Immunohistological Staining Patterns Research Articles

Fibrillary glomerulonephritis mimicking membranous nephropathy – A diagnostic pitfall

Kidney biopsies in 2 females with nephrotic syndrome were suggestive of membranous nephropathy at routine light microscopy and immunohistochemistry. Electron microscopy on re-embedded paraffin tissue, however, revealed that the light microscopic pattern was due to a fibrillary glomerulonephritis with a dominant membranous manifestation. These findings suggest that (a) fibrillary glomerulonephritis may be mis-/under-diagnosed at light microscopy; and (b) in reality, a subset of therapy refractory membranous nephropathies might represent fibrillary glomerulopathies. Therefore, electron microscopy is mandatory in any case of membranous nephropathy with therapy refractory nephrotic syndrome or an unusual immunohistological staining pattern, e.g. with mesangial immunoreactivity.

Merkel cell carcinoma: evaluation of KIT (CD117) expression and failure to demonstrate activating mutations in the C‐KIT proto‐oncogene – implications for treatment with imatinib mesylate

Merkel cell carcinomas (MCCs) express the tyrosine kinase receptor KIT. However, it is not known if MCCs have activating mutations in KIT that would make them responsive to treatment with imatinib mesylate. The purpose of this article is to describe the KIT immunohistological staining pattern (CD117) of MCCs and analyze those MCCs for mutations in areas of KIT where mutations are found in gastrointestinal stromal cell tumors. We evaluated KIT immunostaining in nine MCCs from nine patients. In addition, we extracted DNA from the same MCCs, performed PCR amplification of C-KIT exons 9, 11, 13, and 17, and sequenced those gene products for mutations. Eight of nine (88.8%) MCCs expressed KIT. No mutations were found. The majority of MCCs express KIT but do not contain activating mutations in exons 9, 11, 13, or 17 of KIT. Imatinib mesylate is unlikely to provide effective therapy in MCC unless activating mutations in other areas of KIT or activating mutations in other related genes can be detected.

Differential Expression of MART-1, Tyrosinase, and SM5-1 in Primary and Metastatic Melanoma

The new monoclonal antibody SM5-1 has been shown to have significant advantages in immunohistochemistry of melanoma over currently used antibodies such as HMB-45 or anti-S100. In this study we compared the immunohistological staining pattern of SM5-1 with that of the more recently described antibodies A103 (anti-MART-1) and T311 (anti-Tyrosinase) in 344 paraffin-embedded melanoma specimens, consisting of 101 primary melanomas (77 SSM, 16 NM, 6 ALM, 2 LMM) and 243 melanoma metastases. The overall reactivity of SM5-1 for all the specimens was 92% (318/344) compared with 83% (285/344) for MART-1 and 71% (245/344) for Tyrosinase. Staining of melanoma metastases with SM5-1 was found in 91% (222/243), but only in 77% (187/243) with A103 and 63% (154/243) with T311, respectively. Staining with SM5-1 was more homogenous with 196 of 243 (80%) of metastatic lesions showing 50% or more positively stained cells within the lesions, whereas A103 and T311 did so in 141 of 243 (58%) or 117 of 243 (48%) of the lesions. With regard to staining intensity of SM5-1, 157 of 243 (64%) showed a strong or very strong staining intensity, whereas A103 and T311 did so in 85 of 243 (35%) or 70 of 243 (29%) of the lesions. Staining intensity and percentage positivity correlated well for SM5-1, because from the 58 very strong positive metastases 55 showed staining in more than 75% of the cells within a lesion. Importantly, 52 of 56 MART-1-negative metastases and 81 of 89 Tyrosinase-negative metastases were positive for SM5-1. Thirty-eight metastases (15.6%) were negative for both A103 and T311. Of those, 35 (92.1%) were positive for SM5-1, demonstrating the value of SM5-1 in identifying melanoma-associated antigen-negative lesions. We conclude that SM5-1 could be of value in immunohistochemistry of melanoma.

Processing of cathepsins L, B and D in psoriatic epidermis.

