Paraffin Sections Of Formalin-fixed Tissue Research Articles

The utility of C4d, C9, and troponin T immunohistochemistry in acute myocardial infarction.

Abstract Context.—Full activation and involvement of the complement pathway follows acute myocardial infarction. Complement fragment C4d is a stable, covalently bound marker of complement activation. Troponin T is specific for cardiomyocytes. Objectives.—To determine the specificity of C4d, C9, and troponin T immunoreactivity in necrotic myocytes and to establish whether they can be used to delineate acute myocardial infarction. Design.—Twenty-six autopsy cases with a total of 54 myocardium areas of infarction were reviewed retrospectively. Immunohistochemistry for C4d, C9, and troponin T was used on paraffin sections of formalin-fixed tissue. Controls consisted of 5 cases without evidence of infarction, and histologically normal myocardium functioned as an internal control. Results.—C4d and C9 antibodies reacted strongly and diffusely with necrotic myocytes in all samples of infarctions for up to 2 days (19 of 19; 100%). Adjacent histologically normal myocytes were nonreactive, resulting in a clear delin...

The Utility of C4d, C9, and Troponin T Immunohistochemistry in Acute Myocardial Infarction

Full activation and involvement of the complement pathway follows acute myocardial infarction. Complement fragment C4d is a stable, covalently bound marker of complement activation. Troponin T is specific for cardiomyocytes. To determine the specificity of C4d, C9, and troponin T immunoreactivity in necrotic myocytes and to establish whether they can be used to delineate acute myocardial infarction. Twenty-six autopsy cases with a total of 54 myocardium areas of infarction were reviewed retrospectively. Immunohistochemistry for C4d, C9, and troponin T was used on paraffin sections of formalin-fixed tissue. Controls consisted of 5 cases without evidence of infarction, and histologically normal myocardium functioned as an internal control. C4d and C9 antibodies reacted strongly and diffusely with necrotic myocytes in all samples of infarctions for up to 2 days (19 of 19; 100%). Adjacent histologically normal myocytes were nonreactive, resulting in a clear delineation between damaged and viable myocardium. Reactivity declined with increased duration and was absent in scars. Troponin T showed loss of staining in preinflammatory lesions (8 of 13; 62%); however, nonspecific patchy loss of staining was present in negative controls and in viable myocardium. Immunostains provided new diagnoses in 2 cases, including evidence of reinfarction and a newly diagnosed acute myocardial infarction. C4d and C9 have comparable reactivity and specificity for necrotic myocytes. C4d and C9 staining of necrotic myocytes is apparent before the influx of inflammatory cells, demonstrating utility in early myocardial infarction. Patchy loss of Troponin T in some cases of histologically normal myocardium limited its usefulness as a sole marker of infarction.

Cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues.

Diagnosis of rabies is routinely confirmed by detection of rabies virus antigens in acetone-fixed frozen brain tissues or imprint smears using an immunofluorescence method with commercial antirabies virus antibodies. Since recent molecular analyses disclosed wide heterogeneity in the genome sequences of rabies virus strains and related lyssaviruses, it is necessary to confirm the presence of common epitopes in these lyssaviruses. In this study we confirmed the presence of cross-reactive antigens of various lyssaviruses in paraffin sections of formalin-fixed tissue using a monospecific rabbit antiserum prepared by immunization with a recombinant nucleoprotein of rabies virus. By immunohistochemical application, the antigen was detected predominantly in the cytoplasm of neurons in the brains of mice infected with rabies virus, Duvenhage virus, Mokola virus and European bat lyssavirus-1, while no cross-reaction was observed in uninfected humans and animals including dogs, bats, and raccoons. In addition, we examined one autopsy case that was infected in a rabies-endemic nation and developed the clinical manifestation of rabies after returning to Japan in 1970, and found that the antigen was well preserved in paraffin sections of formalin-fixed tissues. Thus, this suggests that the lyssavirus-specific antigen is recognized by the monospecific antibody against rabies virus nucleoprotein, and that this cross-reactive antigen is detectable on formalin-fixed paraffin-embedded tissues by immunohistochemical analysis.

