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系統識別號 U0026-2308201813234400
論文名稱(中文) 新型可攜式光學凝血測定儀開發與實驗室試劑庫存之品質管理
論文名稱(英文) Quality control on novel portable optical-based coagulation detector and reagent inventory
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 106
學期 2
出版年 107
研究生(中文) 張維志
研究生(英文) Wei-Tsu Chang
學號 T36054026
學位類別 碩士
語文別 英文
論文頁數 124頁
口試委員 指導教授-楊孔嘉
口試委員-傅子芳
口試委員-楊慶隆
召集委員-鄭如茜
中文關鍵字 全血凝血酶原時間  光學檢測  重點醫療照護檢驗  品質管理 
英文關鍵字 whole blood prothrombin time  optical sensor  point-of-care testing  quality management 
學科別分類
中文摘要 品質與效能管理在醫學檢驗領域一直都是非常重要的一個課題。根據美國食品及藥物管理局 (U.S. Food and Drug Administration) 的建議,在凝血測定醫療器材上市前,需要經過干擾物的分析測試來證明儀器的效能。本研究開發了一款新型可攜式光學儀儀器,用來偵測全血檢體的凝血酶原時間 (Prothrombin time, PT)。血液檢體中存在可能的干擾物質為:血比容、血紅素、膽紅素、三酸甘油酯、陽離子、抗生素、肝素、聚凝胺 (polybrene)等。在試片設計方面,PT試劑以冷凍乾燥 (Lyophilization) 的方式附著於試片流道內,與全血接觸時,其有效成分被血液溶解後觸發凝血反應,藉此達到量測PT的功能。文獻顯示,冷凍乾燥壓力可能會造成試劑內的微脂體結構受損,使試劑失去效能,透過低溫電子顯微鏡影像技術,可以觀察試劑內微脂體的結構;此外,同時評估試劑對於熱的耐受程度。世界衛生組織 (World Health Organization, WHO) 的品質管理手冊中建議,實驗室的效率及檢驗準確性,包括儀器的品質管理,及藥品及試劑的庫存管理;因應近年網路及行動裝置技術的進步,本研究針對實驗室藥品試劑庫存的管理提出了新的方法。研究結果顯示:(1) 在全血檢體中加入上述的干擾物,不會影響儀器偵測PT的結果;(2) 電子顯微鏡影像顯示試劑內的微脂體之結構並不會因為冷凍乾燥而受損;(3) 在熱耐受性方面,試劑在90˚C加熱30分鐘後仍保有活性;(4) 試劑凍乾試片的融解速度、與全血之混合度將是試片效能之關鍵,透過優化流道與試片的設計,期望可以增進凍乾試劑的溶解度以及與全血的混合度,進而產生可量測之PT訊號;(5) 透過行動裝置與雲端技術的結合,開發可以即時監控實驗室庫存的應用程式,使管理者及時地得知試劑的庫存及使用狀態,以使實驗室的庫存管理更有效率。
英文摘要 Quality management and efficiency has been considered as an important issue in laboratory diagnosis. As U.S. Food and Drug Administration (FDA) recommended, coagulation analyzer should avoid substance interference before becoming a product. In this study, the effects of potential interferents on a novel portable optical-based coagulation detector were examined, including hematocrit, hemoglobin, bilirubin, triglyceride, cation, antibiotic, heparin and polybrene. The coagulation trigger tissue factor was coated on the chips by lyophilization, which was reported to stress and disrupt the structure of the liposomes imposed during the process. By using cryo-electron microscopy (cryo-EM), the structure of liposome was clearly visualized. Besides lyophilization, heat tolerance of the reagent was also accessed by cryo-EM morphology and PT functional measurement. World Health Organization (WHO) quality management recommended that the supply and reagent inventory management system might facilitate the improvement of the efficiency and quality of diagnostic laboratories. Taking advantage of network and mobile devices, a new approach to manage the supply and reagent was established. The study results included that (1) the addition of interferents might not affect the PT measurement; (2) the structure of liposome remained intact after lyophilization; (3) The PT reagent remained active after heating at 90˚C for 30 minutes; (4) the rate of dissolving and the degree of mixing were the bottleneck for lyophilized-reagent chips to generate a detectable PT signal; (5) an application for real-time inventory management was invented, with the application allowing managers to monitor the inventory real-timely in more convenience and efficient way.
