Profile of intracytoplasmic lysozyme in normal tissues, myeloproliferative disorders, hairy cell leukemia, and other pathologic processes. An immunoperoxidase study of paraffin sections and smears.

Intracytoplasmic lysozyme (muramidase) may be readily identified in paraffin sections of tissues fixed in formalin or Zenker's acetic acid and in smears of peripheral blood or bone marrow using an immunoperoxidase technique. Sites of intracellular lysozyme in normal human tissues and in various specimens from patients with myeloproliferative and lymphoproliferative disorders, hairy cell leukemia, granulomatous diseases, toxoplasmic lymphadenitis, and other pathologic processes were defined by this method. Intracellular lysozyme was demonstrated in mature and immature neutrophilic and eosinophilic myeloid cells, in monocytic cells, and in some types of histiocytes and had a limited distribution in normal tissues. The neoplastic cells of hairy cell leukemia were devoid of intracytoplasmic lysozyme. Identification of intracellular lysozyme, as determined by the immunoperoxidase technique, was compared with various cytochemical methods, particularly chloroacetate esterase and alpha-naphthyl butyrate esterase studies, for detection and characterization of myeloid cells, monocytes, and histiocytes.     

Intracellular hemoglobin-a specific marker for erythroid cells in paraffin sections. An immunoperoxidase study of normal, megaloblastic, and dysplastic erythropoiesis, including erythroleukemia and other myeloproliferative disorders.

Intracellular hemoglobin represents an excellent marker for specific characterization of normal, megaloblastic, or dysplastic erythroid cells in paraffin sections. Using an immunoperoxidase indirect sandwich technique for detection of intracellular hemoglobin, erythroid cells at all stages of maturation were readily identified in bone marrow biopsies (58 specimens total) with a) normal erythropoiesis, b) megalobastic erythropoiesis, and c) various myeloproliferative disorders, including erythroleukemia. In other tissues (6 spleens, 2 lymph nodes, 1 liver) with extramedullary hematopoiesis, erythroid cells were similarly defined on the basis of this immunohistochemical method. Initial fixation in Zenker's-acetic acid solution (employed for bone marrow biopsies), B5 solution, or formalin, appeared equally effective in preserving the antigenicity of intracellular human hemoglobin. This sensitive and specific immunoperoxidase technique for erythroid cell characterization is particularly applicable to tissues with abnormal erythropoiesis, in which precise cell identification generally presents a diagnostic problem.     

Leu-M1 immunoreactivity and phaeochromocytoma.

The aim was to evaluate Leu-M1 immunoreactivity as a prognostic factor in phaeochromocytoma. Anti-Leu-M1 monoclonal antibodies were used to determine the Leu-M1 immunoreactivity in 17 histologically confirmed phaeochromocytomas from 15 patients, using an avidin-biotin technique. Ten patients had a sporadic phaeochromocytoma, and five had multiple endocrine neoplasia type 2A (MEN 2A). Malignancy was diagnosed in three patients by the presence of metastases. Leu-M1 immunoreactivity was shown in 12 (70.5%) phaeochromocytomas. Three patterns of arrangement were observed: isolated (scattered positive cells) (n = 3); focal (aggregates of positive cells) (n = 5), and diffuse patterns (dispersed positive cells) (n = 4). Two cases of malignant phaeochromocytoma were positive (one focal and one isolated pattern). All cases of MEN 2A showed immunoreactivity, although no characteristic pattern was prevalent. A diffuse pattern was observed in all phaeochromocytomas longer than 7 cm. In conclusion, Leu-M1 expression is frequent in phaeochromocytoma. However, Leu-M1 immunoreactivity seems to be useless in predicting malignant behaviour and to be influenced mainly by tumour size.     

Leu-M1--a marker for Reed-Sternberg cells in Hodgkin''s disease. An immunoperoxidase study of paraffin-embedded tissues.