Expression of Telomerase Reverse Transcriptase (TERT) in Malignant Mesotheliomas

To evaluate the usefulness of determinations of telomerase activity for distinguishing malignant from benign mesothelial lesions, immunohistochemical (using a rabbit polyclonal antibody and the peroxidase method; n = 68) and in situ hybridization (using sense and antisense oligonucleotide probes; n = 46) studies were made on malignant mesotheliomas (epithelioid, 39; sarcomatoid, 18, including 2 of the desmoplastic type; and biphasic, 11) and 19 benign mesothelial lesions (benign mesothelial hyperplasia, 3; and reactive pleuritis, 16). In addition, biochemical studies of telomerase activity were made in 9 of the malignant mesotheliomas. Telomerase activity was detected histochemically in all but one of the malignant mesotheliomas, but only in one (pleuritis) of the benign lesions, in which it was present only in activated lymphocytes. Antisense hybridization signals indicated the presence of telomerase mRNA mainly in the cytoplasm of the malignant cells. Sense probes gave negative results. Biochemical determinations revealed a strong telomerase activity in the 9 malignant mesotheliomas examined. This study demonstrates the usefulness of immunohistochemical staining for the evaluation of mesotheliomas. The required immunostaining can be performed using paraffin sections of formalin-fixed tissues.

Failure of ultrasonic vibration as a means of antigen retrieval in routine diagnostic immunocytochemistry.

It was recently reported that certain antigens can be retrieved from paraffin sections of formalin-fixed tissues by ultrasonication using either an expensive and sophisticated ultrasonic cell disrupter probe (cytokeratins 13 and 16) or an inexpensive and generally available ultrasonic cleaning bath (bcl-1). We wished to investigate the routine use of the latter method and therefore tried to retrieve from various tissues 11 antigens that usually require heat-mediated retrieval in citrate buffer. We applied ultrasonic vibration for periods of 30 seconds to 1.5 minutes in a cleaning bath containing citrate buffer or water, with and without the addition of heat, or for 1 minute in hot citrate buffer after microwaving for 10 minutes in the same buffer. Although a slight effect of ultrasound was noted for a few antigens, in no case did the immunostaining reach the level achieved after standard microwave heating in citrate buffer. We conclude that, under the conditions we used, ultrasonic antigen retrieval cannot be used for immunocytochemistry in a routine histopathology laboratory.

Intermediate- and low-molecular-weight keratin detection with the monoclonal antibody MNF116. An immunohistochemical study on 232 paraffin-embedded cutaneous lesions.

Immunohistochemical detection of certain low to intermediate molecular weight keratins often is impaired in routinely processed specimens due to masking of these antigens by formalin fixation. Despite standard enzymatic digestion, AE1:AE3 and CAM 5.2, two of the most currently utilized antikeratin antibody preparations, either stain weakly or fail to stain basal keratinocytes and tumors composed of basaloid keratinocytes in paraffin sections of formalin-fixed tissue. We present here our experience with the monoclonal antibody MNF116 which detects keratins 5, 6, 8, 17, and 19 (DAKO, Carpinteria, CA). We have studied 232 routinely-processed skin lesions with MNF116 and compared the staining with that of AE1:AE3 mixture or CAM 5.2. In normal skin, the staining achieved with MNF116 was particularly strong on the basal cells of the epidermis and adnexae. MNF116 was positive in all 154 epithelial tumors and negative in all but one (a leiomyosarcoma) of 78 mesenchymal and melanocytic tumors. AE1:AE3 mixture was positive in all but four poorly-differentiated squamous cell carcinomas and it was only weakly positive in most basal cell carcinomas. CAM 5.2 was positive in tumors of the sweat apparatus, Merkel cell carcinomas, metastatic carcinomas, and 5/15 basal cell carcinomas. We consider that, in routinely processed specimens, MNF116 is very useful and convenient for detection of cytokeratin expression in cutaneous lesions, and therefore helpful in the evaluation of tumors with small cells and other poorly differentiated neoplasms of the skin.

