Intensity of Smoothelin Immunohistochemical Staining in Bladder Muscularis Mucosae Is Strongly Dependent on Antigen Retrieval – Positive Staining Is Not a Reliable Marker for Muscularis Propria
C Lindh, R Nilsson, MA Lindstrom, L Lundin, G Elmberger. Karolinska University Hospital, Stockholm, Sweden
Background: Distinguishing bladder muscularis propria (MP) from muscularis mucosae (MM) is crucial in bladder cancer staging. Immunohistochemical staining for Smoothelin is reportedly a robust marker in staging bladder cancer since it strongly stains MP muscle but not MM muscle. While performing validation studies of the smoothelin antibody in our laboratory we noted that staining intensity varied with pretreatment protocol. This study was conducted to investigate the variation of Smoothelin staining with different IHC pretreatment protocols.
Design: Smoothelin expression in MP and MM was evaluated by IHC in 18 cystectomy specimens. For each specimen three different IHC pretreatment protocols were used: Protocol 1 (enzyme dilutant), Protocol 2 (HIER in acidic buffer (pH 6)) and Protocol 3 (HIER in alkaline buffer (pH 9)) before immunostaining with Smoothelin (R4A, 1:100 dilution). Smoothelin IHC staining intensity in MM and MP was then graded as absent (0), weak (1+), moderate (2+) or strong (3+).
Results: Optimal Smoothelin expression in the MP was achieved using pretreatment protocol 3 (moderate-strong staining in 18/18 (100%) of the cases) but in this group there was also a moderate-strong staining of the MM in 11/18 (61%) of the cases. Protocol 1 and 2 pretreatment resulted in generally weaker staining for both the MP and MM.
|Intensity||MM (%)||MP (%)|
|Protocol 1 (enzyme dilutant)|
|Absent||0||17/18 (94)||14/18 (78)|
|Weak||1+||1/18 (6)||4/18 (22|
|Moderate||2+||0/18 (0)||0/18 (0)|
|Strong||3+||0/18 (0)||0/18 (0)|
|Protocol 2 (HIER in acidic buffer)|
|Absent||0||10/18 (56)||1/18 (5,5)|
|Weak||1+||7/18 (39)||10/18 (56)|
|Moderate||2+||1/18 (5)||6/18 (33)|
|Strong||3+||0/18 (0)||1/18 (5,5)|
|Protocol 3 (HIER in alkaline buffer)|
|Absent||0||1/18 (6)||0/18 (0)|
|Weak||1+||6/18 (33)||0/18 (0)|
|Moderate||2+||7/18 (39)||1/18 (6)|
|Strong||3+||4/18 (22)||17/18 (94)|