- 學位論文
利用分離血球細胞及修正stutter改善週邊血液幹細胞移植後chimarism分析之準確度
Isolation of blood cells and elimination of stutter improved accuracy of chimerism testing after peripheral blood stem cell transplantation
摘要
Chimerism(嵌合體) 是檢測接受異體因造血幹細胞移植後的病人體內存在的捐助者及受贈者細胞比例的重要分子生物學研究方法，可評估血液幹細胞移植後的復發風險，用來監測的捐贈者血液幹細胞的植入成功與否，評估後續治療或有無復發風險的診斷，已廣泛使用在分析血液幹細胞移植後追蹤監測的一個重要檢測工具，因此嵌合體分子檢測已成為一個在幹細胞移植後不可或缺的監測方法。 血液幹細胞移植後的病人，觀察其嵌合體情況，目前普遍利用PCR擴增短串聯重複序列（Short Tandem Repeats ,STR）方法檢測 ，是目前檢測嵌合狀態最靈敏的方法之一。在實驗反應過程中，嵌合體試驗經常受到許多因素的干擾，其結果可能會影響檢測受贈者/捐贈者的嵌合體比例，因而影響臨床判讀，本研究目的在探討是否末梢血液細胞及電泳時stuttrer peak之校正能取得更準確嵌合體結果。 本實驗由10位隨機選擇正常健康血液捐贈者，由週邊血液分離出單核球細胞與顆粒球細胞、T細胞做STR檢測，分析各種細胞的STR檢測結果，計算出正常人stutter peak平均值，以建立實驗室的正常值。並利用stutter peak正常平均值來校正病人嵌合體檢測結果，使幹細胞移植後病人之嵌合體檢測有更準確的結果。 對於疑似殘存受贈者細胞的檢體，經由細胞分離方法，利用T細胞分離做嵌合體檢測，及stutter peak方式修正結果，改善疑似殘存細胞造成的誤差，提供臨床上分析嵌合體結果的良好品質，對於了解異體造血幹細胞移植的成功與否有很大的幫助，並協助臨床醫師評估後續治療或有無復發的診斷。
並列摘要
Chimeric analysis is an important molecular method to evaluate the ratio of donor and recipient component after allogenetic stem cell transplantation. This analysis can monitor the condition of engraftment, evaluate and predict relapse of underlying disease. This method has been used widely as a mandatory monitoring tool after allogenetic stem cell transplantation. To analyze chimeric status, the most common and accurate method is polymerase chained reaction (PCR)-based analysis to amplify short tandem repeats (STR). However, lots of factors were noted to affect the ratio of donor and recipient and make the result inaccurate, one of these factors is stutter formation. The purpose of this study is to improve accuracy of chimeric analysis by separating T-lymphocyte and adjusting stutter peak. We collected peripheral blood samples from ten health donors. Then we separated lymphocytes, granulocyte and T-lymphocyte, each of these cells was used for chimeric analysis. In addition, we analyzed the result of amplification of STR of these cells and calculated the average value of stutter peak to establish the normal standard value. Then the normal standard value was utilized to adjust the result of chimeric analysis of patient to improve the accuracy of chimeric analysis after allogeneic stem cell transplantation. In conclusion, we improved the error caused by the residual cells by cell-separation method, performing chimeric study from T-cell and adjustment of stutter peak. Our method has offered excellent quality of chimeric analysis and helps clinician to evaluate post-transplantation status or prediction of disease relapse.