Correlation of Regional Lymph Node in Vitro Antitumor Immunoreactivity Histology with Colorectal Carcinoma1

previous series (17); i.e., mean age was 61 years, 70 patients were male, 72 patients were female. All the patients had adenocarcinoma of the large bowel; anal carcinomas were excluded. The specimens were selected only insofar as they were received unfixed and yielded enough tissue to permit immunological testing in tissue culture of the regional lymph node lymphocytes against the primary tumor; histo logical sections were available from the same lymph nodes. These criteria were fulfilled in 200 lymph nodes from the 142 patients; in 126 cases the specimens were accompanied by a preoperative blood sample from which blood lymphocytes were separated and tested against the tumor cells as de scribed below. Specimen and blood collections were as described previously (12, 15). One of the lymph nodes as near the tumor as possible, usually adjacent to a draining mesenteric vein, was taken for immunological testing; in 58 cases, another lymph node was obtained as far from the tumor as possible, generally at the mesenteric resection line. Lymph nodes with macro scopic metastatic tumor deposits were not used. The dis tance between tumor and lymph nodes was recorded. One half of the lymph node was used for tissue culture testing, the other half was fixed in formalin as were other lymph nodes and representative tumor tissue for conventional his tological examination. In 25 cases, sufficiently large lymph nodes not less than 5 mm in diameter were trisected: 1 end section was snap-frozen in liquid nitrogen-isopentane slurry and used for staining with complement receptor markers (EAC')3 as described below; the midsection was used for conventional histological examination and assessment; the other end section provided cells for tissue culture cytotoxic ity tests. Suspensions of tumor cells, blood, and lymph node lym phocytes were prepared as described previously (12, 15). Briefly, tumor tissue and lymph nodes were teased gently in Tissue Culture Medium 199 containing 10% fetal calf serum and washed 3 times in medium. Blood lymphocytes were obtained from heparinized or EDTA-collected blood samples by sedimentation of the erythrocytes at 37°fol lowed by passage through a 2-cm glass wool column. All lymphocyte suspensions were washed 7 times with culture medium before use. Lymphocyte Cytotoxlcity Testing. A modification of the