Monoclonal antibody to Leu-M1, a granulocyte-related differentiation antigen, represents a highly effective reagent for detection of diagnostic Reed-Sternberg (R-S) cells and variants in paraffin-embedded tissues of Hodgkin's disease. In 69 of 73 cases of Hodgkin's disease (41 nodular sclerosis, 25 mixed cellularity, 4 lymphocyte predominance, and 3 lymphocyte depletion types), R-S cells were strongly immunoreactive for Leu-M1. Four cases of lymphocyte predominance Hodgkin's disease (nodular) were uniformly nonreactive for Leu-M1. In most of the positive cases (57/69, 83%), the majority (60-90%) of R-S cells and variants exhibited immunoreactivity for Leu-M1. A characteristic staining pattern included granular and/or vesicular cytoplasmic immunoreactivity, often with a prominent globular paranuclear reaction product, and membrane staining with highly irregular cytoplasmic borders. Evaluation of B-cell (37 specimens), T-cell (20 specimens), and true histiocytic (3 specimens) neoplasms and a case of mastocytosis revealed immunoreactivity for Leu-M1 only in 1 B-cell and 4 T-cell malignancies. The staining patterns in these cases, however, clearly differed from that observed for R-S cells. Studies of nonneoplastic lymphoid tissues (38 total) demonstrated that lymphoid cells were typically nonreactive; histiocytes revealed variable reactivity for Leu-M1. Occasional histiocytes of the sinusoidal network of lymph nodes, particularly in toxoplasmic lymphadenitis, exhibited a staining pattern (membranous/cytoplasmic/paranuclear) similar to that observed for R-S cells. Leu-M1 represents a potentially helpful diagnostic discriminant in the assessment of Hodgkin's disease and its distinction from non-Hodgkin's lymphomas and other lymphoid proliferations.     

Immunostaining for leukocyte common antigen using an amplified avidin-biotin-peroxidase complex method and paraffin sections. A study of 735 hematopoietic and nonhematopoietic human neoplasms

Antibodies to leukocyte common antigen (LCA) are valuable in surgical pathology in the diagnostic separation of malignant lymphomas from poorly differentiated neoplasms of other types. However, several publications have reported difficulty in obtaining adequate LCA labeling in paraffin-embedded specimens. To assess this problem further, we applied an amplified version of the avidin-biotin-peroxidase complex procedure to 315 formaldehyde-fixed hematopoietic malignancies, and 420 nonhematopoietic tumors that had been similarly processed. All non-Hodgkin's lymphomas were LCA-reactive with this method, whereas none of the nonhematopoietic neoplasms were stained. Twelve of 25 cases of Hodgkin's disease displayed LCA-positivity in Reed-Sternberg cells, but all contained reactive, benign inflammatory cells. Hence, the specificity of the amplified anti-LCA/avidin-biotin-peroxidase technique was 100%, and its sensitivity was 96%. This procedure should allow surgical pathologists to obtain reliable, reproducible staining of hematopoietic neoplasms in paraffin-embedded tissues.     

Use of the immunoperoxidase method on paraffin sections

Using an indirect immunoperoxidase technique, the methods of fixation and treatment of paraffin sections were explored for subsequent identification of myosin of smooth and skeletal muscle cells, actin, filamin, and immunoglobulins. Bouin's fluid and ethanol were found to be the most adequate fixators for immunomorphological analysis. Pronase treatment of paraffin sections increased the intensity of specific reactions considerably. Incubation of paraffin sections with normal goat serum and hydrogen peroxide decreased nonspecific staining of the sections.     

Detection of terminal transferase in paraffin sections with the immunoperoxidase technique.

Terminal deoxynucleotidyl transferase (TdT) is an early T-cell differentiation marker in a number of species, including man. We have demonstrated TdT in the nuclei of cortical thymocytes in paraffin-embedded sections of calf, rat, and human thymus by indirect immunoperoxidase techniques. In addition, these techniques have been used to verify and extend enzyme assay results by detecting TdT in blast cells from 10 patients with convoluted T-cell lymphoma/leukemia, 1 patient with acute granulocytic leukemia, 1 patient with chronic granulocytic leukemia in blast crisis, and 1 patient with non-T non-B acute lymphocytic leukemia.     

