiological studies. Holland, N.T., Smith, M.T., Eskenazi, B., Bastaki, M., Mutation Research, 543, 217-234 (2003).
  6. Collection and storage of Human Blood Cells for mRNA Expression Profiling: A 15-Month Stability Study. Marteau, J., Mohr, S., le Pfister, M., Visvikis-Siest, S., Clinical Chemistry, 51, 1250-1252 (2005).
  7. Altered lymphocyte markers and blastogenic responses associated with 24 hour delay in processing of blood samples. Kaplan, J., Nolan, D., Ree, A. J., Immunol. Methods, 50, 187–191 (1982).
  8. 8. 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. Imeri, F., Herklotz, R., Risch, L., Arbetsleitner, C., Zerlauth, M., Risch, G.M., Huber, A.R., Clinica Chimica Acta, 397, 68-71 (2008).
  9. How to optimize multiparameter flow cytometry for leukaemia/lymphoma diagnosis. Paiette, E., Best Practice & Research Clinical Haematology, 16, 671-683 (2003).
  10. Dendritic Cells Cross-Dressed with Peptide MHC Class I Complexes Prime CD8 T Cells. Dolan, B.P., Gibbs Jr, K.D., Ostrand-Rosenberg, S. J., Immunol., 177, 6018-6024 (2006).
  11. Purification of lymphocytes and platelets by gradient centrifugation. Perper, R.J., Zee, T.W., Mickelson, M.M., J. Lab. Clin. Med., 72, 842–848 (1968).
  12. Platelet aggregation technique used in the preparation of lymphocyte suspensions. Vives, J., Parra, M., Castillo, R., Tissue Antigens, 1, 276–278 (1971).
  13. Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using Plasmagel and Ficoll-Hypaque. Alexander, E.L., Titus, J.A., Sega, D.M., J. Immunol. Methods, 22, 263–272 (1978).
  14. Analysis of the lymphocyte distribution during Isopaque-Ficoll isolation of mononuclear cells from human peripheral blood. Hokland, P., Heron, I., J. Immunol. Methods, 32, 31–39 (1980).
  15. The Isopaque-Ficoll method re-evaluated: Selective loss of autologous rosette-forming lymphocytes during isolation of mononuclear cells from human peripheral blood. Hokland, P., Heron, I., Scand. J. Immunol, 11,353–356 (1980).
  16. Lymphocyte imunnophenotyping by flow cytometry in normal adults Comparison of fresh whole blood lysis technique, Ficoll-Paque separation and cryopreservation. Romeu, M.A., Mestre, M., González, L., Valls, A., Verdaguer, J., Corominas, M., Bas, J., Massip, E., Buendia, E., J. Immunol. Methods, 154, 7-10 (1992).
  17. Expression of adhesion molecules on T lymphocytes in young children and infants – a comparative study using whole blood lysis or density gradient separation. Lin, S.-J, Chao, H.-C, Yan, D.-C, Huang, Y.-J., Clin. Lab. Haem, 24, 353-359 (2002).
  18. Reactivity in mixed cultures of mononuclear leucocytes separated on Ficoll-Hypaque. Bain, B., Pshyk, K. Proceedings 7th Leucocyte Culture Conference, (Ed. Daguillard, F.), Academic Press, New York, 29–37 (1973).
  19. Membrane Surface Nanostructures and Adhesion Property of Tlymphocytes Exploited by AFM. Wu, Y., Lu, H., Cai, J., He, X., Hu, Y., Zhao, H., Wang, X., Nanoscale Res. Lett., 4, 942-947 (2009).
  20. A role for interleukin-12/23 in the maturation of human natural killer and CD56 T cells in vivo. Guia, S., Cognet, C., de Beaucoudrey, L., Tessmer, M.S., Jouanguy, E., Berger, C., Filipe-Santos, O., Feinberg, J., Camcioglu, Y., Levy, J., Jumaah, S.A., Al-Hajjar, S., Stephan, J., Fieschi, C., Abel, L., Brossay, L., Casanova, J., Vivier, E., J. Blood, 111, 5008-5016 (2008).
  21. Targeting NF- B activation via pharamacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells. Frelin,C., Imbert, V., Griessinger, E., Peyron, A-C., Rochet, N., Philip, P., Dageville, C., Sirvent, A., Hummelsberger, M., Berard, E., Dreano, M., Sirvent, N., Peyron, J. Blood, 105, 804-911 (2005).
