Immunoperoxidase Staining Patterns Research Articles

Balloon Cell Melanoma: Case Report

Balloon cell melanoma (BCM) is a rare histological variant of amelanotic melanoma. The differential diagnosis of clear cell lesion includes BCM, renal cell carcinoma, clear cell sarcoma as well as a number of histiocytic and infectious processes. We report a case of BCM in a 33 year‐old Indonesian man who presented with a non‐tender, freely movable, non‐pigmented left thigh nodule, which had been present for a year. On microscopic examination tumor consisted of a nodular infiltrate present within the dermis with superficial invasion of subcutaneous fat. Tumor cells formed nested aggregates were relatively bland in appearance and had abundant, clear cytoplasm, enlarged nuclei and prominent eosinophilic nucleoli. Rare mitotic figures were present within the deep portion of the neoplasm. No definitive nests were seen at the dermal epidermal junction or within the epidermis. Histochemical stains AFB, PAS/D, and GMS were negative. Immunoperoxidase studies were positive with antibodies to vimentin, S100, HMB45, CD68, and negative with cytokeratin, Factor XIIIA, and lysozyme. The diagnosis of BCMM was favored based on histological features, and immunoperoxidase staining pattern. We believe this case can provide additional information to help establish diagnostic criteria of this rare variant of melanoma.

Spindle-Cell Epithelioma: “Mixed Tumor” of the Vagina

Spindle-Cell Epithelioma: “Mixed Tumor” of the Vagina

Megakaryocyte c-Mpl expression in chronic myeloproliferative disorders and the myelodysplastic syndrome: immunoperoxidase staining patterns and clinical correlates.

The objectives of this study were to expand on recent observations that have suggested decreased thrombopoietin receptor (c-Mpl) expression in megakaryocytes of patients with polycythemia vera (PV) and agnogenic myeloid metaplasia (AMM). We applied an immunoperoxidase method with anti-c-Mpl antibody to 55 bone marrow sections from previously untreated patients with chronic myeloproliferative disorder (CMPD) or myelodysplastic syndrome (MDS). These included 8 patients with PV, 15 with AMM, 9 with essential thrombocythemia, 5 with chronic myelocytic leukemia, 9 with the 5q-syndrome and 9 with MDS with fibrosis. The findings were compared with those in four patients with reactive erythrocytosis (RE), six with immune thrombocytopenic purpura (ITP) and five normal controls. Staining intensity (SI) was moderate to strong both in normal controls and in patients with RE or ITP. In contrast, SI was weak in variable proportions of the megakaryocytes in every one of the aforementioned clonal myeloid disorders. The staining pattern (SP) was relatively uniform in MDS and heterogeneous in CMPD. Neither SI nor SP was significantly correlated with certain clinical or laboratory parameters. We concluded that altered megakaryocyte c-Mpl expression may be a nonspecific phenomenon in various subtypes of both CMPD and MDS. However, the characteristic staining patterns may complement the morphological distinction between clonal and reactive myeloproliferation.

Characterization of stage-specific tyrosinated α-tubulin immunoperoxidase staining patterns in Sertoli cells of rat seminiferous tubules by light microscopic image analysis

Microtubules are involved in many structural and functional changes that occur in Sertoli cells during the cycle of the seminiferous epithelium. However, few studies have addressed stage-specific changes in the distribution of microtubules that accompany the process of spermatogenesis. This study provides a stage-by-stage immunohistochemical evaluation of Sertoli cell microtubules in paraffin sections of whole rat testes using an antibody to tyrosinated α-tubulin. Microtubules that contain tyrosinated tubulin are considered to be less stable and are therefore expected to populate Sertoli cells undergoing dynamic changes during spermatogenesis. A quatitative method was developed to analyze the relative tyrosinated α-tubulin staining in different stages of the cycle. Immunostaining patterns of the stages were separated into five different groups. Stages VII–VIII had the least amount of tyrosinated α-tubulin, while stages IX–XI contained the greatest amount. The staining patterns were consistent with established structural changes in the seminiferous epithelium, such as the formation of ectoplasmic specializations, the presence of microtubule nucleation sites along the periphery of the apical cytoplasm, and the translocation of elongate spermatids from deep crypts in the Sertoli cell to the tubule lumen. These data should provide improved methods for the evaluation of microtubules in the study of Sertoli cell responses to environmental toxicants and testicular diseases.

