The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.

Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein expressed in cartilage, ligament and tendon. Mutations in the COMP gene cause two dominantly inherited skeletal dysplasias, pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia (MED/EDMI). We report on a novel point mutation D511Y in the seventh calcium-binding repeat of the COMP gene and the resulting iliac crest growth plate pathology. The PSACH iliac crest growth plate is comprised of a large region of resting chondrocytes above a narrow region composed of clusters of disorganized proliferative and hypertrophic chondrocytes. Chondrocytes in all zones show massive intracellular retention of COMP and the surrounding extracellular matrix is deficient in COMP. Moreover, the 511Y COMP mutation selectively affects type IX collagen as little is found in the growth plate matrix whereas type I1 collagen and aggrecan are abundant in the matrix. Chondrocyte remnants are observed in the chondrocyte clusters and dead cells are found throughout the growth plate. Apoptosis studies demonstrate an unusual pattern of TUNEL staining in the PSACH chondrocytes compared to the control growth plate. These in vivo findings support our previous observation that retention of COMP leads to chondrocyte death. These results also add to the increasing evidence that PSACH and E D M l are rER storage diseases and that impaired linear growth and joint erosion are caused by the disruptive effect of massive amounts of COMP within the chondrocytes.

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Purpose-Surprisingly little is known about the cellular composition of the temporomandibular joint (TMJ) disc, which is a crucial piece of the puzzle in tissue engineering efforts. Toward this end, cell types were identified and quantified regionally in the TMJ disc.Materials and Methods-Porcine TMJ discs were examined by histology, electron microscopy, and immunohistochemistry. Histology consisted of hematoxylin and eosin staining to identify regional variation of cell type and cell numbers. Transmission electron microscopy was used to elucidate differences in organelle content and pericellular matrix between TMJ disc cells and chondrocytes from hyaline cartilage. Immunohistochemistry was used to assess the presence of smooth and skeletal muscle character in the TMJ disc.Results-The overall ratio of fibroblasts to chondrocyte-like cells in the TMJ disc was approximately 2.35 to 1, with the highest relative number of chondrocyte-like cells in the intermediate zone. Electron microscopy revealed distinct differences between TMJ disc chondrocyte-like cells and chondrocytes from hyaline cartilage with respect to organelles and the