- 學位論文
微流道多通道光盤系統於血液檢測應用之研究
The Study of Blood Testing Using a System with Microfluidic Multi-Channel Discs
摘要
為了配合光盤式自動化血液試驗系統之開發，本研究目的在發展出適合微流道多通道光盤的血液檢測技術。首先，以U底微孔盤做為光盤血液試驗參數調整之載體，當紅血球 (RBC) 與抗體總體積縮減至7 μL時，仍可進行ABO/Rh D型血之分型，但卻不易以肉眼判讀血塊及價數。為了增加光盤的血液相容性，利用臭氧改質可將聚苯乙烯表面的親水性增加，溶血指數從3.47 % 降低為1.82 % ，血小板貼附率從5.4×104 顆/cm2降低至2.8×104 顆/cm2。檢驗親水性表面改質是否影響RBC及血清之凝集價數，結果發現親水性改質微孔盤對ABO/Rh D血型的凝集價數影響不大。在不規則抗體篩檢方面，親水性改質之微孔盤可略微提升聚乙烯亞胺 (PEI) 手工凝聚胺法 (MP) 對含有Mia+Dia抗體血清樣本的凝集價數，並使以低離子強度鹽水間接抗球蛋白試驗 (LISS-IAT) 檢驗含有C+e、Mia+Dia、E+Mia、E及Mia抗體血清樣本的凝集價數提升。利用離心式微流體監測系統檢測光盤各試劑由試劑槽通過毛細管閥之突破頻率，發現除了反應槽中的MP上清液會提早流入廢液槽外，其餘試劑槽之實際突破頻率均在預設轉速的誤差範圍內。以光盤式自動化血液試驗系統進行101組檢體之血液試驗，結果發現在ABO/Rh D型血鑑定方面，光盤法與試管法判讀一致性為100 %；其中，A型血佔22.8 %、B型血佔35.6 %、AB型血佔9.5 %、O型血佔32.7 %。Rh D型陽性血佔97.0 %、Rh D型陰性血佔2.0 %、Rh 弱D型血佔1.0 %。經由LISS-IAT試管法測得101組檢體中有7組含有不規則抗體，包括：1組c+e型血清、1組D+Fya+s型血清、1組Dia型血清、1組Mia型血清與2組Leb型血清；MP試管法無法檢測到1組Dia型血清；MP光盤法無法檢測到1組Dia型血清及2組Leb型血清。上述結果顯示LISS-IAT試管法檢測的靈敏度仍較其他方法為高，未來微流道光盤將朝向LISS-IAT光盤法開發，以提高微流道光盤在血液試驗的敏感度。
並列摘要
The purpose of the study was to develop blood testing procedures for microfluidic multi-channel discs during the development of a disc-type automated blood typing system for pre-transfusion testing. The blood testing parameters for optical discs were first obtained on U-bottom microplates. It was able to perform ABO/Rh D grouping with some difficulty to estimate the strength of blood agglutination by naked eyes when the mixture volume for red blood cells (RBC) and antibody decreased to 7 μL. In order to increase the disc's blood compatibility, hydrophilicity of the polystyrene surface was increased by ozone treatment. Hemolytic index reduced from 3.47 % to 1.82 % and platelet adhesion decreased from 5.4×104 cells/cm2 to 2.8×104 cells/cm2 after the treatment. Whether hydrophilic modification on the surface modified microplates affected the titer of serum and RBC agglutination was next determined and found no obvious difference in the strength of agglutination for ABO/Rh D typing. Hydrophilic modification slightly enhanced the strength of agglutination for test serum containing Mia+Dia antibody using manual polybrene (MP) technique with polyethyleneimine (PEI) and for serum samples containing C+e, Mia+Dia, E+Mia, E and Mia antibodies using low ionic strength saline- indirect antiglobulin test (LISS-IAT). The burst frequency for the reagent of each reservoir forcing through the capillary valve was examined by centrifugal microfluidic monitoring system and found that most of measured burst frequencies fell within the range of standard deviation for predicted velocities except for that of MP supernatant in the reaction chamber flowing into the waste chamber. Samples from 101 blood donors were tested by blood typing using the disc-type automated blood typing system and standard manual test tube method. There were 100% concordance for ABO/Rh D typing where 22.8 % of A, 35.6 % of B, 9.5 % of AB, 32.7 % O, 97.0 % of D+, 2.0 % of D- and 1 % weak D were found. Seven out of 101 sera samples with irregular antibodies detected by LISS-IAT tube method were anti-c and -e: 1; anti-D, -Fya and -s: 1: anti-Dia: 1; anti-Mia: 2; and anti-Leb: 2. One serum sample with anti-Dia was not detected by MP tube method. One with anti-Dia, and two with anti-Leb were not detected by MP disc method. The above results showed that LISS-IAT tube method had higher accuracy. Therefore, it is necessary to develop a microfluidic disc for LISS-IAT disc method in order to improve the sensitivity of disc-type blood typing.