Use of CD34 and Factor VIII To Diagnose Hepatocellular Carcinoma on Fine Needle Aspirates

The immunohistochemical staining patterns characteristic of hepatocellular carcinoma (HCC) using CD34 and factor VIII antibodies were compared with those of other hepatic lesions to determine if these stainings can be used as a diagnostic criterion. We reviewed 44 fine needle aspirates from the liver to evaluate the immunoperoxidase staining patterns on cell block preparations using CD34 and factor VIII and to determine whether this could distinguish HCC from the other lesions. These included HCC (14 cases), metastatic tumor (14 cases) and nonneoplastic liver lesions (16 cases). This retrospective study showed that in the nine documented cases of HCC, staining for CD34 and/or factor VIII was positive. The pattern of staining was either peripheral, around small clusters of tumor cells, or linear, diffuse and sinusoidal. In all the documented cases (27) of metastatic carcinoma and nonneoplastic lesions staining for CD34 and factor VIII was negative. In addition, there were eight problematic cases. In 7 cases the cytologic diagnosis on Papanicolaou-stained smears was inconclusive about HCC or metastatic carcinoma. CD34 and factor VIII confirmed the final diagnosis on the cell blocks. Based on this staining, 4 were HCC, 2 were metastatic carcinoma and 1 was equivocal, most probably HCC. In one case the differential diagnosis between well-differentiated HCC and a nonneoplastic liver lesion could not be made on cytologic smears, and here, also, CD34 and factor VIII aided in the correct diagnosis of a nonneoplastic liver lesion. We suggest immunoperoxidase staining with CD34 and factor VIII be performed on the cell block sections from FNAs in any problematic hepatic case.

CD11/CD18 and ICAM-1 Expression in a Murine Foreign Body Granulomatous Lung Model

The expression of leukocyte adhesion molecules (LeuCAM; CD11/CD18 family) and ICAM-1 was studied on bronchoalveolar lavage (BAL) cells and lung tissue sections from a mouse lung granulomatous model. When foreign body lung granuloma were induced in mice by intratracheal injection of 1.5 × 10 4 Sephadex G-50 beads, total and differential BAL cell counts demonstrated a maximal recovery of macrophages and neutrophils at 3 days. Indirect immunoperoxidase staining patterns indicated that intratracheal challenge with beads activated BAL cells and upregulated expression of CD11b, ICAM-1, and CD18 antigenic markers on macrophages and neutrophils when compared with BAL cells from saline controls. Immunohistochemical staining also confirmed the presence of CD11b on activated macrophages and neutrophils around the beads and of the ICAM-1 antigen in the lung parenchyma of bead challenged mice.

A rat monoclonal antibody specific for murine type 1 pneumocytes

A rat monoclonal antibody (MAb), 411-52, that binds specifically to murine pulmonary alveolar type 1 cells was developed. The cell-binding specificity of MAb 411-52 was assessed by light microscopy on immunoperoxidase-labeled tissue sections, electron microscopy on immunogold-labeled tissue blocks, and by flow cytometric analysis and fluorescence-activated cell sorting of immunofluorescently labeled cells enzymatically dissociated from murine lungs. The epitope recognized by MAb 411-52 was first detected in immunoperoxidase-stained sections of neonatal lungs of mice approximately 3 weeks after birth. In adult mice, the MAb 411-52-directed, immunoperoxidase-staining pattern was uniform throughout the lung parenchyma, was restricted to the luminal surfaces of alveoli, and was absent from type 2, endothelial, and interstitial cells, as well as from the epithelial cells of conducting airways. Electron microscopic analysis of immunogold-labeled lung tissue confirmed the type 1 cell binding specificity of MAb 411-52. Analysis by multiparameter, laser flow cytometry indicated that MAb 411-52 binds to 4.6 ± 0.5% (mean ± SD) of enzymatically dissociated cells from the lungs of normal adult mice. The absence of immunogold-labeling of type 2 cells suggested that the epitope recognized by MAb 411-52 might be a differentiation marker for the type 1 cell phenotype. With this MAb and standard immunohistochemical techniques, it is possible to visualize directly type 1 cells in paraffin sections.

Poorly differentiated carcinoma of unknown primary site: clinical usefulness of immunoperoxidase staining.

