跳到主要內容

臺灣博碩士論文加值系統

(44.201.99.222) 您好!臺灣時間:2022/12/04 01:07
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:曾華偉
研究生(外文):Hua-Wei Tseng
論文名稱:優化微流體碟盤系統將母體血液中的胎兒細胞分離後作功能性之分析
論文名稱(外文):Optimization of A Disk-Based Microfluidics System to Isolate Fetal Cells from Maternal Blood for Functional Analysis
指導教授:李黛苹李黛苹引用關係
指導教授(外文):Tai-Ping Lee
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:66
中文關鍵詞:胎兒有核紅血球細胞非侵入性的產前診斷微流體人類慢性骨髓白血病細胞株K562全基因體放大
外文關鍵詞:fetal nucleated red blood cellsnon-invasive prenatal diagnosisK562 cellsWhole Genome Amplificationmicrofluidics
相關次數:
  • 被引用被引用:0
  • 點閱點閱:330
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
自西元 1978 年科學家早已發現,胎兒有核紅血球細胞 (fetal nucleated red blood cells; fnRBCs) 存在母體的血液循環內,而這些 fnRBCs 帶有完整的遺傳訊息,因此若可應用在非侵入性的產前診斷 (Non-invasive Prenatal Diagnosis; NIPD),對人類是一大福音。但由於 fnRBCs 在母親體血液循環當中相當稀少 (約23 cells/ mL),因此如何以有效率、高純度的方式將胎兒細胞分離出來就成為應用fnRBCs進行產前診斷的大難題。
近年來微流體 (microfluidics) 系統因所需檢體較少、反應速率快、花費低以及高分離純度的優點而被廣泛應用在稀少細胞的分離。
實驗室之前所建立收取fnRBCs的微流體系統, 其原理主要透過梯度密度離心以及CD71細胞表面抗原篩選,再以epsilon-globin鑑定收取到的 fnRBCs確實為胎兒細胞。但分離出來的 fnRBCs 依舊存在純率的問題,因此我們首先透過改變depleted CD45+細胞的試劑組劑量來提高 CD45+ 細胞的移除效率,當我們使用的劑量由原先的10µl提高到15µl時,CD45+ 細胞的移除效率可由75倍提高到315倍,但純率還是不足以用於後端的基因分析;因此我們希望透過修改微流體系統來增加細胞的回收率,修改的部分主要是 (1) 將原先母血檢體經過去除CD45+細胞及成熟紅血球的前處理,改以PBS 1 : 1 稀釋母血檢體,直接經由微流體碟盤進行分離,(2) 並加大了廢液收集區以容納更多的廢血,(3) 細胞收集區的部分則是由原先固定在碟盤上的方式改用可分離式的chip以利細胞觀察及抽取。目前我們先使用人類慢性骨髓白血病細胞株K562丟入非懷孕組別的血液當中模擬fnRBCs測試回收率的結果,舊系統對細胞的回收率為24.35% (spiked 123-128顆細胞),修改過的系統則分別為45.6% (spiked 100顆細胞)、49% (spiked 200顆細胞),有顯著性意義的改善 。最後將分離出的細胞抽出後使用single cell picking的技術將單顆fnRBC挑選出來以克服純率問題,再進行全基因體放大(Whole Genome Amplification; WGA),接著使用PCR技術成功得到K562基因片段 (BCR/ABL fusion gene)。
未來我們收集孕婦母血進行fnRBCs分離、計數後會和懷孕週數作相關性的統計,同時後端主要會以FISH (Fluorescence In Situ Hybridization) 及STR (Short Tandem Repeat)/ VNTR (Variable Number Tandem Repeat) 來做基因分析,希望可以藉此作為確認分離出的細胞為胎兒細胞的依據,以達到將其遺傳訊息應用在臨床診斷,成為NIPD的目標。

Scientists have discovered that fetal nucleated red blood cells (fnRBCs) exist in maternal blood circulation with complete genetic information since 1978. If fnRBCs can be applied on non-invasive prenatal diagnosis (NIPD), it will become the vital goal of prenatal diagnosis. However, the fnRBCs are rare in maternal blood circulation (about 23 cells / mL), it is necessary to find a high-efficiency and purity technology to isolate. In recent years, microfluidics system have been widely used in rare cells isolation due to less sample, faster reaction time, low cost and high purity separation.