An immunoperoxidase technique for demonstrating membrane localized HBsAg in paraffin sections of liver biopsies

An improved unlabelled antibody enzyme (PAP) method to demonstrate the localization of Hepatitis B surface Antigen (HBsAg) in liver cell membranes of paraffin embedded liver tissue, fixed in Bouin's solution, is described.     

Use of patients' sera for immunoperoxidase demonstration of infectious agents in paraffin sections.

Using patients' sera diluted from 1:10 to 1:1,000 as the primary antibodies in indirect immunoperoxidase staining, the authors visualized a variety of infectious agents in formalin-fixed, paraffin-embedded tissue sections. The target lesions included 1) pyoderma caused by Staphylococcus aureus, 2) cryptococcal infection, 3) dermal sporotrichosis, 4) colon ulcer caused by amebic dysentery, 5) cutaneous leishmaniasis, and 6) chronic liver abscess containing ova of Ascaris lumbricoides. The infectious agents were clearly identified in the respective lesions. Paraffin sections of other kinds of infectious lesions served as controls to clarify the specificity of the immunostaining. While the sera of patients with bacterial and fungal infection showed a wide range of cross-reactivity against bacteria and/or fungi, those with parasitic infection exhibited a relatively good specificity for the pathogen. Almost no immunoreactivity of endogenous human IgG in the paraffin sections was demonstrated under the conditions of this study. This approach can be used in diagnostic pathology, particularly when specific heteroantisera or monoclonal antibodies are unavailable.     

Detection of Leu-M1 immunoreactivity in brain tissue and brain tumors

Immunohistochemical investigations were made of 170 tumours of the central nervous system, using anti-Leu-M1, with electron microscopy being used on 22 of them. At least focal Leu-M1 positive reactivity was established from 10 in 24 astrocytomas, four in 22 oligodendrogliomas, and 9 in 15 ependymomas. Unambiguous Leu-M1 positivity was recorded from 54% of Grade I gliomas and 20% of Grade II gliomas. Other malignant primary tumours of neuroepithelial origin as well as all meningiomas and neurinomas proved to be Leu-M1 negative. However, severe immunopositivity was exhibited in all cases by reactive cerebral tissue adjacent to tumours. Three of four carcinoma metastases were Leu-M1 positive. Investigations, using electron microscopy, have clearly shown that MMA positivity in reactive brain is associated primarily with extracellular space and with plasma membranes of gliocytes. No products of immune reaction were identified, on the other hand, not even ultrastructurally, from neoplastically dedifferentiated cells of anaplastic neuroepithelial tumours. This is likely to suggest that neoplastically transformed gliocytes are no longer capable of expressing lacto-N-fucopentose III. It has proved helpful in distinguishing between the benign and malignant nature of a tumour. More observations along those lines might contribute to more knowledge on dedifferentiation of gliocytes. Also, in electron microscopy, MMA positivity of carcinomas proved to be associated with glycocalyceal material.     

Amylase as an additional marker of salivary gland neoplasms. An immunoperoxidase study

The presence of amylase in normal and neoplastic salivary gland tissue was investigated by immunoperoxidase techniques. Apart from normal and inflamed parotid glands, different kinds of tumours were studied with regard to amylase: acinic cell tumours, adenocarcinomas, adenoidcystic carcinomas, salivary duct carcinomas, mucoepidermoid tumours and squamous cell carcinomas. Amylase could be seen in acinic cell tumours, but not in other neoplasms. The results were discussed with respect to the diagnostic implications.     

Specific identification of intracellular immunoglobulin in paraffin sections of multiple myeloma and macroglobulinemia using an immunoperoxidase technique.

