Effects of fixation and enzymatic digestion on the immunohistochemical demonstration of laminin and fibronectin in paraffin embedded tissue.

The effects of different fixatives and enzymatic digestion procedures on the immunohistochemical demonstration of fibronectin and laminin in paraffin embedded tissues have been compared. None of the fixatives tested enabled staining of these proteins without enzymatic digestion. No intracytoplasmic laminin was found either in fixed or in fresh frozen tissue. Fixation in formol acetic acid was unsatisfactory for demonstration of fibronectin; prolonged fixation in formol sublimate was unsatisfactory for demonstration of laminin. Optimal results were achieved after fixation in routine 10% formol saline. Trypsin was completely ineffective for unmasking laminin antigens except after fixation in ethanol acetic acid; it was only partially effective for showing fibronectin antigens. The best results were obtained with protease digestion, but pepsin was an adequate, although slightly less reliable, alternative. These enzymes may be used at lower concentrations than usually recommended.

Value of CD15 immunostaining in diagnosing Hodgkin's disease: a review of published literature.

The role of antibodies of CD15 as diagnostic markers of Hodgkin's disease was assessed from a review of the literature. A total of 571 cases of Hodgkin's disease and 386 cases of non-Hodgkin's lymphoma were included. The sensitivity of CD15 in detecting cases of Hodgkin's disease was 80% or 91% if cases of lymphocyte predominant Hodgkin's disease were excluded. The specificity of CD15 was only 80.6%, or in other words, 19.4% of cases of non-Hodgkin's lymphoma were CD15 positive. In an ideal test both the sensitivity and specificity would be 100% and if the test performance were no better than chance then they would both be 50%. It is concluded that CD15 immunostaining cannot be regarded as a sensitive or specific marker of Hodgkin's disease. Application of this formal method of analysis to other immunohistological reagents and panels of antibodies is discussed.

Distribution of laminin, fibronectin, and interstitial collagen type III in soft tissue tumours.

The distributions of laminin, fibronectin, and interstitial collagen type III have been investigated in a series of 60 soft tissue tumours by immunochemistry. Positive laminin staining was seen in sites predicted by the distribution of ultrastructurally visible basal lamina. Pericellular laminin was present in all benign tumours of Schwann cell and smooth muscle origin examined, in the two malignant Schwannomas examined, and in six of 13 leiomyosarcomas. It was also evident around nests of cells in an alveolar soft part sarcoma and around malignant endothelial cells in an angiosarcoma. In fibroblastic and fibrohistiocytic tumours it was found only in blood vessel walls. The results of laminin staining led to revision of the original histopathological diagnosis in seven of the 60 cases studied. Fibronectin was abundant in the stroma of most neoplasms, both benign and malignant. It was also found in a distribution parallel to that of laminin. In some tumours this was clearly distinguishable from the distribution of interstitial collagen. Intracellular fibronectin was shown consistently only in mast cell granules. Its demonstration in synovial cells, fibroblasts, and histiocytes was more variable. Interstitial collagen type II had the most irregular distribution of the three proteins. It was as plentiful in tumours of smooth muscle origin as in tumours of fibroblastic origin, but was scanty in fibrous histiocytomas. Its distribution appeared similar to that of laminin and fibronectin in leiomyomas, but differed from these two proteins in Schwann cell tumours and other neoplasms. In one leiomyosarcoma fibronectin, laminin, and type III collagen appeared to be lost concomitantly from tumour cell peripheries.

Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections.

The polymerase chain reaction was applied to the analysis of DNA contained in archival paraffin wax embedded material. DNA suitable for the reaction was obtained from these tissues by simple extraction methods, without previous dewaxing of tissue sections. When compared with unfixed material, the reaction efficiency was compromised, so that an increased number of amplification cycles were required to produce equivalent amounts of amplified product. This in turn led to an increase in amplification artefacts, which can be minimised by a simple modification of the standard reaction. Amplification of relatively large DNA fragments was not always successful, and it seems prudent to bear this in mind when designing oligonucleotide primers which are to be used for the amplification of archival material. The efficiency of the procedure can be improved by dividing the amplification cycles into two parts: this reduces the amount of reagent needed, is relatively simple and inexpensive, and can be performed in one working day.

Detection of single copies of Epstein-Barr virus in paraffin wax sections by non-radioactive in situ hybridisation.