The original microfluidics system was designed in 2013 by Wei-Lun Cheng. The principle of design was the density gradient centrifugation (DGC) and CD71 positive immunomagnetic separation to isolate fnRBCs from maternal blood. In addition the fnRBCs were identified by the presence of the fetal specific marker epsilon-globin gene. But the purity of fnRBCs was poor, we increased the volume of EasySep® Human Whole Blood CD45 Depletion Kit to 15µl for depletion of more CD45+ cells. We found that it had 4.2 times more efficiency than original condition. However, it was too impure to use in gene analysis. Hence, we tried to increase the recovery rate by optimizing the microfluidics system. The parameters were modified into the following points: (1) we diluted whole blood with PBS (1:1 dilution) as loading sample, instead of blood sample predepleted with CD45. (2) We increased the area of waste chamber to accommodate more wasted blood, and (3) the collection chamber could replace the chip to facilitate the observation and extraction.
Next, we spiked K562 cells (100~500 cells) in blood as fnRBC model for recovery rate testing. The recovery rate of chip disc was 45.6%. In addition, the chip disk was more suitable then the original disc. Finally, isolated cells were extracted by the single cell picking technique (collaborate with NTU institute of applied mechanics) to overcome the problem of purity. Then, the single cells were amplified with whole genome amplification (WGA) kit and quantified by genomic PCR of bcr/abl genes.
In the future, we will isolate fnRBC from maternal blood and compare the number of fnRBC with gestational age. On the other hand, we will try to verify the fnRBC by fluorescence in situ hybridization (FISH) or short tandem repeat (STR)/ variable number tandem repeat (VNTR) analysis. Taken together, we believe that the optimized microfluidic chip disk will bring the new NIPD methods in the future.

致謝 I
中文摘要 II
Abstract IV
目錄 VI
縮寫表 1
第一章 導論 3
1.1 產前診斷 3
1.2 母血循環中的胎兒DNA (cffDNA) 4
1.3 母血循環中的胎兒細胞 5
1.4 胎兒細胞分離技術 7
1.5 胎兒細胞分析技術 10
1.6 研究目的 12
第二章 材料與方法 13
2.1 實驗材料 13
2.1.1 實驗藥品 13
2.1.2 細胞培養相關試劑 13
2.1.3 細胞株 13
2.1.4 緩衝液 13
2.1.5 套組試劑 16
2.1.6 實驗抗體 16
2.1.7 引子 17
2.1.8 微流體碟盤製作材料 (本表格由於合作關係不對外公開) 17
2.1.9 其他 18
2.2 實驗方法 18
2.2.1 細胞培養 18
2.2.2 錐形藍染色法 (Trypan Blue Exclusion) 19
2.2.3 Cell pre-labeling 20
2.2.4 微量細胞計數 20
2.2.5 CD45+ 細胞移除 21
2.2.6 微流體碟盤原型機操作 22
2.2.7 微流體碟盤系統測試 22
2.2.8 Cell direct PCR 24
2.2.9 血液樣本於微流體碟盤處理 25
2.2.10 微流體碟盤製作 26
2.2.11 螢光原位雜合技術 (FISH) 26
2.2.12 螢光探針標定 27
2.2.13 WGA (whole genome amplification) 27
第三章 實驗結果 29
3.1 使用微流體碟盤分離、觀察母血中的fnRBCs 29
3.2 母血中的fnRBCs和懷孕周數之相關性 29
3.3 將使用original disc分離出的fnRBCs進行螢光原位雜交 30
3.4 CD45+ 細胞移除效率測試 30
3.5 Cell direct PCR 31
3.6 微流體碟盤優化 32
3.7 以K562細胞株測試chip微流體碟盤對細胞之回收率 32
3.8 以K562細胞株進行完整實驗流程後之基因分析 33
3.9 以男女性PBMC混和並經過single cell isolation後之基因分析 33
第四章 討論 35
第五章 結論 40
第六章 結果圖表 41
圖一、使用微流體碟盤分離、觀察母血中的fnRBCs 41
圖二、母血中的fnRBCs和懷孕周數之相關性 42
圖三、將使用微流體碟盤分離出的fnRBCs進行螢光原位雜交 43
圖四、CD45+細胞移除效率測試 44
圖五、背景細胞對direct PCR結果影響測試 45
圖六、微流體碟盤優化 46
圖七、以K562細胞株測試優化後的微流體碟盤對細胞之回收率 47
圖八、以K562細胞株進行完整實驗流程後之基因分析 48
圖九、以男女性PBMC混和並經過single cell isolation後之基因分析 49
Table 1 Comparison of microfluidics system 50
Table 2 K562 cell model and human PBMC in WGA 51
第七章 參考文獻 52
第八章 附錄 61
附錄一、以K562細胞株測試原始微流體碟盤對細胞之回收率 61
附錄二、使用Hetasep、ACK lysis buffer移除紅血球及PE、PC材質membrane對回收率影響之測試 62
附錄三、以K562細胞株測試原始微流體碟盤對細胞之存活率 63
附錄四、將母血中分離出細胞進行direct PCR 64
附錄五、以VNTR分析男女性DXS52多樣性 65
附錄六、Original disc與chip disc背景細胞數比較、K562細胞標記表現 66


