The value of tumour marker CA 125 in surgical pathology

CA 125 is a tumour marker located primarily on non-mucinous epithelial ovarian tumours and which is recognized by the monoclonal antibody OC 125. In this study the value of CA 125 in surgical pathology was assessed. In fresh frozen material, the expression of CA 125 was demonstrated in 82% of 83 epithelial ovarian neoplasms using the indirect immunoperoxidase technique. In addition, all adenocarcinomas of cervix (n = 5) and endometrium (n = 15) tested expressed CA 125, and 25 of 111 (22%) non-gynaecological malignant tumours were positive. The positive cases included 20 breast carcinomas, one carcinoma of the stomach and one of the colon. Using a commercial kit on routinely fixed, paraffin embedded material, CA 125 positivity was demonstrated in 29 of 36 (80%) serous cystadenocarcinomas after pronase pre-treatment of the sections, in contrast to 100% (n = 25) positivity on frozen tissue sections. CA 125 can, therefore, be demonstrated in routinely fixed paraffin embedded material, although the number of positive results is less than in fresh frozen sections.     

Long-term pathological follow-up of cerebral arteriovenous malformations treated by embolization with bucrylate

We examined 17 intracranial arteriovenous malformations that were resected after treatment by embolization using bucrylate (isobutyl-2-cyanoacrylate). In nine specimens removed 5 days to 16 months after embolization therapy, a series of pathologic changes was seen, including patchy mural angionecrosis (adjacent to bucrylate fragments) up to six weeks after embolization, the presence of bucrylate in vessel walls and fibromuscular intimal cushions, and the occurrence (after several months) of entirely extravascular bucrylate. Occasional parts of recanalized vascular malformations were identified. Bucrylate was present within arteriovenous malformations as late as 16 months after embolization, although the amount appeared to be diminished. These findings suggest a specific sequence of events in the interaction between bucrylate and mural components within the malformations and may explain some important complications of embolization therapy (e.g., delayed hemorrhage after embolization).     

Usefulness of MIB1 monoclonal antibody in assessing the proliferative index in human bladder carcinoma: comparison with Ki-67 antibody

The reactivity of MIB1 antibody on routinely processed paraffin sections was compared with that of Ki-67 antibody on frozen sections of 80 transitional cell carcinomas of the bladder. The percentage of labelled cells was expressed as the labelling index. MIB1 labelling indices were higher than those of Ki-67 but for each case the two values were strongly correlated ( r = 0.91). Ki-67 and MIB1 indices were also correlated to tumour grade and stage (P 0.001). MIB1 indices determined after both formaldehyde and ethanol based Bouin's fluid fixatives did not show any significant difference. MIB1 antibody staining after microwave oven heating of tissue sections is a simple technique for assessing the proliferative fraction of bladder tumours on fixed material. The use of MIB1 antibody permits retrospective studies and should determine whether the proliferation index in bladder carcinoma has the same prognostic value as demonstrated in other types of neoplasms.     

NCL-5D3: a new monoclonal antibody recognizing low molecular weight cytokeratins effective for immunohistochemistry using fixed paraffin-embedded tissue

Production of a new monoclonal antibody designated NCL-5D3 is described. The antibody recognizes several low molecular weight cytokeratins, in particular cytokeratin Moll number 8 as determined by immunoblotting studies, and is highly effective for immunocytochemistry using routinely processed paraffin-embedded material. Staining is enhanced by prior treatment of the sections with trypsin. Assessment using a wide variety of normal and neoplastic tissue indicates reactivity with all tissues of simple or glandular epithelial origin, and in addition with many squamous carcinomas. Thus the antibody should prove of value in diagnostic histopathology.     

A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory. Quality of histologic sections and advantages of microwave processing

Rapid processing of histopathologic material is becoming increasingly desirable to fulfill the needs of clinicians treating acutely ill patients. Traditional techniques for rapid processing of paraffin-embedded tissues require 4 to 5 hours, delaying treatment for some critically ill patients and requiring additional shifts of technologists in the laboratory. Microwave processing further shortens this time, allowing even more rapid histopathologic diagnosis. Few data exist comparing quality of microwave-processed tissue with that processed by more traditional techniques. We randomly selected 158 paired specimens from 111 patients. One member of the pair was processed routinely overnight, while the other was processed by the rapid microwave technique. The slides then were compared for quality of histologic preparation in a blinded fashion by 2 pathologists. Eight routinely processed specimens were judged as suboptimal, while 6 microwave-processed specimens were judged as suboptimal and 1 was considered unsatisfactory for evaluation. In the remaining cases, the material obtained by the 2 techniques was considered of identical quality. Microwave processing considerably shortens the preparation time for permanent histologic sections without a demonstrable decrease in section quality or "readability."     

