Analysis of Epstein-Barr virus in Hodgkin's disease: experience of a single university hospital in Korea.

Hodgkin's disease is known to be associated with Epstein-Barr virus (EBV) infection in Western countries, and viral nucleic acids and proteins have been identified within Reed-Sternberg (RS) cells, which are the histopathologic hallmark of the disease process. Twenty-five cases of Hodgkin's disease from a single university hospital in Korea were studied for evidence of EBV by in situ hybridization for EBV DNA and RNA and immunohistochemistry for an EBV latent protein. EBV nucleic acids were studied by a rapid (60 minutes) in situ hybridization procedure, which utilized biotinylated DNA probes specific for the following nucleic acid sequences: (1) EBV EBER1 RNA (an abundant RNA sequence expressed during latent EBV infection), (2) EBV NotI repeats (a tandemly repeated DNA sequence, which has been established to identify amplified EBV genome in lytic EBV infection), and (3) BAM HI W (a DNA sequence reiterated 11 times within the viral genome). In addition, immunohistochemistry for EBV latent membrane protein, a protein that is capable of inducing cellular transformation in cell culture, was also performed. EBV was identified within the neoplastic RS cells by at least one method in 19/25 cases (76%). The mixed cellularity subtype was the most common subtype associated with EBV infection (11/13-85%). In situ hybridization for EBV EBER1 RNA was the most sensitive method for EBV detection and was present in 17/25 cases. A significant proportion of Korean Hodgkin's disease cases is associated with EBV infection.

Immunomycology: rapid and specific immunocytochemical identification of fungi in formalin-fixed, paraffin-embedded material.

We report the rapid (less than 1 hr), immunocytochemical identification of various fungi in formalin-fixed, paraffin-embedded tissues using antisera originally developed for use in immunodiffusion assays. Primary antisera directed towards fungal genera including Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, and Sporothrix were examined. The specificity of each antiserum was evaluated by the presence or absence of crossreactivity with other morphologically similar fungi in both paraffin-embedded pure fungal cultures and tissues with culture-confirmed fungal infections. Each antiserum reacted strongly with the fungus to which it had been raised, whether examined in pure culture or infected tissues. The antisera raised against Candida, Cryptococcus, and Sporothrix did not exhibit cross-reactivity with any other fungus tested. However, the antisera raised to Aspergillus, Blastomyces, Coccidioides, and Histoplasma demonstrated significant crossreactivity with other genera of fungi, thus precluding their routine use in diagnostic immunocytochemistry. The results indicate that immunocytochemistry may provide an important adjunct to other methods, such as immunodiffusion or complement fixation assays and histochemical stains such as the Grocott methenamine silver or periodic acid-Schiff, when attempts are made to specifically identify certain fungi in formalin-fixed, paraffin-embedded tissues before mycology culture results are available.

A rapid colorimetric in situ mRNA hybridization technique using hyperbiotinylated oligonucleotide probes for analysis of mdr1 in mouse colon carcinoma cells.

We describe the development of a rapid colorimetric in situ hybridization technique utilizing oligonucleotide probes labeled with six biotin molecules at the 3' end to detect mdr1 in mouse colon cancer cells growing in culture and in vivo. mRNA integrity was verified by the use of a multibiotinylated poly d(T) oligonucleotide, and the specificity of the reaction was confirmed by use of labeled sense and anti-sense probes in serial cryostat sections and cultured cells. The multiple biotin label produced a strong signal after a short hybridization time. Avidin-alkaline phosphatase detection and the capillary technology used in the Microprobe Accelerated System allowed completion of the procedure in less than 5 hr. Excellent correlations with the MDR phenotype of the cells, Northern blot analysis, and immunohistochemistry recommend this procedure for identifying cells that express the MDR phenotype in culture and in vivo.

A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas.