論文目次 Chapter I: Introduction 1
1. Coagulation cascade and prothrombin time 2
2. Point-of-care test on coagulation test 3
3. Portable optical-based coagulation detector 4
4. Interferences of coagulation detection 5
5. Liposome contained in PT reagent might be effect by lyophilization 7
6. Laboratory reagent inventory management system 8
7. Aims of the study 9
7.1. Interference analysis of optical-based coagulation detector 9
7.2. Chip design optimization 9
7.3. Developing a laboratory reagent inventory management system 9
Chapter II: Material and Method 11
1. Sample collection 13
2. Prothrombin time test 13
2.1. Commercial thromboplastin 13
2.2. Whole blood PT test by ACL TOP 700 14
2.3. Whole blood PT test by portable optical-based coagulation detector 14
3. Interference analysis of portable optical-based coagulation detector 15
3.1. Interference analysis 15
3.2. Sample preparation of hematocrit interference test 16
3.3. Sample preparation of hemoglobin interference test 16
3.4. Sample preparation of triglyceride interference test 16
3.5. Sample preparation of bilirubin interference test 17
3.6. Sample preparation of daptomycin interference test 17
3.7. Sample preparation of polybrene interference test 17
3.8. Sample preparation of heparin 17
3.9. Sample preparation of LMWH 18
3.10. Ca2+ interference test 18
3.11. Mg2+ interference test 19
3.12. Zn2+ interference test 19
3.13. Cu2+ interference test 19
3.14. Mn2+ interference test 20
3.15. Fe2+ interference test 20
3.16. Fe3+ interference test 21
4. Liposome structure observation by cryo-electron microscopy 21
4.1. Normal control sample 21
4.2. Lyophilized sample 21
4.3. Heated sample 22
4.4. -80˚C treated sample 22
4.5. Room temperature treated sample 22
4.6. Cryo-electron microscopy 22
4.7. Liposome diameter analysis 22
5. Heat tolerance of thromboplastin 23
6. Lyophilized-reagent chips PT detection on Portable optical-based coagulation detector 23
6.1. Chip designs 23
6.2. Lyophilized-reagent chip 24
6.3. Performing lyophilized-reagent chips PT test on optical-based coagulation detector 24
6.4. Reagent dialysis 25
6.5. Saccharide adding 25
7. Laboratory inventory management system 25
7.1. System design 25
7.2. Cloud platform and application building 26
Chapter III: Results 27
1. Interference analysis 29
1.1. Interference of hematocrit 29
1.2. Interference of hemoglobin 30
1.3. Interference of bilirubin 30
1.4. Interference of daptomycin 30
1.5. Interference of triglyceride 31
1.6. Interference of polybrene 31
1.7. Interference of heparin and low molecular weight heparin 32
1.8. Interference of metal cations 32
2. Cryo-EM image analysis of PT reagent 33
2.1. Structure and particle size of different condition treated liposome 33
2.2. Thromboplastin activity after different condition treated 34
3. Heat tolerance of PT reagent 34
4. Designs of lyophilized-reagent chips 34
5. Reagent degas and changing lyophilization parameter stabilized the production of lyophilization 35
5.1. Appearance 35
5.2. Performance 36
6. Dialyzed PT reagent lyophilized chips 37
6.1. Appearance 37
6.2. Performance 37
7. Changing the components of the PT reagent 37
7.1. Appearance 37
7.2. Performance 38
8. Signal analysis 38
8.1. Fake signal analysis 38
8.2. Lyophilized-reagent chip signal analysis 39
9. Fetal bovine serum management system 39
9.1. System design 39
9.2. Automatically recording of the usage of FBS 40
9.3. Reagent assignment by manager 40
9.4. Account management 40
Chapter IV. Discussion 42
1. Interference of coagulation detection 43
1.1. Hemolysis interference 43
1.2. Neutralized the activity of heparin and low molecular weight heparin with polybrene 44
2. Tissue factor activity and phospholipid membrane 45
3. Factors that would interfere the product of lyophilization 46
4. Fake signal appeared in lyophilized-reagent chips 48
5. The practice of reagent management monitoring APP 49
6. Conclusion 49
References 52
Tables and figures 58
參考文獻 Ahmed, I. S., Nafadi, M. M., & Fatahalla, F. A. (2006). Formulation of a fast-dissolving ketoprofen tablet using freeze-drying in blisters technique. Drug development and industrial pharmacy, 32(4), 437-442.
Almgren, M., Edwards, K., & Karlsson, G. (2000). Cryo transmission electron microscopy of liposomes and related structures. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 174(1-2), 3-21.
Ataullakhanov, F. I., Pohilko, A. V., Sinauridze, E. I., & Volkova, R. I. (1994). Calcium threshold in human plasma clotting kinetics. Thrombosis research, 75(4), 383-394.