Silver Labeling of Alzheimer Neurofibrillary Changes and Brain β-Amyloid

Abstract

Alzheimer neurofibrillary changes and brain β-amyloid can be stained selectively by silver impregnation on tissue sections and (in the case of neurofibrillary proteins) on sodium dodecyl sulfate-polyacrylamide gels. The stain can be applied to frozen sections of tissue from 10–150 μm thick, to similarly thick polyethylene glycol sections, or to 5–15 μm thick paraffin sections. The technique can also be applied to routinely fixed autopsy material. The technique takes advantage of the physical development of nucleation sites, thereby permitting tight control of the entire procedure. It is less expensive than immunocytochemical techniques, and facilitates processing of large numbers of sections through entire hemispheres of the human brain. (The J Histotechnol 16:335, 1993)     

Pitfalls in diagnostic molecular pathology – significance of sampling error

Today, molecular diagnostic tests are widely used in clinical medicine with polymerase chain reaction (PCR)-based techniques being of particular interest. In tissue specimens, however, false-positive and false-negative results can be obtained if pathomorphological and processing aspects are not considered. We therefore studied the impact of tissue sampling in three widely used diagnostic tests: (1) assessment of clonality in B-cell non-Hodgkin's lymphoma, (2) analysis of microsatellite instability (MSI) in colorectal neoplasia, and (3) demonstration of mycobacterium tuberculosis. Tissue sections of routinely formalin-fixed and paraffin-embedded diagnostic specimens were taken, and the significance of sampling was systematically investigated using laser microdissection or processing of the entire section. PCR analyses were done according to standard protocols. False-positive pseudo-monoclonality was obtained in small gastrointestinal biopsies and in laser microdissected lymph follicles of non-neoplastic tonsils. False negativity (pseudo-stability) could be demonstrated in a case with hereditary rectal adenoma if whole tissue sections were taken without microdissection of the most dysplastic subareas. Demonstration of mycobacteria failed in tissue sections of a formalin-fixed lymph node that was positive after complete digestion or in fresh frozen material of the same patient. In diagnostic molecular pathology, sampling error is an important source of false-positive and false-negative results, particularly if disease- and tissue-specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity, are ignored.     

Paediatric rhabdomyosarcoma: MyoD1 demonstration in routinely processed tissue sections using wet heat pretreatment (pressure cooking) for antigen retrieval.

AIMS: To investigate wet heat pretreatment (pressure cooking) as a means of antigen retrieval for demonstration of MyoD1 in paraffin wax embedded tissue. METHODS: Routinely processed tissue sections of transmission electron microscope confirmed cases of rhabdomyosarcoma were stained immunohistochemically with the MyoD1 antibody. Antigen retrieval was achieved by wet heat pretreatment of the tissue sections. RESULTS: MyoD1 was stained successfully in all seven cases. The protein was localised to nuclei and cytoplasm depending on the type of tumour cell. CONCLUSIONS: Wet heat pretreatment for antigen retrieval from routinely processed tissue sections permits excellent subsequent immunostaining for MyoD1 in rhabdomyoblasts.     

The diagnosis of fatal pulmonary fat embolism using quantitative morphometry and confocal laser scanning microscopy

The postmortem diagnosis of fat embolism syndrome (FES), traditionally based on the histological demonstration of fat globules, needs a quantitative analysis of both the size and localization of the fat emboli, which is essential for a reliable grading of the pulmonary fat embolism. The clinical data and the autopsy records of 2738 autopsies were retrospectively evaluated, and 21 cases in which FES was pointed out as cause of death were selected and compared with 21 fatal cases referred to as major trauma in which the cause of death was not attributed to fat embolism, and with 47 fatal cases as control group, respectively. The following parameters were investigated: the total area of the embolized tissue; the total number of emboli; the mean area of the emboli; the mean percentage of the embolized tissue area as compared with the total tissue area of each sample; the total percentage of the embolized tissue area as compared with the total tissue area of all slides. The most reliable parameters seem to be the ratio between embolized tissue areas as compared with the total tissue area of each sample. These parameters showed a good correlation with the clinical data.     

Immunofluorescence staining of adenovirus in fixed tissues pretreated with trypsin.