1. Kuo, W.L., et al., Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes. Am J Hum Genet, 1991. 49(1): p. 112-9.
2. Choolani, M., A.P. Mahyuddin, and S. Hahn, The promise of fetal cells in maternal blood. Best Pract Res Clin Obstet Gynaecol, 2012. 26(5): p. 655-67.
3. Caughey, A.B., L.M. Hopkins, and M.E. Norton, Chorionic villus sampling compared with amniocentesis and the difference in the rate of pregnancy loss. Obstet Gynecol, 2006. 108(3 Pt 1): p. 612-6.
4. Choolani, M., et al., FastFISH: technique for ultrarapid fluorescence in situ hybridization on uncultured amniocytes yielding results within 2 h of amniocentesis. Mol Hum Reprod, 2007. 13(6): p. 355-9.
5. Chen, C.P., et al., Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations. Taiwan J Obstet Gynecol, 2011. 50(1): p. 85-94.
6. Soucy, J.F., J. Lavoie, and A.M. Duncan, Locus-specific dual color-probe for the enumeration of chromosome 18 in rapid FISH aneuploidy testing on uncultured amniocytes. Prenat Diagn, 2010. 30(8): p. 811-2.
7. Jia, C.W., et al., Fluorescence in situ hybridization in uncultured amniocytes for detection of aneuploidy in 4210 prenatal cases. Chin Med J (Engl), 2011. 124(8): p. 1164-8.
8. Tabor, A., et al., Randomised controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet, 1986. 1(8493): p. 1287-93.
9. Daffos, F., M. Capella-Pavlovsky, and F. Forestier, Fetal blood sampling during pregnancy with use of a needle guided by ultrasound: a study of 606 consecutive cases. Am J Obstet Gynecol, 1985. 153(6): p. 655-60.
10. Buscaglia, M., et al., Percutaneous umbilical blood sampling: indication changes and procedure loss rate in a nine years' experience. Fetal Diagn Ther, 1996. 11(2): p. 106-13.
11. Wald, N.J., et al., Maternal serum screening for Down's syndrome in early pregnancy. BMJ, 1988. 297(6653): p. 883-7.
12. Rava, R.P., et al., Circulating fetal cell-free DNA fractions differ in autosomal aneuploidies and monosomy X. Clin Chem, 2014. 60(1): p. 243-50.
13. Lo, Y.M., et al., Presence of fetal DNA in maternal plasma and serum. Lancet, 1997. 350(9076): p. 485-7.
14. Lo, Y.M., et al., Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet, 1998. 62(4): p. 768-75.
15. Zhang, J., et al., Determination of fetal RhD status by maternal plasma DNA analysis. Ann N Y Acad Sci, 2000. 906: p. 153-5.
16. Zimmermann, B., et al., Novel real-time quantitative PCR test for trisomy 21. Clin Chem, 2002. 48(2): p. 362-3.
17. Jung, M., et al., Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem, 2003. 49(6 Pt 1): p. 1028-9.
18. Spencer, K., J.B. de Kok, and D.W. Swinkels, Increased total cell-free DNA in the serum of pregnant women carrying a fetus affected by trisomy 21. Prenat Diagn, 2003. 23(7): p. 580-3.
19. Chiu, R.W., et al., Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem, 2001. 47(9): p. 1607-13.
20. Honda, H., et al., Fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum. Hum Genet, 2002. 110(1): p. 75-9.
21. Hahn, S. and W. Holzgreve, Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update, 2002. 8(6): p. 501-8.
22. Schmorl, G., Pathologisch-anatomische Untersuchungen über Puerperal-Eklampsie. Vogel, 1893: p. 138.
23. Walknowska, J., F.A. Conte, and M.M. Grumbach, Practical and theoretical implications of fetal-maternal lymphocyte transfer. Lancet, 1969. 1(7606): p. 1119-22.