Paraffin-embedded tissues of 47 cases of multiple myeloma and 4 cases of macroglobulinemia were studied by the immunoperoxidase indirect sandwich technique for identification of intracytoplasmic immunoglobulin. Specific immunoglobulin with a single heavy and/or light chain was readily detected in the neoplastic cells. Studies were performed on iliac crest bone marrow biopsies which had been fixed and decalcified using Zenker's-acetic acid solution and on tissues from various sites which had been fixed in formalin. An excellent correlation was obtained between the specific immunoglobulin detected intracellularly in the plasma cells or lymphoplasmacytic cells and the monoclonal immunoglobulin found in serum or urine of these cases. The immunoperoxidase technique represents a sensitive and specific method for identifying intracellular immunoglobulin in paraffin-embedded tissues.     

Anti-CD34 immunoperoxidase staining in paraffin sections of acute leukemia: comparison with flow cytometric immunophenotyping.

Anti-CD34 is a monoclonal antibody that reacts with bone marrow progenitor cells and leukemic blasts, and is expressed on 30% to 50% of all acute leukemias. Detection of CD34 has previously been restricted to flow cytometric studies. To expand the utility of CD34, we immunostained 46 paraffin-embedded bone marrow specimens with acute leukemia; results were compared with flow cytometric studies. CD34 reactivity was also evaluated in nine chronic leukemia cases, 27 malignant lymphoma cases (Hodgkin's disease and non-Hodgkin's lymphoma), six normal bone marrow specimens, and three benign, hyperplastic lymph node specimens. All cases that were CD34 positive by flow cytometry (11 of 19 B-cell precursor acute lymphoblastic leukemia cases, one of six T-cell acute lymphoblastic leukemia cases, and seven of 21 acute myeloblastic leukemia cases) were also CD34 positive in paraffin sections. Both cell membrane and cytoplasmic staining was seen. The positivity percentage and fluorescence intensity by flow cytometry correlated with the estimated number of stained cells and the intensity of immunoperoxidase staining in 18 of 19 CD34-positive cases. The remaining bone marrow and lymph node cases studied were CD34 negative; prominent endothelial cell staining, however, was noted. This is the first report of anti-CD34 staining of acute leukemia in paraffin-embedded sections. In contrast to other monoclonal antibodies reactive in bone marrow paraffin sections with leukemia, anti-CD34 immunoperoxidase staining is limited to leukemic blasts and may provide useful diagnostic information when flow cytometric studies are not available.     

Expression of T/NK-cell and plasma cell antigens in nonhematopoietic epithelioid neoplasms. An immunohistochemical study of 447 cases

We studied the expression of CD2, CD3, CD4, CD5, CD7, CD8, CD56, and CD138 in 447 cases of common human neoplasms with epithelioid features. CD2, CD3, CD4, and CD8 antigens were detected in none of 447 cases of nonhematopoietic tumors. CD5 and CD7 antigens were expressed in 12.3% and 19.5% of cases of nonhematopoietic tumors, respectively. Their expression was found primarily in adenocarcinomas from the gastrointestinal tract, breast, and female reproductive organs. The high expression of CD5 and CD7 antigen in pancreatic ductal carcinoma and cholangiocarcinoma and high expression of CD7 in epithelioid sarcoma may have diagnostic value. One quarter of cases were positive for CD56. Overexpression of CD56 antigen was detected mainly in neuroendocrine tumors or adenocarcinomas with neuroendocrine differentiation. Its consistent overexpression in adrenal cortical and thyroid tumors may have diagnostic usefulness. Virtually all tumor types studied were CD138+ with a variable positivity rate. The negative staining of CD138 in malignant mesothelioma may be useful for separating mesothelioma from metastatic adenocarcinoma.     

Accelerated immunogold silver and immunoperoxidase staining of paraffin sections with the use of microwave irradiation. Factors influencing results

With the use of microwave irradiation, incubation times for the immunotechniques used in immunomicroscopy can be shortened significantly. The authors found that factors influencing results are irradiation time, power output, and presence of gold label. Precise temperature control is essential, and techniques to achieve this are discussed. For beta-human chorionic gonadotropin (beta-HCG), the results with the immunoperoxidase technique were inferior to those of the conventional technique, and with the immunogold silver technique an enhancement of labeling results was achieved. Immunogold silver staining for beta-HCG on paraffin sections can be performed with the use of microwave irradiation within one hour.     