We describe an immunofluorescence staining procedure for adenovirus in Formalin-fixed, paraffin-embedded tissue sections pretreated with a 0.25% trypsin solution. In trypsinized tissue sections stained with fluorescein-labeled antibody to adenovirus, viral antigen was brightly fluorescent and easily detected each time specimens were processed, and the nonspecific background fluorescence was always minimal. Viral antigen was nonfluorescent or only weakly fluorescent in tissue sections not pretreated with trypsin. Because pathology laboratories usually receive Formalin-fixed tissues for examination, this rapid and practical procedure can be routinely used to extend the diagnostic capability of conventional histopathology.     

Detection and quantification of hippocampal synaptophysin messenger RNA in schizophrenia using autoclaved,formalin-fixed,paraffin wax-embedded sections

Most in situ hybridization histochemistry studies of messenger RNA in human brain have been carried out on frozen tissue. Recently, autoclaving has been reported to enable routinely processsed material to be used for in situ localization of messenger RNA. We have investigated whether autoclaving also permits in situ hybridization histochemistry to be used quantitatively. To do this, we targeted synaptophysin messenger RNA with a ³⁵S-labelled oligonucleotide probe in autoclaved, formalin-fixed, paraffin wax-embedded sections of the hippocampal formation of 11 schizophrenics and 11 controls. We compared the results with those seen on frozen sections from adjacent blocks, which had been used previously to demonstrate a loss of the messenger RNA in schizophrenia. Synaptophysin messenger RNA was readily detected in the autoclaved sections. The hybridization signal correlated strongly with that seen in the frozen sections. We found a similar pattern and magnitude of decreased synaptophysin messenger RNA in schizophrenia in the autoclaved sections as we had in the frozen sections, including the selective preservation of synaptophysin messenger RNA in CA1. The reduction of synaptophysin messenger RNA was replicated when six subjects with schizophrenia not included in the earlier study were considered separately.We conclude that autoclaving renders formalin-fixed, paraffin wax-embedded sections of human brain suitable for quantitative in situ hybridization histochemistry. This has considerable implications, given the wider availability, better morphology and easier handling of fixed than frozen human brain tissue. Using this material, we confirmed the finding of decreased synaptophysin messenger RNA in the hippocampal formation in schizophrenia, furthering the evidence for synaptic pathology in this region in the disorder.     

Detection of the source of mislabeled biopsy tissue paraffin block and histopathological section on glass slide.

The aim of this study was to identify the source of surgical biopsy tissue in paraffin block and histochemically stained biopsy section on a slide suspected to be mislabeled. Commercially available polymerase chain reaction (PCR)-based human lymphocyte antigen (HLA) DQA1 and Polymarker kits (Roche Molecular Systems, Branchburg, NJ, U.S.A.). were used to generate DNA profiles from biopsy tissue material in paraffin block and on histological slide, and the reference blood sample was collected from the patient. The source of biopsy tissue was detected by HLA DQA1 and polymarker profiling. Profiles obtained from biopsy material were consistent with those obtained from reference blood sample. The PCR-based DNA profiling techniques can determine the source of minute tissue in paraffin block and stained tissue sections on slides routinely prepared for diagnosis of carcinoma.     

Application of domestic microwave for urgent histopathology reporting: an evaluation

Rapid diagnosis of histopathological material is becoming increasingly desirable. In neuropathology, crush smear preparation and frozen section diagnosis of tissues removed during operative procedures, have remained as essential tools for rapid diagnosis. Microwave technology has been introduced into the field of tissue processing and staining in past decade. Now-a-days even automated microwave assisted rapid tissue processors are available. In our study we have analysed the use of a domestic microwave (cost approximately Rs.5000) for urgent histoprocessing (30 minutes). This could be useful in small laboratories or the ones which are in the phase of establishing the department as the procedure is much more economical than obtaining a frozen section (which requires a cryostat worth 3-6 lakhs) and the interpretation of the section obtained does not require any extra experience as these resemble the routinely processed tissue sections. The advantages and limitations of the procedure have been discussed.     

Comparison of in situ hybridization using different nonisotopic probes for detection of Epstein-Barr virus in nasopharyngeal carcinoma and immunohistochemical correlation with anti-latent membrane protein antibody.

