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研究生：	張維志 Chang, Wei-Tsu
論文名稱：	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 Quality control on novel portable optical-based coagulation detector and reagent inventory
指導教授：	楊孔嘉 Young, Kung-Chia
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	124
中文關鍵詞：	全血凝血酶原時間、光學檢測、重點醫療照護檢驗、品質管理
外文關鍵詞：	whole blood prothrombin time, optical sensor, point-of-care testing, quality management
相關次數：	點閱：135 下載：0
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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。

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.

Chapter I: Introduction	1
Coagulation cascade and prothrombin time	2
Point-of-care test on coagulation test	3
Portable optical-based coagulation detector	4
Interferences of coagulation detection	5
Liposome contained in PT reagent might be effect by lyophilization	7
Laboratory reagent inventory management system	8
Aims of the study	9
1.	Interference analysis of optical-based coagulation detector	9
2.	Chip design optimization	9
3.	Developing a laboratory reagent inventory management system	9
Chapter II: Material and Method	11
Sample collection	13
Prothrombin time test	13
1.	Commercial thromboplastin	13
2.	Whole blood PT test by ACL TOP 700	14
3.	Whole blood PT test by portable optical-based coagulation detector	14
Interference analysis of portable optical-based coagulation detector	15
1.	Interference analysis	15
2.	Sample preparation of hematocrit interference test	16
3.	Sample preparation of hemoglobin interference test	16
4.	Sample preparation of triglyceride interference test	16
5.	Sample preparation of bilirubin interference test	17
6.	Sample preparation of daptomycin interference test	17
7.	Sample preparation of polybrene interference test	17
8.	Sample preparation of heparin	17
9.	Sample preparation of LMWH	18
10.	Ca2+ interference test	18
11.	Mg2+ interference test	19
12.	Zn2+ interference test	19
13.	Cu2+ interference test	19
14.	Mn2+ interference test	20
15.	Fe2+ interference test	20
16.	Fe3+ interference test	21
Liposome structure observation by cryo-electron microscopy	21
1.	Normal control sample	21
2.	Lyophilized sample	21
3.	Heated sample	22
4.	-80˚C treated sample	22
5.	Room temperature treated sample	22
6.	Cryo-electron microscopy	22
7.	Liposome diameter analysis	22
Heat tolerance of thromboplastin	23
Lyophilized-reagent chips PT detection on Portable optical-based coagulation detector	23
1.	Chip designs	23
2.	Lyophilized-reagent chip	24
3.	Performing lyophilized-reagent chips PT test on optical-based coagulation detector	24
4.	Reagent dialysis	25
5.	Saccharide adding	25
Laboratory inventory management system	25
1.	System design	25
2.	Cloud platform and application building	26
Chapter III: Results	27
Interference analysis	29
1.	Interference of hematocrit	29
2.	Interference of hemoglobin	30
3.	Interference of bilirubin	30
4.	Interference of daptomycin	30
5.	Interference of triglyceride	31
6.	Interference of polybrene	31
7.	Interference of heparin and low molecular weight heparin	32
8.	Interference of metal cations	32
Cryo-EM image analysis of PT reagent	33
1.	Structure and particle size of different condition treated liposome	33
2.	Thromboplastin activity after different condition treated	34
Heat tolerance of PT reagent	34
Designs of lyophilized-reagent chips	34
Reagent degas and changing lyophilization parameter stabilized the production of lyophilization	35
1.	Appearance	35
2.	Performance	36
Dialyzed PT reagent lyophilized chips	37
1.	Appearance	37
2.	Performance	37
Changing the components of the PT reagent	37
1.	Appearance	37
2.	Performance	38
Signal analysis	38
1.	Fake signal analysis	38
2.	Lyophilized-reagent chip signal analysis	39
Fetal bovine serum management system	39
1.	System design	39
2.	Automatically recording of the usage of FBS	40
3.	Reagent assignment by manager	40
4.	Account management	40
Chapter IV. Discussion	42
Interference of coagulation detection	43
1.	Hemolysis interference	43
2.	Neutralized the activity of heparin and low molecular weight heparin with polybrene	44
Tissue factor activity and phospholipid membrane	45
Factors that would interfere the product of lyophilization	46
Fake signal appeared in lyophilized-reagent chips	48
The practice of reagent management monitoring APP	49
Conclusion	49
References	52
Tables and figures	58
                                    

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