Flow Cytometric Detection of Micro Metastases in Lymph Nodes of Primary Lung Cancer

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15591476
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Thoracic surgery
• Research Institution: Yamaguchi University
• Principal Investigator: KANEDA Yoshikazu Yamaguchi University, University Hospital, Research Associate, 医学部附属病院, 助手 (70325223)
• Co-Investigator(Kenkyū-buntansha): UEDA Kazuhiro Yamaguchi University, University Hospital, Medical staff, 医学部附属病院, 医員(臨床) ; MURAKAMI Tomoyuki NHO Sanyo National Hospital, Clinical Research, Head, 臨床研究部, 形態研究室長 (20200272) ; SUGI Kazuro NHO Sanyo National Hospital, Clinical Research, Director, 臨床研究部, 部長 (70241271)
• Project Period (FY): 2003 – 2004
• Keywords: Detection of micro metastases / Flow cytometer / Primary lung cancer / In traoperative pathologic diagnosis

Research Abstract

In order to develop a new technique for detecting micro metastases of lymph nodes in NSCLC by Flow cytometry (FCM) we performed the following experiments and obtained the results from it.
1.Basic and technical studies
1)Cell dispersion method : Medimachine system ^<TM>(DAKO Cytomation) could collect more sufficient cells than routine scissors dispersion.
2)Cell membrane processing and staining properties : In cell membrane permeability processing, routine ethanol fixation was compared with TX method. TX method is cell membrane was destroyed by detergent (Triton X-100). The destruction of cell membrane by TX method was faster than routine ethanol fixation. Wash staining was compared with non-wash staining. Wash staining is centrifugal washing was repeated and non-wash staining was centrifugal washing was not done. By the use of TX method, wash staining was compared with non-wash staining. Non-wash staining could collect more sufficient cells than wash staining.
3)Detection Sensitivity : Nor mal lymphocytes were mixed with ABC-1 (cultured lung adenocarcinoma cell) in various ratios (0.1-0.001%). These samples were examined by the FCM. The FCM detected, at least, one cancer cells/ 10^6 of lymphocytes.
4)Optimal condition of triple staining (CK/ CEA/ DNA) : By the use of ABC-1 (cultured lung adenocarcinoma cell), optimal condition of triple staining (CK/CEA/DNA) was investigated and was revealed as the following conditions. The dispersed cells were fixed with 70% ethanol for 20 minutes and washed once with antibody diluent. The 800μl antibody diluent was added to the sediment. The half of the cell fliud was the control. Under the light shielding condition, the 400μl cell fluid was reacted with 50μl FITC-conjugated monoclonal mouse anti-human cytokeratin (DAKO, F0859) and 50μl PE-conjugated anti-CEA antibody (BD, 551478) for 20 minutes at the room temperature. The cell fluid was reacted with 50μl 7-AAD (BD, 559925) for 10 minutes at the room temperature.
2.Clinical usefulness : With the use of the resected lymph nodes from the patient with non-small cell lung cancer, The diagnosis of lymph node metastasis was compared between the routine pathological examination and the FCM. All of the lymph nodes that were positive by the pathological examination were also positive by the FCM. There is one lymph node that was positive by the FCM, but negative by the pathological examination.
3.Technical studies for intra-operative pathologic diagnosis : The quicker Medimachine system ^<TM> and the ultra-rapid flow cytometer is expected to be developed for the intra-operative pathologic diagnosis.