BACKGROUND: The detection of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) may be of diagnostic importance, particularly in cases from nonendemic areas. For cellular localization of viral genomes, cold in situ hybridization methods for the demonstration of EBV-associated NPC remain difficult and relatively insensitive for routinely processed tissues. EXPERIMENTAL DESIGN: The aim of the present study was to assess the importance of tissue processing and the hybridization targets to improve the sensitivity of the cold in situ hybridization method. In situ hybridization was performed in six cases of NPC using three biotinylated EBV cDNA probes (BamHI W/IR1, BamHI Y/EBNA2, XhoI/latent membrane protein) and two cocktails of EBER and BHLF1 oligonucleotides labeled with fluorescein isothiocyanate on routinely fixed and paraffin embedded sections. In two cases, in situ hybridization was also performed on specially processed (ModAMeX) sections. Immunohistochemistry was used to detect EBV-induced antigens using monoclonal antibodies against latent membrane protein, EBNA2 and ZEBRA (BZLF1). RESULTS: All cases showed EBV nucleic acids regardless of the tissue preparation with the three cDNA probes and on routinely processed sections with EBER oligonucleotides. By using cDNA probes, the best EBV DNA signal was obtained with BamHI W without heating of slides in tissue sections processed by ModAMeX, which probably gives rise to large amounts of single stranded DNAs. All cases positive with cDNA probes were found to be positive with EBER oligonucleotides and negative with BHLF1. However, on routinely processed paraffin sections, the signals with EBER oligonucleotides were stronger than with BamHI W cDNA probe. Dual labeling with in situ hybridization and immunohistochemistry showed that the hybridization signals were restricted to malignant epithelial cells. Latent membrane protein expression was detectable in four of six EBV nucleic acid-positive cases on both ModAMeX and routinely processed sections. The anti-EBNA2 and anti-ZEBRA antibodies were found to be negative on the two cases processed by ModAMeX. CONCLUSIONS: Cold in situ hybridization, in particular with EBER oligonucleotides, appears to be more reliable than immunohistochemistry with anti-latent membrane protein antibody to detect EBV in NPC in routine pathology. These findings confirm a distinctive phenotype (latent membrane protein +/-, EBNA2-, ZEBRA-) of EBV-positive NPC. The negative staining for BHLF1 oligonucleotides further supports the viral latency.     

Multiparameter immunofluorescence on paraffin-embedded tissue sections.

Immunohistochemical techniques have gained increasing importance in diagnostics and research. While formalin-fixed, paraffin-embedded human tissue retains excellent morphology, the detection of antigens by immunofluorescence in its sections and especially the demonstration of multiple simultaneous antibodies have limitations. Double immunofluorescence labeling of routinely processed paraffin sections has been described previously. The signal intensity observed after triple labeling has been reported to be significantly inferior to that obtained by application of double fluorochromes. The authors show multicolor labeling of three and four primary antibodies in routinely processed paraffin-embedded tissue sections using a standardized immunofluorescence technique. In addition, procedures to reduce background staining and to avoid nonspecific double staining are described.     

Detection of keratin subtypes in routinely processed cervical tissue: implications for tumour classification and the study of cervix cancer aetiology

We investigated the expression of keratin subtypes 7, 8, 10, 13, 14, 17, 18 and 19 in the normal cervix, in cervical intraepithelial neoplasia (CIN) lesions and in cervical carcinomas, using a selected panel of monoclonal keratin antibodies, reactive with routinely processed, formalin fixed paraffin embedded tissue fragments. The reaction patterns derived for each keratin antibody were compared with known expression patterns of the various epithelia, previously examined in frozen tissues. Although the reactivity of the antibodies was generally acceptable, considerable modifications to the manufacturers' staining instructions were often necessary. For some antibodies, which were previously thought to be reactive with fresh frozen tissue only, we developed staining protocols rendering them reactive with routinely processed material. As with previous findings in frozen sections we observed increasing expression of keratins 7, 8, 17, 18 and 19 with increasing grade of CIN. In cervical carcinomas the differences in keratin detectability between the main categories were more pronounced than in frozen sections, probably due to fixation and processing. For routine pathology, keratin phenotyping of cervical lesions may be of value in classification. The fact that keratin 7 was detected for the first time in reserve cells, and that this keratin was also found to be expressed in a considerable number of CIN lesions and cervical carcinomas supports the suggestion that reserve cells are a common progenitor cell for these lesions.     

A new mdr-1 encoded P-170 specific monoclonal antibody: (6/1C) on paraffin wax embedded tissue without pretreatment of sections.