A highly sensitive non-isotopic in situ hybridisation technique was developed for the localisation of Epstein-Barr virus (EBV) in paraffin wax embedded tissue sections. The method uses a repeated sequence of the EBV genome as a probe, labelled with the novel reporter molecule, digoxigenin. The method can identify individual copies of EBV by detection of both EBV DNA and highly localised RNA transcripts. A combination of careful proteolytic digestion of tissue sections, high temperature denaturation of probe and target DNA, and sensitive immunocytochemical detection are used to attain single copy sensitivity. The technique is quicker and simpler to perform than some other methods used for the identification of EBV, and provides simultaneous morphological information which cannot be obtained by methods using tissue extracts. This method permits the investigation of the role of EBV in neoplastic conditions of lymphoid and epithelial cells, and may prove valuable in determining the sites of latent virus in healthy subjects.

Laminin and fibronectin in adenoid cystic carcinoma.

The distribution of fibronectin and laminin was examined by immunohistochemistry in 11 adenoid cystic breast carcinomas, six adenoid cystic carcinomas of mouth and salivary gland, and six cribriform ductal breast carcinomas. Both proteins were present lining cystic lumina and around tumour islands in all the adenoid cystic breast carcinomas and in five of six salivary gland tumours. Abundant laminin and fibronectin were dispersed among adenoid cystic tumour cells arranged in sheets. One adenoid cystic carcinoma from buccal mucosa showed a transition from a cribriform tumour positive for both fibronectin and laminin to a cribriform tumour negative for fibronectin and laminin to undifferentiated carcinoma. Fibronectin and laminin seemed to disappear simultaneously from tumour cell surfaces. Another adenoid cystic carcinoma from buccal mucosa was negative for fibronectin and laminin from the time of initial biopsy. This was the only tumour that gave rise to disseminated metastases, resulting in the death of the patient within two years of surgery. In cribriform invasive ductal breast carcinomas the linings of cystic lumina were always negative for fibronectin and laminin. Varying quantities were present at the tumour boundaries. We suggest that staining for fibronectin and laminin may be a valuable aid to the diagnosis of adenoid cystic carcinomas and that the absence of these proteins may have important prognostic implications.

Spherical connective tissue inclusions in epithelial hyperplasia of the breast ("collagenous spherulosis").

Partial myoepithelial differentiation is common in simple epithelial hyperplasia (epitheliosis) of the breast but functional myoepithelial differentiation with basement membrane production is exceedingly rare. A peculiar change of hyaline globules within benign epithelial hyperplasia has been recognised before as "collagenous spherulosis" and type IV collagen has been shown by immunohistochemistry. Another seven cases are described which show the presence of laminin and collagens IV and III within the proliferation. Electron microscopy examination of two cases using material retrieved from the wax block showed varying degrees of myoepithelial differentiation of the cells immediately surrounding the spherules and basal lamina material, including mature collagen fibrils in one case. The degree of myoepithelial differentiation of the cells surrounding the spherules seemed to correlate with the differing types and amounts of extracellular matrix in the spherule. Histopathologists should be aware of this rare change as it may be misinterpreted as in situ carcinoma.

New marker of B lymphocytes, MB2: comparison with other lymphocyte subset markers active in conventionally processed tissue sections.

The use of the murine monoclonal antibody MB2 for identifying B lymphocytes in routinely processed tissue was evaluated and contrasted with the use of the monoclonal antibody UCHL1 for identifying T cells. One hundred and sixty eight surgical biopsy specimens were immunostained with these antibodies, including a wide range of normal and neoplastic non-lymphoid tissues, as well as normal lymphoreticular tissues and lymphomas. Sixty four non-Hodgkin's lymphomas were also examined, of which 51 had been previously phenotypically defined. In selected cases the results were compared with those obtained using two other monoclonal antibodies MB1 and MT1, used for identifying B and T cells, respectively, in paraffin sections. MB1 stained a smaller proportion of B cell tumours than MB2 and staining was, in general, weaker, except in one case of centroblastic lymphoma. MT1 immunoreactivity was comparable with that of UCHL1, except in one case of T lymphoblastic lymphoma (MT1 positive, UCHL1 negative). None of the antibodies is ideal, but, if used as a panel, they permit the separation of B cells and T cells in paraffin sections.

Rapid technique of DNA-DNA in situ hybridisation on formalin fixed tissue sections using microwave irradiation.

The relative sensitivities of different protocols for detecting cytomegalovirus nucleic acid sequences in histological specimens, using a biotinylated cDNA probe, were assessed. Several commonly used pre-treatment steps were not essential, nor was the use of a highly sensitive detection system. The choice of enzyme used for proteolytic digestion of tissue seems to be important, and increasing the temperature of denaturation of tissue and probe DNA to above 100 degrees C greatly increased the sensitivity of the method. Difficulties in achieving such high temperatures in a controlled manner were overcome by the use of a rapid microwave heating method that can be used routinely in laboratories. This technique detected cytomegalovirus infections in formalin fixed, paraffin processed tissue sections within a single working day.

