臺灣博碩士論文加值系統

顆粒性大淋巴球 (large granular lymphocyte; LGL) 佔周邊血液單核細胞 (peripheral blood mononuclear cells; PBMC) 的10-15%。這些細胞是中到大型的淋巴球，細胞核偏到一邊，細胞質內有藍色顆粒。 PBMC的LGL中有85%是CD3表面抗原陰性的自然殺手細胞（natural killer cell），另外15%是具CD3表面抗原的cytotoxic T細胞。LGL數目升高可能是良性增生，也可能是白血病（血癌）。藉由分析免疫表現型 (immumophenotype) 的異常表現 (aberrant immunophenotype) 和T細胞受器 (T-cell receptor; TCR) 的基因重組 (gene rearrangement) 可鑑別診斷良性T細胞 LGL細胞增生或T細胞 LGL白血病 (T-LGL leukemia)。在本研究中，我們收集25個LGL淋巴球增生 (LGL lymphoproliferation) 的病例，有17個病例是T細胞LGL白血病 (T-LGL leukemia)，8個病例是T細胞LGL淋巴球增生 (reactive LGL lymphoproliferation)。在本研究中，我們用聚合酶連鎖反應 (polymerase chain reaction; PCR) 法來分析TCR基因重組及flow cytometry (FC) 定量TCR-Vβ chain基因重組 (FC-Vβ repertoire)的表現，並比較PCR及FC-Vβ repertoire 此二種技術的敏感度和特異性。分析結果顯示PCR技術的敏感度為94% (16/17)，特異性為75% (6/8)，陽性預測值(positive predictive value; PPV)為89%，陰性預測值 (negative predictive value; NPV)為86%。FC-Vβ repertoire技術的敏感度為 59% (10/17)，特異性為88% (7/8)，PPV為91%，NPV 為50%。這項研究發現PCR的敏感度優於FC-Vβ repertoire技術，但是FC-Vβ repertoire 的特異性優於PCR技術。此二種技術有互補作用,有助於臨床診斷LGL淋巴球增生 (LGL lymphoproliferation) 的病例。

Large granular lymphocytes (LGLs) are medium to large-sized lymphocytes with abundant cytoplasm, eccentric nuclei and presence of azurophilic cytoplasmic granules. These LGL cells account for 10-15% of peripheral blood mononuclear cells (PBMC); 85% of these cells are surface CD3-negative natural killer (NK) cells and 15% are surface CD3-positive cytotoxic T-cells. Increased LGL in peripheral blood might be reactive or neoplastic; and the differential diagnosis between benign and neoplastic could be achieved by determination of immunophenotypic aberrancy and/or polymerase chain reactive (PCR)-based clonality assay of the T-cell receptor (TCR) gene rearrangement. In this study, we collected the specimens from 25 patients with increased LGL count (or LGL lymphoproliferation) including 17 cases of T-cell LGL leukemia and 8 cases of reactive LGL lymphoproliferation. We compared the diagnostic sensitivity and specificity of PCR-based clonality study and flow cytometric immunophenotyping with TCR-Vβ repertoire (FC-Vβ repertoire). We found that PCR-based clonality assay had a high sensitivity for detecting T-LGL leukemia (16/17 cases; 94%) and a modest specificity (6/8 cases; 75%). The positive predictive value (PPV) was 89% and negative predictive value (NPV) was 86%. On the other hand, the sensitivity of FC-Vβ repertoire was lower at 59% (10/17 cases), but the specificity was relatively high at 88% (7/8 cases). The PPV and NPV of FC-Vβ repertoire were 91% and 50%, respectively. Our study showed that PCR-based clonality study was more sensitive but less specific than FC-Vβ repertoire for the diagnosis of T-cell LGL leukemia. These two techniques are supplemental to each other and are useful for diagnosis.

口試委員會審定書…………………………………………………………i
授權書………………………………………………………………………ii
中文摘要 …………………………………………………………………iii
英文摘要 …………………………………………………………………iv
誌謝 ………………………………………………………………………vi
第一章 緒論 ………………………………………………………………1
第一節 研究背景介紹 ……………………………………………………1
第二節 血液學特徵(Hematologic features) …………………………………2
第三節 臨床表現(Clinical Preseentation) ……………………………………3
第四節 細胞免疫分型(immunophenotype) ………………………………… 3
第五節 單源性( Clonality) ………………………………………………… 4
第六節 自體免疫疾病( Autoimmune Disorders) …………………………… 5
第七節 相關疾病……………………………………………………………5
第八節 研究動機……………………………………………………………6
第二章 材料與方法 …………………………………………………….. 8
第一節 檢體來源 ………………………………………………………… 8
1-1 病患來源與檢體採檢 …………………………………………8
1-2 細胞分離與配置細胞懸浮液……………………………………8
第二節 實驗方法…………………………………………………………… 9
2-1流式細胞儀免疫分型(FC immumophenotype)及TCR Vb repertoires分析…………………………………………………………………9
2-1.1 T細胞免疫分型(T-cell immumophenotype) ………………9
2-1.2 TCR-Vβrepertoires分析 …………………………………10
2-1.3流式細胞分析儀細胞圏選及分析 …………………………11
2-1.4 TCR-Vβ表型分析 ………………………………………12
2-2 T細胞受器(T-cell receptor; TCR) 的基因重組(gene rearrangement)分析…………………………………………………12
2-2.1抽取DNA使用QIAamp DNA Blood Mini Ki …………… 12
2-2.2 TCR-G conventional PC ……………………………………13
2-2.3 TCR BIOMED2 PCR ………………………………………14
第三節 統計方法 ………………………………………………………… 14
第三章 結果 …………………………………………………………… 15
第一節 病患實驗室數據 …………………………………………………15
第二節 T-LGL免疫分型異常表現(immunophenotypic aberrancy)數據 ……16
第三節 流式細胞分析TCR Vβ repertoires及T細胞受器的基因重組分析數據 …………………………………………………………………16
第四章 討論………………………………………………………………18
第五章 結論………………………………………………………………21
參考文獻 ……………………………………………………….……… 22
附錄 ……………………………………………………………………….25

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