Immunohistochemically demonstrated expression of HLA‐DR antigen in colorectal adenocarcinomas and its relation to clinicopathological features

We studied 148 colorectal adenocarcinomas to clarify any correlation between HLA-DR antigen expression on tumor cells and histopathological features. Paraffin sections of formalin-fixed tissues were stained with HLA-DR antigen using the indirect immunoperoxidase technique. All the tumor tissues were divided into two groups, depending on the incidence of HLA-DR-positive cells (greater and lesser than 50%). Carcinoma tissues with a higher incidence showed less mural invasion, lymphoductal invasion, venous invasion, lymphonodular metastasis, and peritoneal metastasis. Tissues with a high HLA-DR reactivity were more often observed for Dukes' A and B stages, whereas those with a low HLA-DR positivity were frequently Dukes' C and D stages. As for the cumulative survival rate, the group with high HLA-DR expression demonstrated significantly better survival. We speculate that HLA-DR expression by colorectal cancer cells exerts a favorable influence on clinical course.

Bcl-2 Expression in Melanocytic Nevi

Recently, a new category of oncogenes has been discovered that regulate programmed cell death. The bcl-2 oncogene has been found to inhibit cellular death without affecting cellular proliferation. In the skin, bcl-2 expression is limited to cells along the basal cell layer. However, we also noticed that resting melanocytes appeared to express bcl-2. We examined the expression of bcl-2 and its possible role in the biology of benign melanocytic proliferations. Routine paraffin sections of formalin-fixed tissue were labeled with anti-bcl-2 monoclonal antibody, and expression of bcl-2 was detected by a biotin-avidin-immunoperoxidase procedure. We examined 13 congenital, 11 acquired, and six atypical or dysplastic nevi for expression of bcl-2. Expression of bcl-2 was observed in 11 of 13 congenital nevi. All 11 acquired nevi and 6 nevi with architectural disarray and cytologic atypia expressed bcl-2. Both junctional and intradermal melanocytes expressed bcl-2 in a perinuclear and cytoplasmic pattern. Within neurotized areas, bcl-2 became significantly weaker and totally absent. In mature tissues, bcl-2 expression is quite limited. It appears to be restricted to pluripotential stem cells that serve as a reservoir for tissue that is constantly undergoing renewal, such as the hematopoietic cells and the intestinal mucosa. In the skin, bcl-2 expression has been previously reported to be limited to the epidermal basal cell layer and proliferation zones. Our results indicate that resting melanocytes and melanocytic nevi regularly express bcl-2.

In situ localization of ribosomal RNAs is a reliable reference for hybridizable RNA in tissue sections.

Assessments of RNA integrity and its hybridizability are essential for successful implementation of in situ hybridization (ISH) for mRNA or viral RNA, particularly when paraffin-embedded specimens from surgical, biopsy, and autopsy cases are used. In this study, we examined the suitability of ISH of 28S ribosomal RNA (rRNA) for this purpose. Oligo-DNA with nucleotide sequences complementary to a well-conserved segment of 28S rRNA with auxiliary adenine-thymine-thymine (ATT) repeats at the 3' and 5' ends was synthesized. The oligo-DNA was made antigenic by converting the adjacent thymines to T-T dimers by UV irradiation and was used as a probe for ISH of 28S rRNA. The T-T dimers were detected by enzyme immunohistochemistry. When the results of ISH rRNA staining and that of total RNA staining by methyl green/pyronin Y were compared for various types of sections prepared from rat and human tissues, the staining intensities of total RNA did not always match those of ISH rRNA staining. In paraffin sections of formalin-fixed tissues, the degree of proteinase digestion influenced the ISH rRNA staining intensity, whereas it had no effect on the total RNA staining intensity. The intensities of ISH rRNA staining agreed well with those of various types of mRNA staining by ISH in 10 cases of paraffin-embedded pathological specimens. We therefore believe that ISH rRNA staining is a convenient parameter for evaluation of levels of hybridizable RNAs in tissue sections.

Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections

In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed tissue.

Monoclonal antibodies against recombinant parts of the Ki‐67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave‐processed formalin‐fixed paraffin sections

The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.

Detection of capsid antigen of human papillomavirus (HPV) in benign lesions of female genital tract using anti-HPV monoclonal antibody.

We established a murine monoclonal antibody (K1H8) to human papillomavirus (HPV) using alkaline-disrupted virions of HPV type 1 (HPV-1) as the immunogen. K1H8 recognized a 57 kD capsid protein of HPV-1 and detected the antigen in paraffin sections of formalin-fixed tissue. With K1H8, we examined immunohistochemically 68 biopsy specimens obtained from the female genital tract. The specimens were histologically condyloma acuminatum or koilocytotic lesions with or without dysplasia and each specimen was found to harbour a single type of genital HPV, such as types 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58, by Southern blot hybridization analysis. The antigen was localized in the nuclei and occasionally in the cytoplasm of squamous cells showing koilocytotic changes. Eighty-four per cent of the specimens (57 cases) showed positivity for the antigen, indicating that K1H8 is a broadly-reactive antibody to various genital HPVs. The results suggest that benign mucosal lesions of the female genital tract are more frequently associated with viral production and are a potential source of transmission.

Differential reaction of secretory and non-secretory proteins in hormone-treated Dunning tumor.

Tumor cells from of the Dunning R-3327 PAP tumor were grown in vivo in the flank region of male Copenhagen rats for 4 months. Untreated control animals, castrated animals, and untreated or castrated animals supplemented with testosterone and estramustine (alone or in combination) bearing tumors were used for immunocytochemical studies of the tumor cells. Antibodies against the following secretory proteins were applied to paraffin sections of formalin-fixed tissue: anti-SVS II, anti-PBP, anti-transglutaminase, anti-acid phosphatase (isoenzymes, isoelectric points [pI] 5.6, 7.1, 8.0), non-secretory proteins comprised antiglucocorticoid-receptor, and antibodies against extracellular matrix proteins and intermediate filaments. A differential expression of marker proteins subsequent to the various treatments was observed immunohistochemically. Castration induced a loss of secretory activity, an increase in keratin-immunoreactive cells, and regressive activity in the secretory cells. Immunoreactivity of the glucocorticoid receptor in the nuclei of the basal cells was also decreased. Testosterone substitution only partially restored secretory activity of tumor cells in castrated animals. In experiments where estramustine had been administered to normal or castrated animals, metaplastic transformation of the epithelium, focal reduction, or increase of secretory and/or regressive activity and persistence of glucocorticoid receptor-like immunoreactivity was observed. The findings indicate that different hormonal situations provide conditions for the development of rather heterogeneous reaction patterns of different tumor cells, allowing partial regression of individual clones of tumor cells along with stimulation of others.

Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium.

Poly- and monoclonal antibodies to neoantigens of the human C5b-9 complement complex, as well as polyclonal antibodies to C5, C8, and C9, were used to detect and identify C5b-9 deposits in human myocardial tissue. Immunocytochemical studies were performed on fresh-frozen autopsy material derived from patients with myocardial infarctions; in addition, in 17 of these patients, paraffin sections of formalin-fixed tissue were investigated. Sixteen autopsies from patients with noncardiac diseases were analyzed as controls. Without exception, C5b-9 positivity was registered selectively and exclusively on and in myocardial cells located within the zones of infarction. The selectivity of staining was confirmed by control reactions for succinic dehydrogenase activity performed in adjacent, respective double-stained sections. Most intensive staining with anti-neoantigen antibodies was observed in the peripheral areas of the infarctions. Weak staining for C3d, rather strong staining for C5 and C9, and intermediate staining with anti-C8 antibodies were observed in the same localizations. Stainings for C4 and IgA were negative, whereas immunocytochemical reactions for IgG and IgM revealed an irregular and very weak staining. Only very weak staining was also observed with a monoclonal antibody to complement S-protein, indicating that the terminal complement components were deposited mainly in the form of membrane-damaging C5b-9 complexes. Immunocytochemical staining for C5b-9 was found to represent a most sensitive tool for detection of ischemic myocardial lesions, permitting easy detection even of single cell necroses. As a working hypothesis, we suggest that initial ischemia may cause loss of the ability of the heart muscle cells to regulate complement turnover at the membrane level. The resulting deposition of C5b-9 on the cell membranes may contribute to functional disturbance and irreversible damage of myocardial cells during the infarction process.