24. Bianchi, D.W., et al., Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci U S A, 1990. 87(9): p. 3279-83.
25. Bianchi, D.W., et al., Detection of fetal cells with 47,XY,+21 karyotype in maternal peripheral blood. Hum Genet, 1992. 90(4): p. 368-70.
26. Ganshirt-Ahlert, D., et al., Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am J Reprod Immunol, 1993. 30(2-3): p. 194-201.
27. Bianchi, D.W., et al., PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet, 1997. 61(4): p. 822-9.
28. Hamada, H., et al., Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age. Hum Genet, 1993. 91(5): p. 427-32.
29. Reading, J.P., et al., Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations. Hum Reprod, 1995. 10(9): p. 2510-5.
30. Cheung, M.C., J.D. Goldberg, and Y.W. Kan, Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood. Nat Genet, 1996. 14(3): p. 264-8.
31. Bianchi, D.W., et al., Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn, 2002. 22(7): p. 609-15.
32. Covone, A.E., et al., Trophoblast cells in peripheral blood from pregnant women. Lancet, 1984. 2(8407): p. 841-3.
33. Bertero, M.T., et al., Circulating 'trophoblast' cells in pregnancy have maternal genetic markers. Prenat Diagn, 1988. 8(8): p. 585-90.
34. Hawes, C.S., et al., A morphologic study of trophoblast isolated from peripheral blood of pregnant women. Am J Obstet Gynecol, 1994. 170(5 Pt 1): p. 1297-300.
35. Schueler, P.A., et al., Inconsistency of fetal trophoblast cells in first trimester maternal peripheral blood prevents non-invasive fetal testing using this cell target. Placenta, 2001. 22(8-9): p. 702-15.
36. Hviid, T.V., S. Sorensen, and N. Morling, Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood. Prenat Diagn, 1999. 19(3): p. 271-8.
37. Tjoa, M.L., et al., Antibodies to trophoblast antigens HLA-G, placenta growth factor, and neuroD2 do not improve detection of circulating trophoblast cells in maternal blood. Fetal Diagn Ther, 2007. 22(2): p. 85-9.
38. Beroud, C., et al., Prenatal diagnosis of spinal muscular atrophy by genetic analysis of circulating fetal cells. Lancet, 2003. 361(9362): p. 1013-4.
39. Saker, A., et al., Genetic characterisation of circulating fetal cells allows non-invasive prenatal diagnosis of cystic fibrosis. Prenat Diagn, 2006. 26(10): p. 906-16.
40. Attwood, H.D. and W.W. Park, Embolism to the lungs by trophoblast. J Obstet Gynaecol Br Commonw, 1961. 68: p. 611-7.
41. Filges, I., et al., aCGH on chorionic villi mirrors the complexity of fetoplacental mosaicism in prenatal diagnosis. Prenat Diagn, 2011. 31(5): p. 473-8.
42. Holgado, E., et al., Incidence of placental mosaicism leading to discrepant results between QF-PCR and karyotyping in 22,825 chorionic villus samples. Prenat Diagn, 2011. 31(11): p. 1029-38.
43. Schroder, J. and A. De la Chapelle, Fetal lymphocytes in the maternal blood. Blood, 1972. 39(2): p. 153-62.
44. Lepez, T., et al., Fetal microchimeric cells in blood of women with an autoimmune thyroid disease. PLoS One, 2011. 6(12): p. e29646.
45. Lo, Y.M., et al., Culture of fetal erythroid cells from maternal peripheral blood. Lancet, 1994. 344(8917): p. 264-5.
46. Valerio, D., et al., Culture of fetal erythroid progenitor cells from maternal blood for non-invasive prenatal genetic diagnosis. Prenat Diagn, 1996. 16(12): p. 1073-82.
47. Chen, H., et al., Evaluating the culture of fetal erythroblasts from maternal blood for non-invasive prenatal diagnosis. Prenat Diagn, 1998. 18(9): p. 883-92.
48. Campagnoli, C., et al., Clonal culture of fetal cells from maternal blood. Lancet, 2001. 357(9260): p. 962.
49. Pearson, H.A., Life-span of the fetal red blood cell. J Pediatr, 1967. 70(2): p. 166-71.
50. Thomas, D.B. and J.M. Yoffey, Human foetal haemopoiesis. I. The cellular composition of foetal blood. Br J Haematol, 1962. 8: p. 290-5.