  22. Ficoll-separated mononuclear cells from sepsis patients are contaminated with granulocytes. Van den Akker, E.L.T., Baan, C.C., Van den Berg, B., Russcher, H., Joosten, K., Hokken-Koelega, A.C.S., Lamberts, S.W. J., Koper, J.W., Intensive Care Med, 34, 912-916 (2008).
  23. A novel technique for the enrichment of primary ovarian cancer cells. Chan, J.K., Hamilton, C.A., Anderson, E.M., Cheung, M.K., Baker, J, Husain, A., Teng, N.N., Kong, C.S., Negrin, R.S., Am. J. Obstet. Gynecol., 197, 507. e1—507.e5, (2007).
  24. Non-lymphoid cells obtained by the Bøyum technique and their significance in cancer patients. Kluin-Nelemans, J.C., van Helden, H.P.T., J. Clin. Lab. Immunol., 4, 99–102 (1980).
  25. Gradient separation of normal and malignant cells. II. Application to in vivo tumour diagnosis. Minami, R., Yokota, S., Teplitz, R.L., Acta. Cytol., 22, 584–588 (1978).
  26. Enhancement of human amniotic cell growth by Ficoll-Paque gradient fractionation. Chang, H-C., Jones, O.W., Bradshaw, C., et al., In Vitro., 17, 81–90 (1981).
  27. Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells. Kekarainen, T., Mannelin, S., Laine, J., Jaatinen, T., BMC Cell Biology, 7 (2006).
  28. 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. Briquet, A., Dubois, S., Bekaert, S., Dolhet, M., Beguin, Y., Gothot, A., Haematologica, 95(1), 47-56 (2010).
  29. The active translation of MHCII mRNA during dendritic cells maturation supplies new molecules to the cell surface pool. Malanga, D., Barba, P., Harris, P.E., Maffei, A., Del Pozzo, G., Cell. Immunol., 246, 75-80 (2007).
  30. Reduced number and function of peripheral dendritic cells in celiac disease. Ciccocioppo, R., Ricci, G., Rovati, B., Pesce, I., Mazzocchi, S., Piancatelli, D., Cagnoni, A., Millimaggi, D., Danova, M., Corazza, G.R., Clin. and Exp. Immunol., 146, 487-496 (2007).
  31. Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation. Hattar, K., van Burck, S., Bickenbach, A., Grandel, U., Maus, U., Lohmeyer, J., Csernok, E., Hartung, T., Seeger, W., Grimminger, F., Sibelius, U., J. Leukocytes Biol., 78, 992-1000 (2005).
  32. Modeling the initiation and progression of Human Acute Leukemia in Mice. Lubin, I., Faktorowich, Y., Lapidot, T., Gan, Y., Eshhar, Z., Gazit, E., Levite, M., Reisner, Y., Science. New Series, 252, 427-431 (1991).
  33. Noncanonical Wnt11 Signaling Is Sufficient to Induce Cardiomyogenic Differentiation in Unfractionated Bone Marrow Mononuclear Cells. Flaherty, M.P., Abdel-Latif, A.,Li, Q., Hunt, G., Ranjan, S., Ou, Q., Tang, X., Johnson, R.K., Bolli, R., Dawn, B., Circulation, 117, 2241-2252 (2008).
  34. Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium. Kawka , D.W., Ouellet, M., Hétu, P., Singerm I.I., Riendeau, D., Biochimica et Biophysica Acta., 1771, 45–54 (2007).
  35. Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. Zhang, Y., Lin, H., Frimberger, D., Epstein, R.B., Kropp, B.P., BJU International, 96, 1120-1125 (2005).
  36. Expression of mRNA for multiple serotonin (5-HT) receptor types/subtypes by the peripheral blood mononuclear cells of rhesus macaques. Yang, G., Qiu, C., Zhao, H., Liu, Q., Shao, Y., J. Neuroimmunol., 178, 24-29 (2006).
  37. Functional Analysis of Neuron-like Cells Differentiated from Neural Stem Cells Derived from Bone Marrow Stroma Cells in vitro. Xu, R., Jiang, X., Guo, Z., Chen, J., Zou, Y., Ke, Y., Zhang, S., Li, Z., Cai, Y., Du, M., Qin, L., Tang, Y., Zeng, Y., Cell Mol. Neurobiol., 28, 545-558 (2008).