Sequential Histochemical Staining for Resident and Recruited Macrophages

A new histochemical technique is described that permits differentiation of resident from recruited macrophages by staining of paraffin sections of tissues from rats and mice. Resident macrophages are identified by their ability to phagocytose and retain intravenously injected colloidal Prussian blue. New macrophages that emigrate into tissue are identified by phagocytosis of a second colloid, iron dextran. Paraffin sections of formalin-fixed tissues are sequentially stained for the presence of the two colloids with different chromogens, the endogenous pseudo-peroxidase activity of colloidal Prussian blue used to catalyze the polymerization of diaminobenzidine and after conversion of iron dextran to Prussian blue, the second colloid used to catalyze the polymerization of tetramethylbenzidine. The staining results in resident macrophages staining brown while newly recruited macrophages stain blue. The studies have shown that colloidal Prussian blue is stable in vivo and neither loses its catalytic activity nor undergoes extensive redistribution. They also show that the technique can be used to measure Kupffer cell recruitment stimulated by complete Freund's adjuvant in rats and tumor-associated macrophage recruitment in subcutaneous and spontaneous liver metastases in mice.

The Selective Staining of Curious Bodies in Lymph Nodes of Patients as a Means for Diagnosis of Sarcoid

Paraffin sections of formalin-fixed tissue from some human lymph nodes manifesting the characteristic lesions of sarcoid contain minute bodies 3-15 μ in diameter. These bodies can be stained by direct application -of Schiff's reagent adjusted to pH 3-3.5, without prior hydrolysis or oxidation. They react positively to the naphthoic acid hydrazide procedure for aldehyde, and their reactivity to both these reagents was blocked by aniline and dimedone. These reactions indicate that the peculiar bodies in sarcoid tissues contain reactive aldehydes, present either before or after the processing of the tissue.

Ammoniacal silver hydroxide solution: a one-month test period on its usability for impregnating connective tissue fibers.

An aging solution of ammoniacal silver hydroxide was used to impregnate paraffin sections of formalin-fixed tissue by a procedure based on Gordon and Sweet's method (Amer. J. Path., 12: 545-51, 1936). The solution was first used when it was 1 day old, and subsequently used at 2 day intervals for a total period of 30 days. On the final using it was found that although the staining was slightly reduced in intensity, the stain was still adequate for diagnostic purposes.

Selective demonstration of histidine.

Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color.

The Combined Gram-Pappenheim Stain as Modified for Films and Paraffin Sections

A combination of the Gram-Pappenheim stains for the examination of gonorrheal pus, cellular exudate and paraffin sections of formalin-fixed tissues has been described elsewhere (Scudder and Lisa, 1931). The crystal violet solution was made stable for the first time by employing phosphate buffers on the acid side of neutrality, and a stable counterstain was prepared for the first time from National Aniline dyes, ethylated methyl green and pyronin yellowish. Original findings were demonstrated by means of color plate I and II (Scudder, 1931) to show gonococci, pneumococci and cells in smears, and formalin-fixed tissue brought down to water in the usual way. A new color plate is published herewith to show the microscopic appearance of cells, Gram-positive and Gram-negative bacteria, higher bacteria, fungi and spermatozoa in the study of genitourinary and gynecological cases. The method has a value in the field of medical jurisprudence. Crystals were well demonstrated, especially those resulting from sulfa dr...