AIMS: The generation and characterisation of a monoclonal antibody that specifically recognises the mdr-1 encoded protein, P-glycoprotein (P-170), on routinely processed formalin fixed, paraffin wax embedded tissue sections. METHODS: The monoclonal antibody, designated 6/1C, was produced following a combination of in vivo and in vitro immunisation regimens in Balb/c mice with a synthetic 12 amino acid peptide that corresponds to amino acids 21-32 (believed to be intracellularly located) of P-170 and has insignificant homology with the mdr-3 encoded P-170. Antibody 6/1C was characterised by western blotting and immunocytochemistry on cytospins of paired multidrug resistant or sensitive cell lines, including mdr-1 and mdr-3 transfected cells, and by immunohistochemistry on normal and malignant formalin fixed paraffin wax embedded tissue sections. RESULTS: Antibody 6/1C showed a single band at 170 kDa on western blots of multidrug resistant cell lysates and mdr-1 transfected cell lysates that was absent on similar preparations of drug sensitive cells and mdr-3 transfected cells. Immunocytochemical studies on cytospins of multidrug resistant cells and mdr-1 transfected cells revealed strong inner plasma membrane/cytoplasmic staining. Staining was negligible on drug sensitive cells and cells transfected with the mdr-3 gene. Immunohistochemical studies on formalin fixed, paraffin wax embedded normal adult kidney, liver, and breast tissue and a range of fetal tissues exhibited staining patterns of a variety of secretory surfaces consistent with documented mdr-1 specific staining. Specific staining of malignant cells in similarly treated sections of breast tumours was seen also with antibody 6/1C. Staining on paraffin wax embedded tissue with this antibody did not require any pretreatment of tissue sections. CONCLUSIONS: This new monoclonal antibody, chosen for its specificity with the mdr-1 encoded P-170 and its reactivity on routinely fixed paraffin wax embedded tissue samples without pretreatment, appears to be useful for the investigation of P-170 in archival material. It is especially useful for retrospective studies on pretreatment and post-treatment tissue sections, and could help establish when and how rapidly mdr-1 associated drug resistance develops during chemotherapeutic regimens. Immunohistochemical assessment of P-170 expression in many cancers has potential for diagnostic purposes and may influence the choice of chemotherapeutic drugs used in the treatment of refractory tumours.     

DEVELOPMENT OF AN OPTIMAL PROTOCOL FOR ANTIGEN RETRIEVAL: A ‘TEST BATTERY’ APPROACH EXEMPLIFIED WITH REFERENCE TO THE STAINING OF RETINOBLASTOMA PROTEIN (pRB) IN FORMALIN-FIXED PARAFFIN SECTIONS

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin–paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a ‘test battery’ approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120°C, 100°C, and 90°C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100°C proved most effective. Both fresh and routinely processed formalin–paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin–paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin–paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.     

Development of a sensitive and specific in situ hybridization technique for the cellular localization of antisense oligodeoxynucleotide drugs in tissue sections

A sensitive method has been developed for the identification and assessment of phosphorothioate oligonucleotide accumulation in dosed animal tissues using an in situ hybridization approach, which is both sequence specific yet adaptable to every antisense oligonucleotide (ASO), which has been tested to date. Hybridization is accomplished using a digoxigenin-tailed oligonucleotide probe complementary to the ASO target sequence on routinely processed paraffin sections which have been pretreated with a mild target retrieval solution. The DIG-labeled probe is amplified first with an anti-DIG:FITC antibody conjugate followed by an anti:FITC Alexa 488 antibody, then visualized using FITC epifluorescence microscopy. Fluorescent labeling of ASO drug in tissue sections by this method confirms that H&E basophilia previously observed in dosed tissues represents largely intact ASO. However, the fluorescent method enables a wider assessment of tissue distribution in a variety of tissue types due to increased sensitivity and lower signal to noise than can be obtained through an examination of H&E stained tissue sections alone.     

The utilization of radioimmunoassay antibodies for the immunohistologic staining of polypeptide hormones on paraffin-embedded tissue.

A method is described whereby commercially available radioimmunoassay-grade antibodies specific for the polypeptide hormones calcitonin, gastrin, glucagon, and somotastatin are used to detect these antigens on paraffin sections of routinely fixed tissue. The hormone antibodies are applied to deparaffinized tissue sections as the primary specific immune sera using the standard peroxidase technic. The use of these hormone antibodies to detect their respective antigens has proved valuable in demonstrating polypeptide forming tumor cells in pathologic specimens.