51. Huang, Z., et al., Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child. J Cell Biochem, 2011. 112(6): p. 1475-85.
52. Liou, J.D., et al., Fetal cells in the maternal circulation during first trimester in pregnancies. Hum Genet, 1993. 92(3): p. 309-11.
53. Huie, M.A., et al., Antibodies to human fetal erythroid cells from a nonimmune phage antibody library. Proc Natl Acad Sci U S A, 2001. 98(5): p. 2682-7.
54. Zheng, Y.L., et al., Search for the optimal fetal cell antibody: results of immunophenotyping studies using flow cytometry. Hum Genet, 1997. 100(1): p. 35-42.
55. Pembrey, M.E., D.J. Weatherall, and J.B. Clegg, Maternal synthesis of haemoglobin F in pregnancy. Lancet, 1973. 1(7816): p. 1350-4.
56. Papayannopoulou, T., E. Vichinsky, and G. Stamatoyannopoulos, Fetal Hb production during acute erythroid expansion. I. Observations in patients with transient erythroblastopenia and post-phlebotomy. Br J Haematol, 1980. 44(4): p. 535-46.
57. Olivieri, N.F., The beta-thalassemias. N Engl J Med, 1999. 341(2): p. 99-109.
58. Chung, S.W., et al., Human embryonic zeta-globin chains in adult patients with alpha-thalassemias. Proc Natl Acad Sci U S A, 1984. 81(19): p. 6188-91.
59. Sorensen, M.D., et al., Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis. J Biomed Biotechnol, 2009. 2009: p. 659219.
60. Simpson, J.L. and S. Elias, Isolating fetal cells from maternal blood. Advances in prenatal diagnosis through molecular technology. JAMA, 1993. 270(19): p. 2357-61.
61. Zheng, Y.L., et al., Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting. J Med Genet, 1993. 30(12): p. 1051-6.
62. Sohda, S., et al., The proportion of fetal nucleated red blood cells in maternal blood: estimation by FACS analysis. Prenat Diagn, 1997. 17(8): p. 743-52.
63. Troeger, C., et al., Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod, 1999. 5(12): p. 1162-5.
64. Hahn, S., et al., Allele drop-out can occur in alleles differing by a single nucleotide and is not alleviated by preamplification or minor template increments. Genet Test, 1998. 2(4): p. 351-5.
65. Samura, O., et al., Comparison of fetal cell recovery from maternal blood using a high density gradient for the initial separation step: 1.090 versus 1.119 g/ml. Prenat Diagn, 2000. 20(4): p. 281-6.
66. Ganshirt, D., et al., Enrichment of fetal nucleated red blood cells from the maternal circulation for prenatal diagnosis: experiences with triple density gradient and MACS based on more than 600 cases. Fetal Diagn Ther, 1998. 13(5): p. 276-86.
67. Bischoff, F.Z., et al., Cell-free fetal DNA and intact fetal cells in maternal blood circulation: implications for first and second trimester non-invasive prenatal diagnosis. Hum Reprod Update, 2002. 8(6): p. 493-500.
68. Bhat, N.M., M.M. Bieber, and N.N. Teng, One-step enrichment of nucleated red blood cells. A potential application in perinatal diagnosis. J Immunol Methods, 1993. 158(2): p. 277-80.
69. Sekizawa, A., et al., Improvement of fetal cell recovery from maternal blood: suitable density gradient for FACS separation. Fetal Diagn Ther, 1999. 14(4): p. 229-33.
70. Troeger, C., W. Holzgreve, and S. Hahn, A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS. Prenat Diagn, 1999. 19(6): p. 521-6.
71. Prieto, B., et al., Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient. Prenat Diagn, 2001. 21(3): p. 187-93.
72. Choolani, M., et al., Characterization of first trimester fetal erythroblasts for non-invasive prenatal diagnosis. Mol Hum Reprod, 2003. 9(4): p. 227-35.
73. Herzenberg, L.A., et al., Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Natl Acad Sci U S A, 1979. 76(3): p. 1453-5.
74. Price, J.O., et al., Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry. Am J Obstet Gynecol, 1991. 165(6 Pt 1): p. 1731-7.
75. Simpson, J.L. and S. Elias, Isolating fetal cells in the maternal circulation. Hum Reprod Update, 1995. 1(4): p. 409-18.