To assess the clinical utility of immunoperoxidase tumor-cell staining in patients with poorly differentiated carcinoma of unknown primary site, we performed a battery of stains on tumors from 87 patients treated between August 1978 and April 1983. Poorly differentiated carcinoma or poorly differentiated adenocarcinoma was diagnosed on the basis of light microscopic examination, and all patients were treated before the technology of immunoperoxidase staining was routinely used. Therefore, results of immunoperoxidase staining can be correlated with clinical outcome in this group of similarly treated patients with a long median follow-up. Immunoperoxidase staining confirmed the diagnosis of poorly differentiated carcinoma in 49 patients (56%) and yielded other diagnoses in 14 patients (16%): melanoma, eight; lymphoma, four; prostatic carcinoma, one; and yolk sac carcinoma, one. In 24 patients (28%) the immunoperoxidase staining pattern was inconclusive; electron microscopy was usually helpful in clarifying the diagnosis in these patients. Seventy-five patients (86%) received combination chemotherapy with a cisplatin-based regimen, and 24 patients (28%) had a complete response. Nine of these patients were later given specific diagnoses by immunoperoxidase staining (lymphoma, four; melanoma, four; yolk sac tumor, one). All patients with an immunoperoxidase diagnosis of lymphoma also had clinical features compatible with lymphoma and are long-term survivors. Patients with immunoperoxidase features suggesting melanoma were surprisingly responsive to chemotherapy, with three of seven complete responses and two long-term survivors. Patients with melanoma diagnosed by immunoperoxidase staining should not be excluded from a trial of cisplatin-based therapy. Immunoperoxidase staining is useful in the routine evaluation of metastatic poorly differentiated carcinoma of unknown primary site, as it can occasionally suggest the lineage of the tumor and have specific therapeutic implications.

The prognostic value of immunoperoxidase staining with monoclonal antibodies NCRC-11 and 3E1.2 in breast cancer

The variation in survival of women with clinically similar breast cancers may lead to difficulty in clinical management so it is important to recognise factors which indicate the prognosis. Immunoperoxidase staining patterns of primary breast tumours using monoclonal antibody NCRC-11 have been shown to relate to overall survival (Ellis et al., 1985) but the results have not been reproducible in other centres. In this study paraffin sections of 483 primary breast cancers were stained with NCRC-11 and 3E1.2 using an immunoperoxidase system. The tumour staining patterns were compared with overall survival using life tables and tested for relative prognostic significance by Cox's multivariate analysis. NCRC-11 related to survival in all 483 cases (chi 2 5.8, P = 0.02) but both antibodies achieved maximum prognostic significance in lymph node negative patients (chi 2 9.4, P less than 0.002 and chi 2 10.7, P less than 0.001) in whom no other factor was more significant. Immunoperoxidase staining patterns produced by monoclonal antibodies NCRC-11 and 3E1.2 are important prognostic factors in breast cancer.

Immunocytochemical evaluation of central nervous system tumors obtained by the cavitron ultrasonic surgical aspirator

The Cavitron ultrasonic surgical aspirator (CUSA) is a dissecting system that allows quick and effective removal of CNS tumors without traction or excessive manipulation of normal tissue. In this article, the immunoperoxidase staining patterns of cytology specimens obtained with the CUSA are compared with those from their corresponding resected surgical specimens employing a battery of monoclonal and polyclonal antibodies. Eleven cases of meningioma, three cases of glioblastoma multiforme, one astrocytoma, and two schwannomas were evaluated. In both CUSA cytologic biopsies and surgical biopsies, all the meningiomas showed strong staining for vimentin and epithelial membrane antigen, while two showed focal staining for cytokeratins. The glioblastoma multiforme and astrocytoma cases showed positivity for vimentin, S-100 protein, and glial fibrillary acidic protein, while the schwannomas stained positively for vimentin and S-100 protein. With only rare exceptions, the immunocytochemistry of the CUSA and surgical specimens correlated well in all of these cases in terms of strength of reaction and localization. There were no false-positive staining reactions in the CUSA material. This study suggests that reliable morphologic and immunoperoxidase studies can be performed on cytologic material obtained by the CUSA, which could aid in making an accurate and specific diagnosis of a variety of CNS tumors.

Distribution of HBcAg in hepatitis B detected by immunoperoxidase staining with three different preparations of anti-HBc antibodies.