Baheti, A., Kumar, L., & Bansal, A. K. (2016). Excipients used in lyophilization of small molecules. Journal of Excipients and Food Chemicals, 1(1), 1135.
Bedu-Addo, F. K. (2004). Understanding lyophilization formulation development. Pharmaceutical Technology, 20, 10-19.
Betageri, G. V., & Makarla, K. R. (1995). Enhancement of dissolution of glyburide by solid dispersion and lyophilization techniques. International journal of pharmaceutics, 126(1-2), 155-160.
Bom, V. J., & Bertina, R. M. (1990). The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII. Biochemical Journal, 265(2), 327-336.
Bom, V. J., & Bertina, R. M. (1990). The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII. Biochemical Journal, 265(2), 327-336.
Bozzuto, G., & Molinari, A. (2015). Liposomes as nanomedical devices. International journal of nanomedicine, 10, 975.
Briggs, C., Guthrie, D., Hyde, K., Mackie, I., Parker, N., Popek, M., ... & Stephens, C. (2008). Guidelines for point‐of‐care testing: haematology. British Journal of Haematology, 142(6), 904-915.
Brucato, C., Birr, C., Bruguera, P., Ruiz, J., & Sanchez-Martinez, D. (2000). U.S. Patent No. 6,100,072. Washington, DC: U.S. Patent and Trademark Office.
Butenas, S. (2013). Comparison of natural and recombinant tissue factor proteins: new insights. Biological chemistry, 394(7), 819-829.

Chaimoff, C., Creter, D., & Djaldetti, M. (1978). The effect of pH on platelet and coagulation factor activities. The American Journal of Surgery, 136(2), 257-259.
Chen, C., Han, D., Cai, C., & Tang, X. (2010). An overview of liposome lyophilization and its future potential. Journal of Controlled Release, 142(3), 299-311.
Davie, E. W., Fujikawa, K., & Kisiel, W. (1991). The coagulation cascade: initiation, maintenance, and regulation. Biochemistry, 30(43), 10363-10370.
Dimeski, G. (2008). Interference testing. The Clinical Biochemist Reviews, 29(Suppl 1), S43.
Edsall, J. T., & Lever, W. F. (1951). Effects of ions and neutral molecules on fibrin clotting. Journal of Biological Chemistry, 191(2), 735-756.
Ellis, V., Scully, M., & Kakkar, V. (1983). The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 747(1-2), 123-129.
Ellis, V., Scully, M., MacGregor, I., & Kakkar, V. (1982). Inhibition of human factor Xa by various plasma protease inhibitors. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 701(1), 26-31.
Fatah, K., & Hessel, B. (1998). Effect of zinc ions on fibrin network structure. Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis, 9(7), 629-635.
Favaloro, E. J., Funk, D. M., & Lippi, G. (2015). Pre-analytical variables in coagulation testing associated with diagnostic errors in hemostasis. Laboratory Medicine, 43(2), 1-10.
Filipe, V., Hawe, A., & Jiskoot, W. (2010). Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharmaceutical research, 27(5), 796-810.
Fiore, M. M., Neuenschwander, P. F., & Morrissey, J. H. (1994). The biochemical basis for the apparent defect of soluble mutant tissue factor in enhancing the proteolytic activities of factor VIIa. Journal of Biological Chemistry, 269(1), 143-149.
Grafmeyer, D., Bondon, M., Manchon, M., & Levillain, P. (1995). The influence of bilirubin, haemolysis and turbidity on 20 analytical tests performed on automatic analysers. Clinical Chemistry and Laboratory Medicine, 33(1), 31-52.
Gram, J., Mercker, S., & Bruhn, H. D. (1988). Does protamine chloride neutralize low molecular weight heparin sufficiently?. Thrombosis research, 52(5), 353-359.
Griffith, M. J., Beavers, G., Kingdon, H. S., & Lundblad, R. L. (1980). Effect of monovalent cations on the heparin-enhanced antithrombin III/thrombin reaction. Thrombosis research, 17(1), 29-39.
Gross, J., Sayle, S., Karow, A. R., Bakowsky, U., & Garidel, P. (2016). Nanoparticle tracking analysis of particle size and concentration detection in suspensions of polymer and protein samples: influence of experimental and data evaluation parameters. European Journal of Pharmaceutics and Biopharmaceutics, 104, 30-41.
Hoffmann, J. J. M. L., & Meulendijk, P. N. (1980). Evaluation of a heparin neutralizer. Thrombosis research, 18(6), 897-900.
