Prognostic Significance of Lewis y Antigen in Resected Stage I and II Non-small Cell Lung Cancer: Capillary gap action
By capillary gap action (uptake or removal of a reagent resulting from the surface tension caused by two Probe-on Plus slides being paired together vertically), the excess blocking solution was removed and the primary monoclonal blood group antibody, BG-8 (murine IgM), which detects the Ley (type 2 chain) antigen, was used. The concentration is 0.125 mg/mL (Bio-genex; San Ramon, CA and Signet; Dedham, MA). The antibody was used at a dilution of 1:50 and was incubated at room temperature for 30 min. After each incubation or wash, the vertically paired slides were moved to a pad where the reagent could drain down between the slides.
The slides were then washed in 1 X Automation buffer (Biomeda) incubated sequentially with a 1:200 dilution of goat biotinylated anti-mouse IgM (Vector; Burlingame, CA) in hybrid diluent for 10 min at 42°C, washed in 1 X Automation buffer, and then incubated with 1:40 dilution of peroxidase-conjugated streptavidin (Biogenex) in hybrid diluent for 5 min at 42°C. The slides were then stained as specified using a liquid diaminoben-zidine substrate pack (Biogenex), washed in a solution of 25% absolute methanol in distilled water, counterstained with hematoxylin (Biomeda), dehydrated, cleared with xylene, and covered with a coverslip and a synthetic mounting media. Canadian health & care mall this A negative control slide was included in which 1 X Automation buffer was substituted for the primary antibody. Endothelium and red blood cells were used as internal positive control.
The sections were then evaluated by a pathologist (A.H.T.) without knowledge of patient outcome. The results were divided into five groups (0 through 4) by percentage of cells that were positive as follows 0, 0%; 1, < 25%; 2, 25 to < 50%; 3, 50 to < 75%; 4, 75 to 100%. Analysis was also performed by the intensity of the staining, which was found to be homogeneous and was not considered for the final analysis. Analyses were performed for each group individually (ie, percentage of positive cells 0, 1, 2, 3, 4) as well as combined (ie, 0 vs 1 through 4, 0 and 1 vs 2 through 4, etc). For final data analysis, we combined groups 0 through 2 and compared them with groups 3 and 4, since there was no difference in survival among all five groups (0 through 4). This divided the patients into two relatively equal groups. Therefore, cases with > 50% of cells staining were considered positive.