Proteinase activity is increased in psoriatic epidermis. To elucidate the involvement of enzymes in psoriatic epidermis, the expression of cathepsins, L, B and D was investigated by Western blotting and immunohistological studies. Normal epidermis contained abundant inactive precursors (39 kDa) of cathepsins L and B and an inactive intermediate form (45 kDa) of cathepsin D. Cathepsin L in psoriasis was processed to a variable extent from the precursor to a single-chain form (30 kDa) and a mixture of single- and heavy-chain (25 kDa) forms of the active mature enzyme, corresponding to the immunohistological staining patterns 'diffuse dense', 'small granular', and unevenly distributed 'condensed granular'. Cathepsin B showed a mixture of precursor form (39 kDa) and single-chain (30 kDa) forms and was expressed as a 'diffuse dense' staining pattern in the mid-spinous layer and as a 'condensed' pattern in the upper spinous and granular layers. Cathepsin D was processed to the heavy-chain (31 kDa) form of activated mature enzyme with small granular staining and a mixture of heavy-chain and degraded protein (28 kDa) with larger and more condensed granular staining. The distribution patterns of 'small granular' cathepsin L, and of cathepsins B and D expression in suprabasal keratinocytes were very similar to that of involucrin. After complete clinical resolution of psoriasis by 8-methoxypsoralen plus UVA treatment, the expression of the three cathepsins was normalized. These results suggest that cathepsins L, B and D in forms activated to a variable extent may be involved in the pathology of psoriasis.

Effects of heat shock on the pattern of fibronectin and laminin during somitogenesis in Xenopus laevis.

Heat shock causes partial disruption of the segmentation pattern during somitogenesis in Xenopus as well as in other vertebrates. However, Xenopus undergoes a different type of somite formation than that of most vertebrates: Somites are formed by rotation of cell blocks out of the paraxial mesoderm. We attempted to determine whether or not the segmentation disorder following heat shock is caused by an altered pattern of fibronectin and/or laminin, that could then effect the rotation of cell blocks. Therefore, we carried out heat shock experiments and analyzed the distribution of both ECM proteins in correlation to the position of somitic cells. Our results reveal that heat shock causes an incorrect deposition of fibronectin as well as laminin during somite formation. This leads to an intermingling of cells from different segments and to an anchorage of cells at the lateral matrix. Immunoblots show that the defects in the pattern of these ECM proteins do not correlate with a decrease of both proteins. However, immunohistological staining patterns demonstrate that oversized blocks of 20-cell width, instead of the normal ones of about 9-cell width, are separated out of the paraxial mesoderm following heat shock treatment. This indicates that the altered pattern of fibronectin and laminin might be a secondary effect caused by incorrect segregation and detachment of cell blocks during somitogenesis. Since anchorage of somitic cells is mostly affected by the altered distribution of fibronectin and laminin, it is more likely that both ECM proteins function in anchorage of migrating presomitic cells and in maintaining of segment borders rather than in stimulating cell rotation movements.

Expression of carbohydrate epitopes L2/HNK-1 and L3 in the larva and imago of Drosophila melanogaster and Calliphora vicina

The carbohydrate epitopes L2/HNK-1 and L3 belong to two overlapping families of adhesion molecules in the vertebrate, and probably the invertebrate nervous systems. To investigate their pattern of expression during the development of insects, cryosections of late third instar larvae and imagoes of Drosophila melanogaster and Calliphora vicina were studied by indirect immunofluorescence using several monoclonal antibodies to the L2/HNK-1 and one monoclonal antibody to the L3 epitope. Each monoclonal antibody to the L2/HNK-1 epitope showed a different immunohistological staining pattern, which differed from that of the L3 monoclonal antibody. In both insect species the immunohistological staining patterns for the two carbohydrate epitopes were similar at the two developmental stages, with immunoreactivity not confined to the nervous system. In larvae, immunoreactivities of the monoclonal antibodies L2.334 and L3.492 were predominantly associated with the extracellular matrix as indicated by co-localization with laminin, particularly in the imaginal discs, while L2.349 revealed a more cell surface-associated distribution. In imagoes, immunoreactivities were detectable in most organs studied.

