流式細胞儀在兔子胸腺瘤與胸腺淋巴瘤診斷之應用

Abstract

胸腺瘤與胸腺淋巴瘤為兔子縱膈腔腫塊常見的區別診斷，然而臨床診斷上卻有其困難，如：雖然組織病理是診斷的黃金準則，但胸腔的生檢採樣對兔子而言風險較高，而單獨細胞學檢驗有時卻又無法確診，如何使用侵入性較低的採樣方式來診斷此兩種腫瘤，是我們此次實驗研究的目的；目前流式細胞儀與核酸檢測已廣泛的運用在人類與犬隻的胸腺腫瘤診斷，此實驗嘗試檢驗流式細胞儀是否也可應用在兔子胸腺腫瘤的診斷，實驗中會在臨床胸腺腫塊案例進行細針採樣，於新鮮大體進行組織學採樣，樣本會在48小時內進行流式細胞儀的分析，檢驗其細胞膜表面的細胞標誌比例；臨床案例在死亡後會採其胸腺組織送病理學檢驗，並與先前流式細胞儀的結果作比對；在此次研究中，總共記錄了23個年齡分布於4至12歲的案例（平均年齡為7.7歲），其中11隻因缺乏最終診斷而被排除，剩餘的12隻中，有10隻被細胞學或組織病理學診斷為胸腺瘤（10/12, 83.3%），1隻被組織病理學診斷為B細胞淋巴瘤（1/12, 8.3%），1隻經組織病理學診斷為正常淋巴結組織（1/12, 8.3%）；此實驗中隨著時間推進，共衍生出4種版本的抗體組合，其中D版本為最新版本，在使用版本D抗體組合染色時，發現如同犬隻的研究結論，兔子的胸腺瘤樣本同時表現CD4與CD8標誌的細胞會占較高的比例(範圍：28.4-43.8%，平均：36.43%)，並且比對病理組織學、細胞學及流式細胞儀實驗結果，顯示此次流式細胞儀的抗體染色組合可以有效的區分出B細胞淋巴瘤，然而最新版的抗體組合是否能區分T細胞來源的胸腺淋巴瘤，未來仍需更多案例來佐證，並建立可信的參考值；此外，因類固醇的治療會影響組織病理學的結果，因此若臨床案例在生前無法採到組織送病理檢驗，則建議在死後再對組織進行一次流式細胞儀的檢驗。在我們的認知中，此次的實驗是首次針對兔子前縱膈腔腫瘤進行流式細胞儀診斷能力之探討，未來仍需更多的案例與研究來檢視此次的實驗結果，期望此次研究能對前縱膈腔腫瘤的患兔帶來實質幫助，並成為未來研究的基石。

Thymoma and thymic lymphoma are the top two differential diagnoses of mediastinal masses in rabbits. However, biopsy may be too invasive for rabbits and sometimes cytology alone is inconclusive. In dogs and humans, flow cytometry and PCR for antigen receptor rearrangement (PARR) had been used to differentiate mediastinal neoplasms routinely. To achieve the goal of diagnosing thymoma and thymic lymphoma with a less invasive approach in rabbits, we try to investigate the potential of flow cytometry to distinguish these two neoplasms in clinical cases. Fine needle aspiration samples (FNA) and surgical biopsy will be collected from visiting patients and carcass with mediastinal mass respectively, and samples would be tested by cytology and flow cytometry within 48 hours. The results of the tests would be compared with final diagnosis of histopathology once a patient passed away. In this study, total 23 cases aged from 4 to 12 years old (mean=7.7 years old) were documented. 11 of 23 cases without final diagnosis of cytology or histopathology results were excluded. 10 out of 12 cases (10/12, 83.3%) were diagnosed as thymoma by cytology (4/12, 33.3%) or histopathology (6/12, 50%). One case (1/12, 8.3%) was diagnosed as B-cell lymphoma, and one case (1/12, 8.3%) was diagnosed as a normal lymph node in histopathology. 4 versions of antibody panels were developed with time and the version D was the latest one. As the research in canine species, higher ratios of CD4+ and CD8+ co-expressing lymphocytes (range=28.4-43.8%, average = 36.43%) was noted in thymoma samples of rabbits by using version D antibody panels. Also, by matching histopathology diagnosis with flow cytometry and cytology, our study demonstrate that our flow cytometric panel is an effective tool for discriminating B-cell origin thymic lymphoma and thymoma. Nevertheless, more cases are still needed for establishing referable references and inspecting whether our version D antibody panels could distinguish between thymic T-cell lymphoma and thymoma. Furthermore, since corticosteroid could lead to histopathological change, a second time of flow cytometric test of necropsy samples was suggested in clinical cases, if the tissue sample was not available in patients’ lifetime. In our knowledge, this study was the first research focusing on mediastinal masses diagnosis via flow cytometry in rabbits. With more cases and research, we hope this technique and antibody panels could create practical benefits for rabbits suffering from mediastinal neoplasms and become a cornerstone for further research in the future.