76. Zheng, Y.L., et al., Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: preliminary observations on maternal samples. Prenat Diagn, 1995. 15(10): p. 897-905.
77. Lewis, D.E., et al., Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry. Cytometry, 1996. 23(3): p. 218-27.
78. Wang, J.Y., et al., Fetal nucleated erythrocyte recovery: fluorescence activated cell sorting-based positive selection using anti-gamma globin versus magnetic activated cell sorting using anti-CD45 depletion and anti-gamma globin positive selection. Cytometry, 2000. 39(3): p. 224-30.
79. DeMaria, M.A., et al., Improved fetal nucleated erythrocyte sorting purity using intracellular antifetal hemoglobin and Hoechst 33342. Cytometry, 1996. 25(1): p. 37-45.
80. de Graaf, I.M., et al., Enrichment, identification and analysis of fetal cells from maternal blood: evaluation of a prenatal diagnosis system. Prenat Diagn, 1999. 19(7): p. 648-52.
81. Ganshirt-Ahlert, D., et al., Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood. Am J Obstet Gynecol, 1992. 166(5): p. 1350-5.
82. Busch, J., et al., Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis. Prenat Diagn, 1994. 14(12): p. 1129-40.
83. Borgatti, M., et al., New trends in non-invasive prenatal diagnosis: applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells. Int J Mol Med, 2008. 21(1): p. 3-12.
84. Loken, M.R., et al., Flow cytometric analysis of human bone marrow: I. Normal erythroid development. Blood, 1987. 69(1): p. 255-63.
85. Telen, M.J., Red blood cell surface adhesion molecules: their possible roles in normal human physiology and disease. Semin Hematol, 2000. 37(2): p. 130-42.
86. Durrant, L., et al., Non-invasive prenatal diagnosis by isolation of both trophoblasts and fetal nucleated red blood cells from the peripheral blood of pregnant women. Br J Obstet Gynaecol, 1996. 103(3): p. 219-22.
87. Zhong, X.Y., et al., High levels of fetal erythroblasts and fetal extracellular DNA in the peripheral blood of a pregnant woman with idiopathic polyhydramnios: case report. Prenat Diagn, 2000. 20(10): p. 838-41.
88. Valerio, D., R. Aiello, and V. Altieri, Isolation of fetal erythroid cells from maternal blood based on expression of erythropoietin receptors. Mol Hum Reprod, 1997. 3(5): p. 451-5.
89. Bianchi, D.W., et al., Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood. Prenat Diagn, 1993. 13(4): p. 293-300.
90. Campagnoli, C., et al., Noninvasive prenatal diagnosis. Use of density gradient centrifugation, magnetically activated cell sorting and in situ hybridization. J Reprod Med, 1997. 42(4): p. 193-9.
91. Andrews, K., et al., Enrichment of fetal nucleated cells from maternal blood: model test system using cord blood. Prenat Diagn, 1995. 15(10): p. 913-9.
92. Mohamed, H., J.N. Turner, and M. Caggana, Biochip for separating fetal cells from maternal circulation. J Chromatogr A, 2007. 1162(2): p. 187-92.
93. A. Manz, N.G., H.M. Widmer, Miniaturized total chemical analysis systems: A novel concept for chemical sensing. Sensors and Actuators B: Chemical, 1990. 1: p. 244–248.
94. Liu Lin , L.K., Hu Zhimin, Progress in Analytical Techniques of Microfluidic Chip on Cell Sorting. Chinese Journal of Cell Biology, 2013. 35: p. 727–733.
95. Kersaudy-Kerhoas, M., R. Dhariwal, and M.P. Desmulliez, Recent advances in microparticle continuous separation. IET Nanobiotechnol, 2008. 2(1): p. 1-13.
96. Pamme, N., Continuous flow separations in microfluidic devices. Lab Chip, 2007. 7(12): p. 1644-59.
97. Panaro, N.J., et al., Micropillar array chip for integrated white blood cell isolation and PCR. Biomol Eng, 2005. 21(6): p. 157-62.
98. Lee, D., et al., Separation of model mixtures of epsilon-globin positive fetal nucleated red blood cells and anucleate erythrocytes using a microfluidic device. J Chromatogr A, 2010. 1217(11): p. 1862-6.
99. Huang, R., et al., A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women. Prenat Diagn, 2008. 28(10): p. 892-9.
