Cultured human fibroblasts in agarose gel as a multi-functional control for immunohistochemistry. Standardization of Ki67 (MIBI) assessment in routinely processed urinary bladder carcinoma tissue

Immunohistochemistry (IHC) in clinical practice is hampered by lack of stan dardization and by subjectivity in interpretation and quantitation, This st udy aimed to develop a control system for IHC in routinely fixed and histop rocessed tissues. Such a system should be easy to handle in clinical practi ce and should reflect variations in fixation time, section thickness, secti on storage conditions, and staining protocols. In addition, in image analys is quantitation of immunostained tissues, when using classifiers computed o n IHC-control images, the control system should be very stable. Cultured hu man fibroblasts were suspended in agarose, transferred into a length of tub ing and stored at 4 degrees C, Three pieces of the cellgel control were sep arately fixed, histoprocessed, and paraffin-embedded as external controls. One piece was prepared together with each of 18 bladder carcinoma biopsies as internal controls. Slides with sections from the biopsy and all types of cellgel controls were stored at different temperatures and then stained us ing three different IHC protocols. The fibroblasts were homogeneously distr ibuted in the agarose gel. Variation in section thickness did not influence immunostaining as evaluated by the MIB1 labelling index (MIB1 LI). The ext ernal controls decreased notably in MIB1 LI with increased fixation time, T his was not seen in the 18 internal controls that were each fired with a fr esh biopsy. However, section storage and immunostaining conditions influenc ed the MIB1 expression equally in all control types and to a similar degree to the biopsies. Furthermore, colour-based image analysis quantitation of MIBI LI in biopsies proved stable and independent of the control type used to compute the classifier. Copyright (C) 2000 John Wiley & Sons, Ltd.