We have developed a rapid colorimetric in situ mRNA hybridization procedure to analyze epidermal growth factor receptor (EGF-R) transcripts in paraffin-embedded surgical specimens of human colon carcinomas. This technique is based on the use of 24-base oligonucleotide probes labeled with 6 biotin molecules at the 3' end. mRNA integrity was verified using a hyperbiotinylated 30-residue-long deoxythymidylate oligonucleotide probe, and the specificity of the reaction was confirmed by using labeled EGF-R-specific sense and antisense probes. Avidin alkaline phosphatase detection and the capillary technology used in the Microprobe System allowed for completion of the procedure in under 5 h. The human A431 epidermoid carcinoma cells growing in culture and fixed with formalin as well as paraffin-embedded sections of this tumor growing s.c. in nude mice served as positive controls. In situ hybridization with antisense EGF-R oligonucleotide probes directly correlated with EGF-R mRNA and protein levels observed by Northern blot and immunohistochemistry, respectively. In situ hybridization of paraffin-embedded sections of primary human colon carcinoma and metastases from liver and lymph node revealed cell-specific staining with EGF-R antisense oligonucleotide probes that correlated directly with Northern blot and immunohistochemistry analyses. Since this rapid and sensitive in situ mRNA hybridization technique can be used in properly preserved paraffin-embedded tissue, it allows for retrospective analyses of human tumor specimens using archival material.

Rapid colorimetric detection of epidermal growth factor receptor mRNA by in situ hybridization.

We describe a rapid, formamide-free, random oligomer-enhanced in situ hybridization method in formalin-fixed, paraffin-embedded tissue sections using a biotinylated oligonucleotide probe for colorimetric detection of the mRNA transcript of the epidermal growth factor receptor (EGFR) gene, a putative protooncogene. Transitional cell carcinomas (TCC) of the urinary bladder and oral squamous cell carcinomas (SCC) were examined. A431, a human squamous carcinoma cell line that overexpresses EGFR mRNA, and mature skeletal muscle, known to express EGFR, served as control tissues. A biotinylated poly-T oligonucleotide probe was used to evaluate the preservation of mRNA in the formalin-fixed, paraffin-embedded tissues. The EGFR mRNA and poly-T oligonucleotide probes were constructed with a target-specific, 5' region and a 3' non-hybridizing, biotinylated tail. Random sequence oligomers nine bases long added to the probe cocktail eliminated the need for formamide and sheared carrier DNA in the method. The assay produced strong, specific staining for EGFR mRNA in all cases of TCC and SCC, and in the A431 tumors, but not in skeletal muscle. The assay was completed in approximately 90 minutes. This method may have widespread utility for rapid and specific detection of other mRNA sequences.

Dual organism infection in biopsy specimens from immunocompromised patients: two cases demonstrated by immunocytochemistry.

We report two cases in which immunocytochemistry demonstrated simultaneous infections within a single biopsy specimen obtained from each of two immunocompromised patients in whom multiple organism infection was not suspected. Using a rapid immunocytochemical technique, Pneumocystis carinii and Cryptococcus neoformans were demonstrated in a transbronchial lung biopsy from the first patient; Candida and cytomegalovirus were demonstrated in an endoscopic biopsy of the esophagus from the second patient. Routine hematoxylin and eosin as well as special stains failed to establish the presence of more than one infectious agent in either case. Mycology cultures ultimately confirmed the presence of Cryptococcus in the first case, but cultures were not performed in the second case due to the scant amount of tissue obtained from the biopsy. The results indicate that concurrent infection by more than one opportunistic organism in immunocompromised patients can easily remain unrecognized. The use of immunocytochemistry may thus provide an important adjunct to routine and special stains or specific cultures.

Complete one-hour immunocytochemistry based on capillary action.

We describe a less than one-hour manual method for immunocytochemical analyses of B5 or formalin-fixed, paraffin-embedded tissue sections. The method employs capillary action to sequentially apply, incubate and remove liquid reagents from apposed pairs of up to 20 glass microscope slides and allows for simultaneous immunocytochemical analyses of as many as 10 different antigens. The method described here uses a) positively charged glass slides to rapidly immobilize tissue sections; b) rapid deparaffinization techniques; c) multipurpose reagents; d) ethanol-enriched buffer washes to improve capillary action and reduce nonspecific background; e) a single broad spectrum streptavidin-peroxidase or streptavidin-alkaline phosphatase detection system that identifies most primary monoclonal and polyclonal antibodies; and f) specific immunocytochemical signal amplification by cyclic chromogen enhancement.

Immunocytochemical identification of Rochalimaea henselae in bacillary (epithelioid) angiomatosis, parenchymal bacillary peliosis, and persistent fever with bacteremia.

