臺灣博碩士論文加值系統

凝血酶原時間(PT)試驗，可以用來檢測凝血路徑中的外在途徑及共同途徑的凝血因子的功能是否正常，也可以用來監控口服抗凝劑病患之用藥劑量，以免服用過多而導致產生自發性出血。利用重點醫療照護試驗(POCT)，在病患身邊立即進行PT檢驗，可提供給口服抗凝劑的病人一個更有效率的方式來監控用藥情況。本篇實驗中設計出一個利用光學原理的可攜式POCT凝血偵測儀來偵測全血PT，並與自動化ACL TOP凝血分析儀作比較。設計的凝血偵測儀可記錄全血在凝血過程中透光度的改變，並利用一階微分找出凝血時透光度改變速度最快的時間點，定義它為PT時間。實驗結果顯示，全血PT經由調整過檢體體積之後，結果與血漿PT(r=0.996, p〈0.001, n=20)及ACL TOP plasma PT(r=0.980, p〈0.001, n=60)呈現高度相關性，同時也發現了當低血比容的全血(〈35%)，全血PT會顯著地縮短(n=32, 平均=-2.2±2.2s)，而正常血比容(36-50%)則差異小(n=28, 平均=-0.1±0.6s)。在儀器設計上，使用黑色壓克力製作成一個黑盒子來隔絕外在環境光的影響，並優化檢測區的流道高度，以及全血和反應試劑的比例。最後使用167個全血樣本，實際測試可攜式光學凝血偵測儀。結果顯示，在167個樣本當中，有153個檢體(91.6%)成功地測出全血PT時間，且所測得的結果與手工全血PT (r=0.985, p〈0.001)及自動化ACL TOP plasma PT (r=0.948, p〈0.001)的結果呈現高度相關性。此外，實驗中利用纖維蛋白原及完全血球計算的結果（包含白血球、紅血球、血紅素、血比容、平均血球容積、平均血球血紅素、平均血球血紅素濃度、紅血球分佈幅及血小板）與凝血圖形進行分析，發現了紅血球及纖維蛋白原會影響全血的凝血圖形:紅血球數量增加的聚集效應會使凝血曲線的轉折點變大，而纖維蛋白原的增加會使凝血時間的最大速度點顯著。另外，紅血球數量會造成全血與血漿凝血時間的不同，這就表示，使用全血凝血試驗可能可以更精確的預估活體內的凝血功能。綜合實驗結果：實驗中所計設的可攜式光學凝血偵測儀，可成功地檢測全血PT時間，並與自動化儀器結果呈現相關性。在臨床上也值得評估全血凝血對於凝血功能檢測的應用。

Prothrombin time (PT), testing the function of coagulation factors in the extrinsic and common pathways, is used to monitor the safe range of anticoagulants for avoiding spontaneous hemorrhage. Point-of-care testing (POCT) devices monitoring PT on-site might be much effectively benefit to the patients receiving anticoagulant therapy. In this study, a novel portable optical-based coagulation detector was designed for POCT whole blood (WB) PT test and was compared to the automatic ACL TOP coagulation analyzer. The portable coagulation detector detected the light transmittance of WB, which was decreased during coagulation process and the PT time was determined as the time of the maximum speed point from first-order derivative of coagulation curve. The results showed that the manual WB PT after the adjustment of the sample volume was highly correlated with plasma PT by either the manual method (r=0.996, p〈0.001, n=20) or the ACL TOP coagulation analyzer (r=0.980, p〈0.001, n=60). The result also showed that WB PT was significantly faster in the low (〈35%) hematocrit (HCT) samples (n=32 average difference=-2.2±2.2s) comparing to normal (36-50%) HCT samples (n=28 average difference=-0.1±0.6s). An acrylic black box encircled the electric circuit was made to reduce the environmental effect. Additionally, the parameters for testing chamber and the ratio of WB to reagent were optimized. Finally, 167 WB samples were tested with our portable WB coagulation detector. It showed that PT of 153 of 167 samples (91.6%) was determined successfully, and WB PT results obtained from portable coagulation detector were highly correlated with manual WB PT (r=0.985, p〈0.001) and ACL TOP plasma PT (r=0.948, p〈0.001). Fibrinogen (FIB) and complete blood count data, including white blood cell, red blood cell, hemoglobin, HCT, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width and platelet, were used to discover the substances in WB that affected the detection of WB coagulation. Only RBC and FIB were found to affect the pattern of coagulation curve. Increasing RBC number might enhance RBC aggregation which causes a more conspicuous turning point on coagulation curve, and increasing FIB enlarged the signal at maximum speed of clotting time. Moreover, the amount of RBC also cause the difference of PT between WB and plasma sample, which indicates WB coagulation test might be able to predict the coagulation function in vitro more precisely than plasma. In conclusion, the designed portable optical coagulation detector can determine the WB PT time, and the results were highly comparable with clinical reports. WB coagulation tests might be suitable for evaluation of coagulation function.

中文摘要 I
Abstract III
Acknowledgement V
Contents VII
Index of tables and figures IX
Abbreviations XI
Chapter I: Introduction 1
1. Coagulation pathway and hemostasis 2
2. Prothrombin time and other clinical coagulation tests 4
3. Point-of-care testing 6
4. Principle of detecting coagulation 9
5. Aim of this study 10
Chapter II: Materials and methods 13
1. Sample collection and clinical results 14
2. Prothrombin time test 14
2.1. Commercial thromboplastin 14
2.2. Plasma PT test by manual method 15
2.3. WB PT test 16
2.4. Modified WB PT 16
3. WB PT test with portable optical coagulation detector 17
3.1. Electric circuit and experimental setup 17
3.2. Coagulation chips and detecting area 18
3.3. Procedures 19
3.4. Determination of PT and interpretation of WB coagulation curve 19
4. Coomassie blue stain of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 20
5. Observation of RBC aggregation in micro-channel 21
6. Statistics 22
Chapter III: Results 23
1. Establishing WB PT test 24
2. Designing WB coagulation detector 26
2.1. Testing WB coagulation with designed optical coagulation detector 26
2.2. Optimization of coagulation curve 27
2.3. Optimization of light source and detector 28
2.4. Determination of PT clotting time 29
3. Comparing Dade Innovin and HemosIL Recombiplastin 30
4. Testing WB PTs of 167 samples with the portable optical coagulation detector 31
5. Discovery the effect of RBC in coagulation detection 34
Chapter IV: Discussion 37
1. Development of WB PT and portable coagulation detector 38
2. Difference between WB PT and plasma PT 39
3. The factors that affects] detecting WB coagulation with portable optical coagulation detector 40
3.1. RBC aggregation 40
3.2. Fibrinogen and platelet 42
4. Conclusion 43
References 45
Tables and figures 51

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