Demonstration of lymphoid antigens in decalcified bone marrow trephines.

A panel of antibodies recognising lymphoid and epithelial antigens in formalin fixed, paraffin embedded sections was applied to a series of 54 bone marrow trephines decalcified by formic or edetic acids. Normal trephines and cases infiltrated by myeloid, lymphoid, and epithelial tumours were included. Patterns of reactivity were distinct and allowed the different diseases to be distinguished. All lymphoid tumours expressed leucocyte common antigen, with B cell tumours staining with MB1 and MB2, and T cell tumours staining with MT1 and UCHL1. T cell acute lymphoblastic leukaemia (ALL)/lymphoblastic lymphoma all stained with MT1, but some were negative with UCHL1. B cell ALL/lymphoblastic lymphoma also stained with MT1, but could be distinguished by its reactivity with MB1 and MB2. Reed-Sternberg cells did not stain with any reagent. Normal and neoplastic myeloid cells stained with MT1. Carcinomas stained with CAM 5.2 but were negative for lymphoid markers except MB2 staining in some cases. A case of neuroblastoma could be distinguished from ALL/lymphoblastic lymphoma by its lack of reactivity with all antileucocyte antibodies and its staining with antineurone specific enolase. Although not ideal, if used together, this panel of reagents may usefully be applied to routinely fixed and processed, decalcified bone marrow trephines.

Immune complex nephritis in alcoholic cirrhosis: detection of Mallory body antigen in complexes by means of monoclonal antibodies to Mallory bodies.

A Mallory body (alcoholic hyaline) antigen (JMB2) which is also present in intermediate filaments of epithelial origin was demonstrated immunohistochemically in renal glomeruli of three out of eleven patients with alcoholic liver damage. In two of these patients, both of whom had alcoholic cirrhosis with Mallory bodies, it was associated with mesangial deposits of IgA and C3. JMB2 was not found in glomeruli of normal controls, nor in a series of cases of glomerulonephritis in non-alcoholic patients. It is concluded that JMB2 is present in immune complexes in renal glomeruli of patients with renal disease consequent on alcoholic liver disease.

Fibronectin and type III collagen in epithelial neoplasms of gastrointestinal tract and salivary gland.

The distribution of fibronectin (FN) and collagen type III (IIIC) have been compared in a series of epithelial neoplasms from the gastrointestinal tract and salivary gland. The difficulty of distinguishing between FN of epithelial and fibroblastic origin is emphasised and evidence is presented for the validity of this distinction. In carcinomas FN was sometimes, but not invariably, lost from epithelial cell surfaces. Both FN and IIIC were increased in reactive connective tissue stroma. It is concluded that loss of cell surface FN is unlikely to be a useful diagnostic marker for malignancy, but that the occurrence of this phenomenon in vivo as in vitro indicates that it is biologically significant.

Cyclosporin and renal graft histology.

The histology of renal allografts was compared in a series of 107 biopsies from patients receiving cyclosporin and 126 biopsies from patients receiving azathioprine and prednisolone. Patients receiving cyclosporin were converted to azathioprine and prednisolone 90 days after transplantation. Biopsies were taken routinely at 7, 21, 90, and 365 days, irrespective of clinical graft function and were examined "blind" by two independent observers. Interstitial haemorrhage was more common in patients treated with azathioprine and prednisolone corresponding with their poorer graft survival. Analysis of glomerular, tubular, vascular, and interstitial changes showed no other important differences between the two groups despite clinical evidence of reversible cyclosporin nephrotoxicity. Quantitation of interstitial fibrosis in 90 day biopsies showed it to be equal in prevalence after treatment with azathioprine and prednisolone and cyclosporin. It was preceded by diffuse interstitial cellular infiltration, a common finding in early biopsies. Diffuse cellular infiltrates were generally associated with higher serum creatinine concentrations and, if persistent, a poorer graft prognosis than focal infiltrates, but they were not always associated with renal dysfunction.

Persistence of Epstein-Barr virus in Reed-Sternberg cells throughout the course of Hodgkin's disease.