We report the immunocytochemical identification of Rochalimaea henselae, a newly recognized fastidious, Gram-negative, Warthin-Starry-positive organism, as the common pathogen in bacillary angiomatosis (BA), bacillary peliosis (BP) of the liver and spleen, and persistent fever with bacteremia in immunocompromised patients. Immunogenic proteins of the R. henselae strain isolated from the blood of a febrile immunocompromised patient with BP of the liver were used to produce primary immune serum in rabbits. Using immunocytochemical procedures, the polyclonal antiserum reacted strongly not only with the immunizing strain of the bacteria, but also with other blood isolates of R. henselae (five cases) from both immunocompromised and immunocompetent patients and with the organisms present in the tissue lesions of cutaneous BA (five cases) and BP of the liver (two cases) and spleen (one case). The blood isolates and BA and BP tissue samples were obtained from widely separated geographic areas. The antiserum was weakly cross-reactive with cultures of Rochalimaea quintana, an organism closely related to R. henselae, but this reactivity was eliminated by specific adsorption. The antiserum did not cross-react with the Warthin-Starry-positive organisms associated with cat scratch disease (Afipia felis), syphilis (Treponema pallidum), Lyme disease (Borrelia burgdorferi) or chronic active gastritis (Helicobacter pylori). Likewise, the antiserum did not identify organisms in eight cases of Kaposi's sarcoma, a disorder of immunocompromised patients that is clinically similar to BA. Further studies are needed to determine the prevalence of this newly recognized organism as well as its possible involvement in other angioproliferative diseases.

Detection of Epstein-Barr virus genomes by in situ DNA hybridization with a terminally biotin-labeled synthetic oligonucleotide probe from the EBV Not I and Pst I tandem repeat regions.

We describe a case of acute, disseminated Epstein-Barr virus (EBV) infection which was analyzed for the cellular distribution of viral replication by automated, colorimetric in situ DNA hybridization using a single, synthetic, terminally biotin-labeled oligonucleotide probe composed of 23 consecutive nucleotides selected from the EBV Not I region. The GC-rich, Not I region is a 125-base pair sequence that is repeated in tandem an average of 12.6 times in the EBV genome. The synthetic sequence had 91% base homology with another EBV genomic tandem repeat, the 102-base pair Pst I region, which is also GC-rich, has an overall 70% homology with the Not I region, and is reiterated about 25 times in the viral DNA. Disseminated EBV infection was detected in nuclei of atypical lymphocytes in several organs, including lung, bronchus, trachea, spleen, liver, and stomach, with the probes. In addition, the synthetic oligomer compared favorably with a significantly more expensive, nick translated, biotinylated probe cloned from the BAM HI-V (W), large internal repeat region. This 3.0-kilobase pair (kbp) sequence is repeated an average of 11 times in EBV. Although both probes identified regions repeated multiple times in the virus, and each confirmed an identical tissue distribution for the infection, the signal obtained with the Not I/Pst I probe was more intense and confined to the nuclei of fewer lymphocytes than the general, more weakly distributed signal obtained with the probe from the large internal repeat region. Consistent positive cellular staining was obtained with the Not I/Pst I probe in both EBV-infected control tissue culture cells and in formalin-fixed, paraffin-embedded tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Quality control in immunohistochemistry. Report of a workshop sponsored by the Biological Stain Commission.

Although immunohistochemical methods are increasingly applied in diagnostic histopathology, there has been little standardization or quality control of immunoreagents; and published reports have not standardized Material and Methods for meaningful comparisons of results among clinicians. The Biological Stain Commission-sponsored workshop was convened to address the following issues: a manufacturers' testing program for probity of commercial antibodies, development of a manual for performance criteria and quality control assurance procedures, standardization of package inserts, standardization of information provided in the Materials and Methods sections of publications, establishment of a reagent and procedure clearing house, study of the effects of different fixation regimes on tissue antigens, and investigation of the environmental conditions needed for antigen-antibody interaction. The recommendations of the ad hoc committee and their implications for the future are discussed.

Anatomic viral detection is automated: the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques.

This paper presents the first automated system for simultaneously detecting human papilloma, herpes simplex, adenovirus, or cytomegalovirus viral antigens and gene sequences in standard formalin-fixed, paraffin-embedded tissue substrates and tissue culture. These viruses can be detected by colorimetric in situ nucleic acid hybridization, using biotinylated DNA probes, or by indirect immunoperoxidase techniques, using polyclonal or monoclonal antibodies, in a 2.0-hour assay performed at a single automated robotic workstation.

Colorimetric in-situ hybridization in clinical virology: development of automated technology.