Characterization of an activity-dependent, neuronal surface proteoglycan identified with monoclonal antibody Cat-301

Monoclonal antibody Cat-301 was previously shown to recognize a surface-associated antigen on subsets of mammalian CNS neurons whose expression is regulated by neuronal activity early in an animal's postnatal life. We now present the partial purification and characterization of the Cat-301 antigen and demonstrate that it is a chondroitin sulfate proteoglycan. Extracellular localization of the Cat-301 epitope is demonstrated by staining live, intact neurons in situ. Extraction of the antigen from membranes in the absence of detergent indicates that it is either a peripheral membrane protein or a component of an extracellular matrix. The Cat-301 antigen migrates on Western blots of SDS gels with a molecular weight of ∼680,000 dalton and is purified by DEAE chromatography and Sepharose gel filtration in 8 M urea (pH 4.9) buffer. The antigen is sensitive to chondroitinase ABC, indicating that it is a chondroitin sulfate proteoglycan. Furthermore, we provide strong evidence that the biochemically characterized antigen is indeed the histologically detected species by using a second antibody, Cat-304, that produces, immunohistological staining patterns identical to those of Cat-301 and reacts with the purified antigen, but at a distinct epitope. Our earlier developmental findings and the present localization and biochemical results suggest that the antigen may play a role in the maturation of functional connections between neurons, perhaps through stabilization of axosomatic and axodendritic synapses.

In situ immunological characterization of the infiltrating cells in positive patch tests

Skin biopsies from positive allergic patch tests were analysed by immunoenzymatic labelling of frozen sections with monoclonal antibodies. In seventeen patients the cellular infiltrate consisted of T cells admixed with Langerhans cells/indeterminate cells, but in two patients there were also many B lymphocytes. The B cells were accompanied by dendritic reticulum cells forming B-cell follicles, indistinguishable from those of normal and hyperplastic lymph nodes. There was no correlation between these two immunohistological staining patterns and the sensitizing antigen, the extent of local reaction or the time from epicutaneous application of allergen to examination (2 to 16 days). The ratio between T-helper and T-suppressor cells varied considerably, and showed no correlation with these variables. In all patients the infiltrating T cells expressed HLA-DR antigen. Transferrin receptors were identified on the infiltrating T cells in biopsies from nine patients. These data indicate activation of T cells in the infiltrate from positive patch tests, and support the functional significance of Langerhans cells in the initiation and maintenance of cutaneous contact allergy. An involvement of B cells and B-cell accessory cells in the pathogenesis of contact allergic reactions is also suggested. The presence of dendritic reticulum cells in skin infiltrates from positive patch tests may reflect a functional implication of the skin in the development of B-cell memory.

Patterns of chronic liver disease in Kuwait with special reference to localisation of hepatitis B surface antigen.

Two hundred and fifty six consecutive liver biopsy specimens (without secondary malignancy) collected over five years were reviewed to characterise the pattern of liver diseases encountered in the Kuwait region. A relatively high proportion of chronic active hepatitis (19%) and cirrhosis (40%) was found. Localisation of HBsAg was carried out by the histochemical orcein method and the immunohistological peroxidase-antiperoxidase (PAP) procedure. The PAP technique was superior to orcein both in quality and quantity in addition to its specificity. Three immunohistological staining patterns were observed: diffuse pancytoplasmic , partial perinuclear, and peripheral cytoplasmic. The positivity rate of HBsAg in chronic hepatitis was 29% and 27%, in all cases of cirrhosis. The results of immunohistology and serology of HBsAg were compared in 52 patients in whom both tests were carried out; almost one third of chronic active liver diseases were positive by both methods. Our data clearly show the sensitivity of immunohistology and its value in detecting HBsAg, especially in retrospective studies where serology is not always available. Additionally, the data show that hepatitis B infection is often associated with the development of chronic liver disease in Kuwait.

The diversity of the immunohistological staining pattern of Sternberg-Reed cells.

Lymph node tissue of eight cases of Hodgkin's disease of all different subtypes was studied with an immunoperoxidase technique for the detection of immunoglobulin G (IgG), J chain, kappa and lambda light chains, and alpha-1-antitrypsin in different types of Sternberg-Reed cells. It was found that L&H type Sternberg-Reed cells of the nodular lymphocyte predominance type contained IgG, J chain, and one type of light chain per individual cell. It is concluded that these findings indicate that L&H type Sternberg-Reed cells produce IgG and, consequently, are B immunoblasts. Typical and lacunar type Sternberg-Reed cells of mixed cellularity and nodular sclerosis subtypes were found to contain IgG and both types of light chains per individual cell. J chain was absent from these cells are alpha-1-antitrypsin was found in some of them in a paranuclear pattern, comparable to that in histiocytes. It is concluded that these findings exclude the production of IgG by these types of Sternberg-Reed cells and it is suggested that these Sternberg-Reed cells may be related to histiocytes on the basis of the similarity in the staining pattern for alpha-1-antitrypsin.