Hole, P., Sillence, K., Hannell, C., Maguire, C. M., Roesslein, M., Suarez, G., ... & Cooke, L. (2013). Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA). Journal of nanoparticle research, 15(12), 2101.
Hull, R. D., Garcia, D. A., & Burnett, A. E. (2018) Heparin and LMW heparin: Dosing and adverse effects. UpToDate.
Ingvarsson, P. T., Yang, M., Nielsen, H. M., Rantanen, J., & Foged, C. (2011). Stabilization of liposomes during drying. Expert opinion on drug delivery, 8(3), 375-388.
James, M. F. M., & Neil, G. (1995). Effect of magnesium on coagulation as measured by thrombelastography. BJA: British Journal of Anaesthesia, 74(1), 92-94.
Jelis, E., Kristol, D., Arora, R. R., & Spillert, C. R. (2004, April). The effect of copper ion on blood coagulation. In Bioengineering Conference, 2004. Proceedings of the IEEE 30th Annual Northeast (p. 127). IEEE.
Kamal, A. H., Tefferi, A., & Pruthi, R. K. (2007, July). How to interpret and pursue an abnormal prothrombin time, activated partial thromboplastin time, and bleeding time in adults. In Mayo Clinic Proceedings (Vol. 82, No. 7, pp. 864-873). Elsevier.
Kroll, M. H., & Elin, R. J. (1994). Interference with clinical laboratory analyses. Clinical chemistry, 40(11), 1996-2005.
Laposata, M. (2001). Point-of-care coagulation testing: stepping gently forward.
Lawson, J. H., Butenas, S., & Mann, K. G. (1992). The evaluation of complex-dependent alterations in human factor VIIa. Journal of Biological Chemistry, 267(7), 4834-4843.
Lee, T. C., D'agostino, P. M., & Gorman, A. J. (2001). U.S. Patent No. 6,183,979. Washington, DC: U.S. Patent and Trademark Office.
Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and environmental microbiology, 61(10), 3592-3597.
Lever, R., Mulloy, B., & Page, C. P. (Eds.). (2012). Heparin-a century of progress (Vol. 207). Springer Science & Business Media.
Lippi, G., & Ippolito, L. (2014). Interference of spurious haemolysis on prothrombin time, activated partial thromboplastin time, and fibrinogen. New Zealand Journal of Medical Laboratory Science, 68(2), 51.
Lippi, G., Montagnana, M., Salvagno, G. L., & Guidi, G. C. (2006). Interference of blood cell lysis on routine coagulation testing. Archives of pathology & laboratory medicine, 130(2), 181-184.
Lippi, G., Plebani, M., & Favaloro, E. J. (2013, April). Interference in coagulation testing: focus on spurious hemolysis, icterus, and lipemia. In Seminars in thrombosis and hemostasis (Vol. 39, No. 03, pp. 258-266). Thieme Medical Publishers.
Lippi, G., Salvagno, G. L., Montagnana, M., Brocco, G., & Guidi, G. C. (2006). Influence of hemolysis on routine clinical chemistry testing. Clinical Chemistry and Laboratory Medicine (CCLM), 44(3), 311-316.
Long, W. F., & Williamson, F. B. (1982). Potentiation by calcium ions of the antithrombin III inhibition of thrombin. Biochemical and biophysical research communications, 104(2), 363-368.
Lundblad, R. L., Roberts, J. C., Johnston, D. K., Featherstone, G. L., & Griffith, M. J. (1981). Acceleration by calcium of the inactivation of thrombin by plasma proteinase inhibitors. Thrombosis research, 22(1), 135-146.
Luppa, P. B., Müller, C., Schlichtiger, A., & Schlebusch, H. (2011). Point-of-care testing (POCT): Current techniques and future perspectives. TrAC Trends in Analytical Chemistry, 30(6), 887-898.
Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., & Reynolds, J. A. (1981). Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry, 20(4), 833-840.
Muller, Y. A., Ultsch, M. H., & de Vos, A. M. (1996). The crystal structure of the extracellular domain of human tissue factor refined to 1.7 Å resolution. Journal of molecular biology, 256(1), 144-159.
Nair, C. H., & Dhall, D. P. (1991). Studies on fibrin network structure: the effect of some plasma proteins. Thrombosis research, 61(3), 315-325.
Nair, C. H., Shah, G. A., & Dhall, D. P. (1986). Effect of temperature, pH and ionic strength and composition on fibrin network structure and its development. Thrombosis research, 42(6), 809-816.


Nireesha, G. R., Divya, L., Sowmya, C., Venkateshan, N., Babu, M. N., & Lavakumar, V. (2013). Lyophilization/freeze drying—an review. International journal of novel trends in pharmaceutical sciences, 3(4), 87-98.