Automation of in situ hybridization is an important first step toward practical implementation of the widely recognized diagnostic potential of nucleic acid hybridization. Our laboratory has concentrated its efforts towards automating colorimetric in situ hybridization on formalin-fixed paraffin-embedded tissue sections. We have capitalized upon the technology developed for the automation of immunohistochemistry (Brigati et al. 1988) and are collaborating with Fisher Scientific in modifying the Fisher Code-On Stainer to achieve successful automated in situ hybridization. Preliminary results are encouraging. We feel that the capillary gap technology has the potential to be modified to automate other hybridization assay formats such as dot and sandwich blot hybridizations. While specifically developed for colorimetric hybridization, the instrumentation is self-contained and could be safely adapted to the use of radio-labeled probes if necessary.

Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method.

An improved method of colorimetric in situ hybridization for the diagnosis of viral infections in standard formalin-fixed, paraffin-embedded tissue sections has been developed. This method employs a 2-hour hybridization with biotin-labeled DNA probes followed by direct colorimetric detection with avidin-alkaline phosphatase complexes. Visual results are obtained within 8 h of cutting the tissue section. Specific histologic localization of cytomegalovirus and adenovirus genetic information has been achieved in infected lung tissues from autopsy or biopsy. Simultaneous denaturation of tissue and probe DNA at elevated temperature (100-105 degrees C) resulted in increased signal. It is our suggestion that these denaturing conditions may be required to denature more fully formalin cross-linked tissue DNA and favor penetrance of probe into the tissues. Comparison of the results of hybridization and viral culture for the diagnosis of CMV infections suggest that in clinical situations hybridization will allow specific diagnosis of productive viral infection more rapidly than viral culture with some loss in sensitivity. Colorimetric in situ DNA hybridization offers the surgical pathologist a powerful new technique that provides an alternative to immunocytochemistry and electron microscopy in the diagnosis of viral pathogens.

A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.

Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.

Correlation of immunohistochemical markers with patient prognosis in breast carcinoma: a quantitative study.

In a series of 46 cases of primary mammary ductal carcinoma, immunohistochemical markers of differentiation (casein, human placental lactogen, alphalactalbumin, pregnancy specific beta-1 glycoprotein, secretory component, CEA, and peanut lectin agglutinins [PLA]), were quantitated via point-counting. An immunoperoxidase bridge (PAP) was used to identify all except the PLA, in which an avidin-biotin complex with alkaline phosphatase development was employed. For none of the markers was there any difference in the quantity present in tumors of patients who had recurred versus the tumors of patients who had enjoyed a minimum of five years disease-free survival. Nonneoplastic epithelium was only rarely positive for these markers. Although eventually surmounted, technical problems significantly hampered application of morphometry to this histochemical material. The authors conclude that these markers have little relationship to differentiation toward mammary duct epithelium and that they do not provide significant prognostic information in patients with breast cancer.

Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.

Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.

Ultrastructural localization of viral antigen in nuclear inclusions of cytomegalovirus infected guinea pig cells.

Intranuclear localization of viral antigens in guinea pig cytomegalovirus (GPCMV) infected guinea pig embryo (GPE) cells was investigated by cross-reactive indirect immunoperoxidase and immunoferritin techniques utilizing guinea pig antisera to GPCMV. Following primary fixation with 4 percent paraformaldehyde, a brief treatment of infected cells with 0.25 percent trypsin was found to enhance penetration of antibodies and the conjugates. Ferritin or horseradish peroxidase conjugated goat anti-rabbit IgG was used as a secondary antibody that cross reacted with guinea pig immunoglobulins in order to reduce non-specific immunochemical reactions. Using light microscopy following immunoperoxidase staining, GPCMV antigens in an intranuclear location were not discernable when the infected cells were stained without pretreatment with trypsin, however intranuclear GPCMV antigens could be visualized after the fixed cells were treated with trypsin for 2-4 minutes prior to addition of the antiserum. Electron microscopic examination following indirect immunoferritin staining revealed viral antigens localized on viral capsids and on scattered electrondense amorphous matrices but not on the surrounding tubular structures on fibrils. The possibility that tubular structures may be a host cell product produced in response to GPCMV infection is discussed.

Primary gastric choriocarcinoma. An immunohistological study.

Two cases of extragenital choriocarcinoma arising in association with gastric adenocarcinoma are described. Using immunohistochemical methods, cells containing human chorionic gonadotropin, beta-subunit (hCG-beta) were localized exclusively to the choriocarcinomatous portion of the tumor. One patient whose tumor appeared to confined to the stomach at the time of surgery, had elevated serum hCG-beta postoperatively. Metastases became evident subsequently.