Non-isotopic in situ hybridization employing digoxigenin-labelled DNA probes has been used to localize Epstein-Barr virus (EBV) in 55 cases of Hodgkin's disease (HD). The virus was found in Reed-Sternberg (RS) and mononuclear Hodgkin's cells in nine patients (16 per cent). Further samples taken at different times from three patients also showed the presence of EBV in the malignant cell population. Estimations of the number of EBV genomes present per cell suggested wide variations between different patients, but relatively constant amounts in different samples from the same patient. These findings are compatible with a stable infection of the neoplastic cells and support the notion that EBV may play a role in the development of HD in these patients. We also found evidence for the presence of EBV in a small percentage of non-neoplastic cells in 8 of the 55 samples. This suggests that isolation of EBV from HD tissue does not always signify a pathogenetic role for the virus. Furthermore, it is apparent that a high percentage of HD tissues do not contain demonstrable EBV, and the virus is therefore unlikely to be a causative agent for all cases of HD.

Ultrastructural localization of antigens recognized by the monoclonal antibodies MB1, MB2, MT1, UCHL1, and TAL 1B5.

In this study the ultrastructural localization of antigens recognized by novel antibodies that allow the recognition of lymphoid antigens in conventionally fixed and wax-embedded sections was investigated. MT1, MB1, and UCHL1 recognize antibodies restricted to the cell membrane, whereas the antigen recognized by MB2 is present only in the cytoplasm. These distributions are different from that of immunoreactivity with TAL 1B5 (anti-HLA DR), which is present both on the cell membrane and in the cytoplasm. The significance of these findings is discussed.

Paucity of fibronectin in invasive lobular carcinoma of breast.

Fifty-four cases of invasive carcinoma of breast were immunostained for fibronectin and laminin. They included 36 cases of invasive ductal carcinoma and 18 cases of invasive lobular carcinoma. Although there was some heterogeneity within tumours, it was found that whilst the majority of ductal carcinomas (31/36) had abundant fibronectin at cell/stroma boundaries or diffusely throughout stroma, a substantial proportion of lobular carcinomas (12/18) had very little (P less than 0.001). This difference could not be related to differences in laminin immunoreactivity, which was most commonly scanty or absent in both tumour types. It is postulated that the characteristic infiltration pattern of lobular carcinoma may be attributed in part to paucity of stromal fibronectin.

Paraffin section immunohistochemistry. II. Hodgkin's disease and large cell anaplastic (Ki1) lymphoma.

A panel of antibodies that recognize antigens that survive fixation and conventional processing have been applied to 43 cases of Hodgkin's disease and five cases of large cell anaplastic lymphoma. Reed-Sternberg cells in all five cases of nodular lymphocyte predominance Hodgkin's disease were positive with leucocyte common (CD45) and B-cell antibodies, and negative with LeuM1 (CD15) and BerH2 (CD30) antibodies. In other types of Hodgkin's disease, Reed-Sternberg cells were positive with BerH2 in all cases, positive with LeuM1 in 63% of cases (with enzymic predigestion), positive with at least one B-cell antibody in 29% of cases and positive for CD45 in 8% of cases. In 19% of all cases, Reed-Sternberg cells were positive for epithelial membrane antigen and in 93% they were positive with TAL1B5 (anti-class II MHC). No case showed immunoreactivity with anti-T-cell antibodies. The patterns of immunoreactivity of large cell anaplastic lymphoma were similar, except that none was positive with B-cell antibodies and three were positive with T-cell antibodies. All five were positive with BerH2 (CD30) and TAL1B5. Comparison of the results with those seen in other cases of non-Hodgkin's lymphoma indicates that, with the currently available reagents, this immunohistological profile cannot be used as the sole diagnostic discriminant of these conditions; this must still be based upon careful morphological assessment.

In situ demonstration of renal tubular regeneration using the monoclonal antibody Ki67.

The murine monoclonal antibody Ki67 recognizes a nuclear antigen present in all phases of the cell cycle except Go and can be used with a simple indirect immunohistochemical technique to demonstrate cell proliferation in tissue sections. This antibody was applied to 37 unselected renal biopsies showing a wide variety of histological appearances. Ki67-positive nuclei were seen most frequently in tubular epithelium in acute tubulo-interstitial pathology, particularly in renal allograft rejection. Tubular epithelial staining ranged from 0 to 10% of cells. In chronic nephropathies few tubular cells were stained. Staining was seen in glomerular crescents, but was rare in glomerular tufts except those that showed mesangial proliferation where occasional cells stained. This study demonstrates that information regarding cellular proliferation in renal biopsies can be easily obtained using Ki67 immunostaining. This is likely to be a useful investigative tool and may provide clinically useful information.