以 Ficoll-Paque 方法分離的單核細胞的特性

自 1968 年問世以來，Bøyum 所描述的單核細胞分離方法已被廣泛用於許多免疫學研究中。^{1, 2} 這種廣泛的應用表明，使用這種技術可以獲得優異的結果，而且不會損害淋巴細胞的功能。

據報導，使用 Ficoll-Paque PLUS 分離會導致親細胞的 IgG 被吸附到單核白細胞上，導致對含 Ig 淋巴細胞數量的估計偏高，而對含 Fc 受體細胞數量的估計過低。 ¹³這種干擾可以在分離前用平衡鹽溶液清洗血細胞，從而去除血漿中存在的引起這些假象的 IgG 來避免。

有報告指出，使用標準程序會發生選擇性遺失與自體紅細胞形成玫瑰花簇的淋巴細胞群  而且有證據顯示這是特定淋巴細胞與紅細胞互作的結果，而非非特異性誘捕。^14,15 研究發現這群淋巴細胞約佔血液樣本中最初存在的淋巴細胞的 6%，只要將紅細胞團重懸於培養基，再以比正常密度稍高的梯度重新離心（即 1.083 g/m2），就可以幾乎定量地回收這群淋巴細胞。¹⁵

新鮮的全血樣本和使用 Ficoll-Paque PLUS 純化的樣本在表型組成上有輕微的差異。^16、17 與在 Ficoll-Paque PLUS 梯度上純化的新鮮淋巴細胞相比，新鮮全血樣本中 CD4+、CD19+ 和 CD4+CD45RA+ 細胞的百分比較低，而 CD8+ 和 CD4+CD29+ 子集的百分比較高。¹⁵

Ficoll-Paque密度梯度分離（FDS）與幼兒和成人的CD3+/CD62L+和CD3+/CD11b+淋巴細胞百分比明顯較高有關，而成人的CD3+/CD54+細胞百分比不受FDS影響。兒童和成人的 T 細胞中 CD54、CD62L 和 CD11b 的表達百分比在 FDS 後都顯著增加，幼兒的 T 細胞中 CD11b 表達增加更多，達到成人的水平。¹⁶

有報告指出，與「富含白細胞血漿」（未接觸 Ficoll-Paque PLUS）中的淋巴細胞相比，經 Bøyum 程序分離的淋巴細胞在混合淋巴細胞培養物中顯示出增強的刺激作用。據推測，這種增強的反應至少部分取決於 Ficoll-Paque PLUS 方法中嗜中性粒細胞的去除，而嗜中性粒細胞似乎對混合淋巴細胞反應有抑制作用。

參考資料

1. 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.

2. Bøyum A. 1968. Isolation of leucocytes from human blood – further observations. (Paper II). Scand. J. Clin. Lab. Invest. 21 Suppl, . 9731-50.

3. BØYUM A. 1976. Isolation of Lymphocytes, Granulocytes and Macrophages. 59-15. https://doi.org/10.1111/j.1365-3083.1976.tb03851.x

4. Almeida AP, A. Beaven M. 1980. Gel formation with leucocytes and heparin. Life Sciences. 26(7):549-555. https://doi.org/10.1016/0024-3205(80)90318-5

5. 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. https://doi.org/10.1016/s1383-5742(02)00090-x

6. 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. https://doi.org/10.1373/clinchem.2005.048546

7. 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. https://doi.org/10.1016/0022-1759(82)90224-1

8. 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. https://doi.org/10.1016/j.cca.2008.07.018

9. Paietta E. 2003. How to optimize multiparameter flow cytometry for leukaemia/lymphoma diagnosis. Best Practice & Research Clinical Haematology. 16(4):671-683. https://doi.org/10.1016/s1521-6926(03)00070-7

10. 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. https://doi.org/10.4049/jimmunol.177.9.6018

11. 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.

12. VIVES J, PARRA M, CASTILLO R. Platelet Aggregation Technique Used in the Preparation of Lymphocyte Suspensions. 1(6):276-278. https://doi.org/10.1111/j.1399-0039.1971.tb00106.x

13. 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. https://doi.org/10.1016/0022-1759(78)90034-0

14. 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. https://doi.org/10.1016/0022-1759(80)90114-3

15. 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. https://doi.org/10.1111/j.1365-3083.1980.tb00245.x

16. 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. https://doi.org/10.1016/0022-1759(92)90206-9

17. 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. https://doi.org/10.1046/j.1365-2257.2002.00462.x

18. 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.

19. 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. https://doi.org/10.1007/s11671-009-9340-8

20. 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. https://doi.org/10.1182/blood-2007-11-122259

21. 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. https://doi.org/10.1182/blood-2004-04-1463

22. 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. https://doi.org/10.1007/s00134-007-0989-0

23. 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. https://doi.org/10.1016/j.ajog.2007.05.006

24. 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.

25. 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.

26. 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. https://doi.org/10.1007/bf02618035

27. Kekarainen T, Mannelin S, Laine J, Jaatinen T. 2006. BMC Cell Biol. 7(1):30. https://doi.org/10.1186/1471-2121-7-30

28. 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. https://doi.org/10.3324/haematol.2009.008524

29. 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. https://doi.org/10.1016/j.cellimm.2007.06.003

30. 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. https://doi.org/10.1111/j.1365-2249.2007.03431.x

31. 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. https://doi.org/10.1189/jlb.0902442

32. 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. https://doi.org/10.1126/science.1826797

33. 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. https://doi.org/10.1161/circulationaha.107.741066

34. 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. https://doi.org/10.1016/j.bbalip.2006.09.015

35. 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. https://doi.org/10.1111/j.1464-410x.2005.05741.x

36. 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. https://doi.org/10.1016/j.jneuroim.2006.05.016

37. 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. https://doi.org/10.1007/s10571-007-9174-9

38. 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. https://doi.org/10.1016/0022-1759(79)90252-7

39. 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.

40. 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): https://doi.org/10.1186/1756-9966-28-48

41. 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.

42. 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. https://doi.org/10.1016/j.cryobiol.2009.09.004

43. Blaxhall PC. 1981. A comparison of methods used for the separation of fish lymphocytes. J Fish Biology. 18(2):177-181. https://doi.org/10.1111/j.1095-8649.1981.tb02812.x

44. 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. https://doi.org/10.1016/0022-1759(82)90194-6

45. 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. https://doi.org/10.1016/0022-1759(80)90329-4

46. 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. https://doi.org/10.1016/0022-1759(82)90327-1

47. 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. https://doi.org/10.1002/stem.5530130513

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. Leene W, Roholl PJ, De Groot C. 1976. Lymphocyte differentiation in the rabbit thymus. In Annales d'immunologie. 127911.

51. 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. https://doi.org/10.1111/j.1365-3083.1991.tb01594.x

52. 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.

53. 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. https://doi.org/10.1084/jem.177.3.851

54. 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. https://doi.org/10.3109/14653240903313966

55. 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. https://doi.org/10.1038/leu.2009.65

56. 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. https://doi.org/10.3109/14653241003649510

57. 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. https://doi.org/10.1111/j.1365-2141.2008.07492.x

58. Miltenyi S, Müller W, Weichel W, Radbruch A. 1990. High gradient magnetic cell separation with MACS. Cytometry. 11(2):231-238. https://doi.org/10.1002/cyto.990110203

59. 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. https://doi.org/10.1161/circulationaha.107.741314

60. 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.

61. 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. https://doi.org/10.1016/j.jim.2008.09.019