100. Takabayashi, H., et al., Development of non-invasive fetal DNA diagnosis from maternal blood. Prenat Diagn, 1995. 15(1): p. 74-7.
101. Sekizawa, A., et al., Prenatal diagnosis of Duchenne muscular dystrophy using a single fetal nucleated erythrocyte in maternal blood. Neurology, 1996. 46(5): p. 1350-3.
102. Sekizawa, A., et al., Fetal cell recycling: diagnosis of gender and RhD genotype in the same fetal cell retrieved from maternal blood. Am J Obstet Gynecol, 1999. 181(5 Pt 1): p. 1237-42.
103. Sin, A., et al., Enrichment using antibody-coated microfluidic chambers in shear flow: model mixtures of human lymphocytes. Biotechnol Bioeng, 2005. 91(7): p. 816-26.
104. Peng, W., H. Takabayashi, and K. Ikawa, Whole genome amplification from single cells in preimplantation genetic diagnosis and prenatal diagnosis. Eur J Obstet Gynecol Reprod Biol, 2007. 131(1): p. 13-20.
105. Camaschella, C., et al., Prenatal diagnosis of fetal hemoglobin Lepore-Boston disease on maternal peripheral blood. Blood, 1990. 75(11): p. 2102-6.
106. Watanabe, A., et al., Prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood. Hum Genet, 1998. 102(6): p. 611-5.
107. Samura, O., et al., Diagnosis of trisomy 21 in fetal nucleated erythrocytes from maternal blood by use of short tandem repeat sequences. Clin Chem, 2001. 47(9): p. 1622-6.
108. Poon, L.L., et al., Presence of fetal RNA in maternal plasma. Clin Chem, 2000. 46(11): p. 1832-4.
109. Lee, C.N., et al., Clinical utility of array comparative genomic hybridisation for prenatal diagnosis: a cohort study of 3171 pregnancies. BJOG, 2012. 119(5): p. 614-25.
110. Wachtel, S., et al., Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction. Hum Reprod, 1991. 6(10): p. 1466-9.
111. Abdul Vahab Saadi , P.K., PM Gopinath and Kapaettu Satyamoorthy, Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR) for Prenatal Diagnosis of Chromosomal Aneuploidies Int J Hum Genet, 2010. 10: p. 121-129
112. Hromadnikova, I., et al., Prenatal detection of trisomy 21 on nucleated red blood cells enriched from maternal circulation by using fluorescence in situ hybridization. Prenat Diagn, 2002. 22(9): p. 836-9.
113. de la Cruz, F., et al., Low false-positive rate of aneuploidy detection using fetal cells isolated from maternal blood. Fetal Diagn Ther, 1998. 13(6): p. 380.
114. Cacheux, V., et al., Detection of 47,XYY trophoblast fetal cells in maternal blood by fluorescence in situ hybridization after using immunomagnetic lymphocyte depletion and flow cytometry sorting. Fetal Diagn Ther, 1992. 7(3-4): p. 190-4.
115. Bischoff, F.Z., et al., Detection of low-grade mosaicism in fetal cells isolated from maternal blood. Prenat Diagn, 1995. 15(12): p. 1182-4.
116. Babochkina, T., et al., Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character. Haematologica, 2005. 90(6): p. 740-5.
117. Mergenthaler, S., et al., FISH analysis of all fetal nucleated cells in maternal whole blood: improved specificity by the use of two Y-chromosome probes. J Histochem Cytochem, 2005. 53(3): p. 319-22.
118. Elias, S., et al., First trimester prenatal diagnosis of trisomy 21 in fetal cells from maternal blood. Lancet, 1992. 340(8826): p. 1033.
119. Bischoff, F.Z., et al., Prenatal diagnosis with use of fetal cells isolated from maternal blood: five-color fluorescent in situ hybridization analysis on flow-sorted cells for chromosomes X, Y, 13, 18, and 21. Am J Obstet Gynecol, 1998. 179(1): p. 203-9.
120. Colls, P., et al., Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos. Reprod Biomed Online, 2012. 24(6): p. 621-9.
121. Gossett, D.R., et al., Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem, 2010. 397(8): p. 3249-67.
122. Park, J.M., et al., Fully automated circulating tumor cell isolation platform with large-volume capacity based on lab-on-a-disc. Anal Chem, 2014. 86(8): p. 3735-42.
123. Cima, I., et al., Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives. Biomicrofluidics, 2013. 7(1): p. 11810.


連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top