Olson, S. T., & Chuang, Y. J. (2002). Heparin activates antithrombin anticoagulant function by generating new interaction sites (exosites) for blood clotting proteinases. Trends in cardiovascular medicine, 12(8), 331-338.
Oshima, G., & Nagasawa, K. (1986). Effects of different ions on the interactions of heparin with bovine antithrombin III and thrombin. Thrombosis research, 42(3), 375-382.
Owen, W. G., Esmon, C. T., & Jackson, C. M. (1974). The conversion of prothrombin to thrombin I. Characterization of the reaction products formed during the activation of bovine prothrombin. Journal of Biological Chemistry, 249(2), 594-605.
Pai M., Crowther M.A. (2012) Neutralization of Heparin Activity. In: Lever R., Mulloy B., Page C. (eds) Heparin - A Century of Progress. Handbook of Experimental Pharmacology, vol 207. Springer, Berlin, Heidelberg
Palta, S., Saroa, R., & Palta, A. (2014). Overview of the coagulation system. Indian Journal of Anaesthesia, 58(5), 515–523.
Plebani, M. (2009). Does POCT reduce the risk of error in laboratory testing?. Clinica chimica acta, 404(1), 59-64.
Plug, T., & Meijers, J. C. (2016). Stimulation of thrombin-and plasmin-mediated activation of thrombin-activatable fibrinolysis inhibitor by anionic molecules. Thrombosis research, 146, 7-14.
Prabhu, S., Ortega, M., & Ma, C. (2005). Novel lipid-based formulations enhancing the in vitro dissolution and permeability characteristics of a poorly water-soluble model drug, piroxicam. International journal of pharmaceutics, 301(1-2), 209-216.
Preston, F. W., Hohf, R., & Trippel, O. (1956). The neutralization of heparin with polybrene. Quarterly Bulletin of the Northwestern University Medical School, 30(2), 138.
Ratnoff, O. D., & Potts, A. M. (1954). The accelerating effect of calcium and other cations on the conversion of fibrinogen to fibrin. The Journal of clinical investigation, 33(2), 206-210.
Reddy, L. H., & Ghosh, B. (2002). Fast dissolving drug delivery systems: A review of the literature. Indian journal of pharmaceutical sciences, 64(4), 331.
Sekiya, F., Yoshida, M., Yamashita, T., & Morita, T. (1996). Magnesium (II) Is a Crucial Constituent of the Blood Coagulation Cascade POTENTIATION OF COAGULANT ACTIVITIES OF FACTOR IX BY Mg IONS. Journal of Biological Chemistry, 271(15), 8541-8544.
Singh, P. (2001). U.S. Patent No. 6,248,353. Washington, DC: U.S. Patent and Trademark Office.
Smith, S. A., & Morrissey, J. H. (2004). Rapid and efficient incorporation of tissue factor into liposomes. Journal of Thrombosis and Haemostasis, 2(7), 1155-1162.
Tantanate, C., Teyateeti, M., & Tientadakul, P. (2017). Influence of plasma interferences on screening coagulogram and performance evaluation of the automated coagulation analyzer Sysmex® CS-2100i. Siriraj Medical Journal, 63(5), 151-156.
Tientadakul, P., Kongkan, C., & Chinswangwatanakul, W. (2013). Use of an automated coagulation analyzer to perform heparin neutralization with polybrene in blood samples for routine coagulation testing: practical, rapid, and inexpensive. Archives of Pathology and Laboratory Medicine, 137(11), 1641-1647.
Van der Pol, E., Coumans, F., Varga, Z., Krumrey, M., & Nieuwland, R. (2013). Innovation in detection of microparticles and exosomes. Journal of Thrombosis and Haemostasis, 11, 36-45.
Van Winden, E. C. (2003). Freeze-drying of liposomes: theory and practice. Methods in enzymology, 367, 99.
World Health Organization. (2011). Laboratory quality management system handbook. World Health Organization, Lyon, France.
Yamada, T., Kato, R., Oda, K., Tanaka, H., Suzuki, K., Ijiri, Y., ... & Tamai, H. (2016). False prolongation of prothrombin time in the presence of a high blood concentration of daptomycin. Basic & clinical pharmacology & toxicology, 119(4), 353-359.
Yang, C. L., Huang, S. J., Chou, C. W., Chiou, Y. C., Lin, K. P., Tsai, M. S., & Young, K. C. (2013). Design and evaluation of a portable optical-based biosensor for testing whole blood prothrombin time. Talanta, 116, 704-711.
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