To evaluate the role of the expression of hepatitis B core antigen (HBcAg) in liver cell damage the immunoperoxidase staining pattern of cryostat liver biopsy specimens from 16 chronic carriers of hepatitis B surface antigen (HBsAg) was investigated using three different kinds of anti-HBc antibodies. Polyclonal antibody prepared from recombinant HBcAg seemed to be more sensitive in detecting HBcAg than did monoclonal antibody from the same antigen. The topographical distribution of HBcAg detected by these two antibodies was similar, showing a close correlation to the histological activity of disease. Furthermore, the predominant localisation of cytoplasmic HBcAg usually reflected an active and severe ongoing hepatitis. On the other hand, monoclonal antibody prepared from purified Dane particles resulted in the prominent cytoplasmic staining for HBcAg regardless of histological severity of the hepatitis. The quantitative expression and topographical distribution of HBcAg depended on the type of anti-HBc antibodies used.

Immunohistochemical reactivity of a monoclonal antibody against cell membranes of human mammary carcinoma cells (125B4) with rat normal tissues and DMBA-induced mammary tumors.

Immunoperoxidase staining patterns with a monoclonal antibody against human breast cancer cell membrane, 125B4, were investigated in frozen sections of normal tissues from adult rats of both sexes and of various mammary tumors from 7, 12-dimethylbenz (α) anthracence (DMBA)-treated female rats. In normal tissues, 125B4 preferentially recognizes myoepithelial layers of the mammary, sweat and salivary glands, basally situated epithelial cells of the skin, vagina, esophagus, urethra, urinary bladder, seminal vesicle and epididymis, and endothelial cells of blood vessels. It does not react with luminal cells of the abovementioned organs, epithelial cells of the stomach, intestine, liver, pancreas and uterus, and mesenchymal cells. The reactivity in benign mammary tumors is comparable to that in normal mammary tissue, but it is observed on some adenoma cells scattered spottedly in proliferating fibroadenoma. In malignant mammary tumors, the reactivities are not restricted to the banally localized carcinoma cells and distributed abundantly in the carcinoma foci of squamous cells carcinoma, adenocarcinoma and carcinosarcoma. Neoplastic mesenchymal cells cannot be stained with 125B4, which stains endothelial cells within the tumor stroma. Except for the endothelium, therefore, our data indicate that this antibody may react to the antigens present in basal and myoepithelial cells of rat mammary parenchyma.

HLA-DR antigen expression on keratinocytes in highly inflamed parts of psoriatic lesions

We have investigated the immunoperoxidase staining pattern in the epidermis and dermal infiltrates of highly inflamed portions of psoriatic lesions, selecting for biopsy early pinpoint lesions or margins of active plaque lesions. We found positive intercellular staining for HLA-DR antigens in localized areas of the epidermis in about half of the patients tested. In contrast, OKT6 antigen was found only on the dendritic cells in the epidermis and dermis in all cases. These findings support the hypothesis that an active cellular immune reaction involving the epidermis, possibly associated with the expression of HLA-DR antigens on keratinocytes, occurs in the highly inflamed areas of psoriatic lesions, particularly in early pinpoint lesions or at the edges of spreading plaque lesions.

Localization of a Normal Prostatic Secretory Product Using the Monoclonal Antibody KR-P8

We previously reported that the monoclonal antibody KR-P8 detects a prostate organ-specific antigen that is distinct from other markers such as prostatic acid phosphatase and prostate specific antigen. In this report we demonstrate that the antigen recognized by KR-P8 is among the secretory products of both normal and malignant prostatic epithelium. Immunoperoxidase staining patterns showed that the antigen was concentrated on the luminal surfaces of the glandular epithelial cells of prostate, and also that the antigen was localized within secretory vacuoles of cells of the prostate tumor line PC3. In addition, using an immunoblotting assay the KR-P8 binding antigen was detected in the growth media of PC3 cells and also found to be present in human urine and seminal plasma. The data suggest that the antigen recognized by KR-P8 may be a useful marker for studying the secretory processes of the prostate gland.

HLA-DR antigen expression on the keratinocyte surface in dermatoses characterized by lymphocytic exocytosis (e.g. pityriasis rosea).