  38. The bovine lymphoid system: Binding and stimulation of peripheral blood lymphocytes by lectins. Pearson, T.W., Roelants, G.E., Lundin, L.B., et al., J. Immunol. Methods, 26, 271–282 (1979).
  39. Acid a-naphthyl acetate asterase: presence of activity in bovine and human T and B lymphocytes. Yang, T.J., Jantzen, P.A., Williams, L.F., J. Immunol., 38, 85–93 (1979).
  40. The effect of blue light exposure in an ocular melanoma animal model. Di Cesare, S., Maloney, S., Fernandes, B.F., Martins, C., Marshall, J., Antecka, E., Odashiro, A.N., Dawson, W.W., Buriner Jr, M.N., J. Exp. and Clin. Cancer Res., 28:48, 1-9 (2009).
  41. Comparative study of six methods for lymphocyte isolation from several mammalian sources and determination of their carbohydrate composition. Hueso, P., Rocha, M., (Article in Spanish) Rev. Esp. Fisiol., 34, 339–344 (1978).
  42. Effect of cryopreservation on IL-4, IFNc and IL-6 production of porcine peripheral blood lymphocytes. Li, X., Zhong, Z., Liang, S., Wang, X., Zhong, F., Cryobiology, 59, 322-326 (2009).
  43. A comparison of the methods used for the separation of fish lymphocytes. Blaxhall, P.C., J. Fish Biol, 18, 177–181 (1981).
  44. Separation of human peripheral blood monocytes on continuous density gradients of Polyvinylpyrrolidone-coated silica gel (Percoll). Brandslund, I., Møller-Rasmussen, J., Fisker, D., et al., J. Immunol. Methods, 48, 199–211 (1982).
  45. Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (Percoll). Feucht, H.E., Hadam, M.R., Frank, F., et al., J. Immunol. Methods, 38, 43–51 (1980).
  46. An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood. Mizobe, F., Martial, E., Colby‑Germinario, S., et al., J. Immunol. Methods, 48, 269–279 (1982).
  47. Quantitative and Qualitative Comparative Analysis of Gradient Separated Hematopoietic Cells from Cord Blood and Chemotherapy-Mobilized Peripheral Blood. Sato, J., Kawano, Y.F., Takaue, Y., Hirao, A., Makirnoto, A., Okarnoto, Y., Abe, T., Shimokawa, T., Iwai, A., Kurodaa, Y., Stem Cells, 13, 548-555 (1995).
  48. EC Guide to GMP (Good Manufacturing Practice), annex 1 “Manufacture of Sterile Medicinal Products”.
  49. United States Pharmacopeia. Recommendations for ancillary materials, chapter <1043>
  50. Lymphocyte differentiation in the rabbit thymus. Leene, W., et al., Ann. Immunol. (Paris), 127, 911-921 (1976).
  51. Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Bøyum, A., et al., Scand. J. Immunol., 34, 697-712 (1991).
  52. Isolation of bovine colostral lymphocytes: in vitro blastogenic responsiveness to concanavalin A and bovine rotavirus. Archambault, et al., Ann. Rech. Vet., 19, 169-174 (1988).
  53. Characterization and species distribution of high affinity GTP-coupled receptors for human rantes and monocyte chemoattractant protein. Van Riper et al., JEM, 177, 851-856 (1993).
  54. Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy, Chin et al., Cytotherapy, 12, 31-37 (2010),
  55. Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors, Garayoa et al., Leukemia, 23, 1515-1527 (2009).
  56. GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion, Grisendi et al., Cytotherapy, 12, 466-477 (2010).
  57. Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials. Brooke et al., British J. of Haematology, 144, 571-579 (2008).
  58. High gradient magnetic cell separation with MACS, Miltenyi et al., Cytometry, 11, 231-238 (1990).
  59. Reduced expression of ATP-binding cassette transporter G1 increases cholesterol accumulation in macrophages of patients with type 2 diabetes mellitus. Mauldin, J. P. et al., Circulation, 117, 2785-2792 (2008).
  60. Defined serum-free culturing conditions for neural tissue engineering of human cord blood stem cells. Hamad A. et al., Acta. Neurobiol. Exp., 69, 11-23 (2009).
  61. High purity and yield of natural Tregs from cord blood using a single step selection method, Figueroa-Tentori et al., J. Immunol. Methods, 339, 228-235 (2008).


Related Links