Lymphoma immunotyping by paraffin immunoperoxidase and cell suspension methods--a comparative study

Immunoperoxidase staining incorporating an enzyme digestion step was performed on paraffin sections of 84 biopsy cases of lymphoproliferative disorders. Monoclonality was demonstrated in 100% of plasmacytomas and related tumours, and in 66% of non-Hodgkin's lymphomas. In 83% of lymphomas the immunoglobulin class was IgM and the light chain distribution was kappa 64% and lambda 36%. Polyclonality was found in 89% of cases of reactive lymphoid hyperplasia and within Reed-Sternberg cells in 55% of cases of Hodgkin's disease. Similar results were obtained by dispersed cell studies in 56 overlapping cases. The concordance rate between the two methods in 40 cases of non-Hodgkin's lymphoma was 67.5%. Reasons for the inconsistencies are discussed. Immunoperoxidase staining of enzyme digested paraffin sections is useful in the diagnosis of B cell lymphoproliferative disorders with a particular role in centres where cell suspension studies are not available or when there is no access to fresh tissue.     

Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity

Different protocols of protease (pepsin) treatment were compared in the immunostaining for intermediate filament (IF) proteins in formaldehyde-fixed and paraffin-embedded tissues. Without protease treatment, the immunoreactivity for all IF proteins was poor in such material. Appropriate pepsin pretreatment improved the immunoreactivity of formaldehyde-fixed tissues for all types of IF proteins, except for the 68 K neurofilament protein, which could not be immunostained with the antibody used. The optimal time for the pepsin treatment was varying in different tissues, and too long treatment caused progressive loss of immunostaining. Thus, for instance when demonstrating cytokeratins, renal adenocarcinomas were more sensitive to protease and needed a shorter treatment than other carcinomas. Therefore, a nonoptimal protease treatment protocol may cause false negative results and false cell type selective IF immunostaining. Prolonged fixation made it necessary to prolong the protease treatment. In tissues fixed up to four years in formalin, cytokeratin immunoreactivity could still be restored by a long pepsin treatment (up to 2-3 hours). For most tissues fixed for 24 hours in formaldehyde, an optimal protocol was the following: 0.05% pepsin (2 U/ml) in HCl, pH 1.8, at +37 degrees C for 20-30 minutes. The protease treatment did not produce false positive results. Alcohol-fixed material was good for IF immunostaining without any protease treatment, but such tissue blocks mostly lost the immunoreactivity during long term storage.     

Histomorphometry in paraffin sections of thyroid tumors.

Planimetric features of cell nuclei in paraffin-embedded histological sections of benign and malignant thyroid tumors, as well as normal thyroid tissue as control, were determined by means of a semiautomatic system. The main aim was to objectify possible quantitative differences between adenomas and carcinomas of the thyroid gland, which had recently been reported by several authors. For each nuclear profile, the area, the maximum diameter as well as two form factors were calculated. Statistical analyses of morphometric differences between normal controls, oxyphilic adenomas and carcinomas, and between follicular adenomas and carcinomas were performed using the T-test, a multivariate test, and a discriminant analysis. The tests revealed significant differences between controls and all other groups. The most striking result, however, was the total discrimination between follicular adenomas and carcinomas, with no false reclassification. Carcinomas had a higher mean nuclear area and diameter and a lower form factor. A similar reliability of discrimination could be obtained by comparing these morphometric values in oxyphilic adenomas and carcinomas. When using a test set of 9 cases (4 adenomas, 5 carcinomas), only one adenoma was falsely reclassified as a carcinoma by the discriminant analysis. Our results thus allow the conclusion that planimetric nuclear measurements indeed seem to be useful for the objectivation of cytomorphologic differences between adenomas and carcinomas of the thyroid gland.