We have investigated the immunoperoxidase staining pattern of the epidermis in several dermatoses characterized by exocytosis of mononuclear cells into the epidermis. We found that HLA-DR antigens showed an intercellular distribution in localized areas of the epidermis in nine of ten cases of pityriasis rosea, and in all four cases of spontaneously regressing flat warts, two cases of pityriasis lichenoides chronica, two of Schamberg's disease, and one case of lichen striatus. Lichen planus and mycosis fungoides cases were used as positive controls. OKT6 antigen was recognized only on the dendritic cells of the epidermis in all these cases. Judging from the distribution of Langerhans cells, the epidermal intercellular HLA-DR antigen seems to be expressed on the keratinocytes in such diseases, and this feature was confirmed by immunoelectron microscopy. These findings support the hypothesis that the expression of HLA-DR antigen on keratinocytes in these dermatoses is linked to cellular immune reactions involving the epidermis.

Immunohistochemical studies on carcinoembryonic antigen in adenocarcinomas of the uterus.

In order to distinguish endocervical adenocarcinoma from endometrial adenocarcinoma, an immunoperoxidase stain for carcinoembryonic antigen (CEA) was tried. All of 10 endocervical adenocarcinomas revealed CEA and an adenosquamous carcinoma in the uterine cervix also showed it, while a mesonephroid adenocarcinoma in the uterine cervix did not. The immunohistochemical reaction products for the antigen were not observed in the glandular structures of 20 endometrial adenocarcinomas, although CEA was detected in all foci of squamous epithelial metaplasia occurred within 7 endometrial adenocarcinomas. CEA was detected in the endocervical type of glandular epithelium within a special endometrial adenocarcinoma containing predominantly endocervical type glandular epithelium. The immunoperoxidase staining pattern for CEA in the endocervical adenocarcinoma was related to the degree of histological differentiation of tumors, as follows; in the well differentiated glandular structure CEA was located on the luminal surface, while it was detected in the whole cytoplasm of tumor cells within the moderately and poorly differentiated areas. In conclusion, the immunoperoxidase stain for CEA would be useful for estimating malignancy of glandular structures within the uterus, distinguishing endocervical adenocarcinoma from endometrial adenocarcinoma, and grading of histological differentiation of endocervical adenocarcinoma.

Monoclonal antibodies identify novel neural antigens.

Monoclonal antibodies (Mabs) were raised against synaptic plasma membranes from rat cerebellum. The hybridomas were screened with a solid-phase immunoassay, the positive lines were characterized by their immunoperoxidase staining pattern on cerebellum, and the specific polypeptide antigens were identified on protein blots. Among the Mabs described are some that stain only neurons or only glia and others that react with specific parts of cells, such as axons, dendrites, and synapses. Many Mabs reveal novel relationships between antigens and the cells in which they occur. For example, a Mab designated 7D5 reacts with a family of greater than 30 proteins but stains only glial cells. Several Mabs stain punctate sites of synaptic size and distribution in the cerebellar cortex but each reacts with a different subset of polypeptides. One of the most restricted cytological staining patterns is given by 12D5, which stains punctate sites in the granular layer of the cerebellar cortex and reacts with a single polypeptide band of apparent Mr 270,000. These results illustrate the feasibility of raising Mabs that can be used to follow the expression of specific gene products during brain development.

An immunohistologic study of multiple myeloma and related conditions, using an immunoperoxidase method.

This study illustrates both some of the uses and the limitations of the immunoperoxidase method, and emphasizes the broad range of immunochemical findings that may be seen in B-cell neoplasia. Paraffin-embedded sections of bone marrow from 29 cases of various types of monoclonal gammopathy were examined by an immunoperoxidase method that permits demonstration of cytoplasmic immunoglobulin and yet preserves cellular morphology. In each case serial sections were stained for the presence of light and heavy chains, and the resulting staining pattern was compared with the serum and urinary immunoglobulin findings. In IgG and IgA monoclonal gammopathies and in macroglobulinemia there was good correlation between the observed pattern of cytoplasmic staining and the serum–urine monoclonal protein type. Anomalous staining patterns, in which there was no clear correlation between serum–urine monoclonal protein type and the immunoperoxidase staining pattern, were encountered in cases of IgD, light-chain and nonsecretory myeloma, and malignant lymphoma. Thus, using immunoperoxidase methods, antisera that clearly discriminate between the different immunoglobulin light and heavy chains in reactive plasma cell populations, and in the majority of cases of multiple myeloma, fail to recognize and distinguish the immunoglobulin components produced by certain neoplastic plasma cells. Several possible explanations for this observation are considered.