Syphilitic placentitis: Demonstration ofTreponema pallidum by immunoperoxidase staining

Virchows Archiv - We report a case of early congenital syphilis in which the placenta showed diffuse proliferative villitis andTreponema pallidum was identified by indirect immunoperoxidase stain...     

Neutralization test for BK virus: plaque reduction detected by immunoperoxidase staining

We developed an immunoperoxidase staining test to detect structural antigens of BK virus (BKV) in Vero cell cultures. This test was used to examine the neutralizing activity of human and immunized animal sera. It was shown that sera positive for BKV antibodies measured by hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) were able to prevent expression of BKV structural antigens in cell cultures. The correlation between titers in the hemagglutination inhibition test, levels of BKV IgG measured by ELISA, and the titers assayed by the immunoperoxidase neutralization test was high. We suggest that this type of test may be used instead of conventional neutralization tests for other viruses with slowly developing cytopathogenic effects.     

Rapid immunoperoxidase staining of lymphocyte antigens using microwave irradiation

The innovative application of microwave irradiation in the immunoperoxidase staining of a wide range of labile lymphocyte antigens is described. Fifteen second irradiation at 320 watts produced excellent adherence of the frozen sections to the glass slides without loss of surface antigen staining. This brief procedure eliminated the need for much longer periods of freeze drying or drying at 4 degrees C. Microwave irradiation was also used to accelerate the incubation of the primary antibody. Thirty seconds of irradiation at 320 watts produced specific antigen staining of an intensity which was as good as that obtained with much longer durations of incubations at room temperature. Cytomorphologic preservation was also good and background staining was minimal.     

Diagnosis of Junin virus in cell cultures by immunoperoxidase staining

Summary Vero cells grown in Leighton tubes were infected with blood taken from Argentine Hemorrhagic Fever patients. In 11 out of 12 cases, and between the 2nd–8th days p.i. of the monolayers, Junin viral antigen was detected by the PAP method.With 1 Figure     

Comparison of immunofluorescence and immunoperoxidase staining for identification of rubella virus isolates.

To explore possible advantages which immunoperoxidase (IP) staining might have over immunofluorescence (IF) staining for identifying rubella virus isolates, direct comparative studies were done on the same coded clinical materials using the same rubella immune rabbit serum as the primary antiserum in both systems. The rubella immune rabbit serum and conjugated anti-rabbit immune globulins could be used more dilute in the IP system than in the IF system. Both IP and IF staining detected rubella antigen in all specimens which were positive by interference. IP staining also detected low levels of rubella antigen in a few additional specimens which had originally been positive for rubella virus, but which on retesting were negative by interference and IF staining. With second-cell-culture-passage material, IP and IF staining showed comparable specificity, and the few specimens which reacted nonspecifically generally did so in both systems. Cell cultures inoculated directly with urine specimens showed greater nonspecificity by IP than by IF, but this activity could be abolished by pretreatment with sodium azide and peroxide; other methods tried for inactivating endogenous peroxidase activity destroyed rubella antigen as well. The intensity of staining for positive specimens was comparable in the two systems. However, more antigen was demonstrable in both systems when BHK-21 cells were inoculated as a cell suspension and then permitted to grow into monolayers than when the same specimens were inoculated into preformed monolayers. IP staining was considered to be a highly satisfactory alternative to IF staining for identification of rubella virus isolates.     

Rapid immunoperoxidase staining of axons for frozen section diagnosis of nerve biopsy specimens.

A new method of freezing and embedding a nerve biopsy specimen and staining it with the immunoperoxidase technique for neurofilaments was developed to overcome the difficulties normally encountered in the assessment of tiny portions of nerve. The method clearly shows the architecture of the nerve, the exact number and size of all axons present, and the degree of fibrosis present. The entire procedure may be accomplished in 20 minutes.     

A rapid and simple immunoperoxidase staining procedure for blood and bone marrow samples

A fast and simple indirect immunoperoxidase staining technique is described, which can also be used for the characterization of cells with high endogenous peroxidase activity such as many haemopoietic cells. It is based on the combination of a newly developed glucose-oxidase plus glucose procedure for the inhibition of endogenous peroxidase activity with a standard 2 or 3 layer immunoperoxidase staining protocol. Glucose-oxidase plus glucose mixture completely inhibits endogenous peroxidase activity without having a detectable deleterious effect on any of the cellular antigens so far studied. In many instances this permits the use of only 1 layer of horseradish peroxidase (HRP)-labelled antibodies. The glucose-oxidase plus glucose mixture can also be added to cells together with the HRP-labelled antibody solution without losing its inhibitory effect for endogenous peroxidase activity and without leading to a visually detectable loss of the activity of the HRP conjugate. Consequently, the separate incubation step previously necessary for the inhibition of endogenous peroxidase activity becomes superfluous.     

Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: comparison with flow cytometric immunophenotyping

CD10 is common in B-precursor acute lymphoblastic leukemia (ALL) but is rare in acute myeloid leukemia (AML). However, until recently, analysis for CD10 has generally required fresh or frozen tissue. 56C6 is a monoclonal antibody that is now commercially available for the detection of CD10 in routinely processed paraffin-embedded tissue. Immunoperoxidase stains for CD10 on paraffin-embedded bone marrow core biopsy specimens (B5-fixed, decalcified) and marrow aspirate clots (formalin-fixed) were compared with flow cytometric immunophenotyping for CD10 on fresh cell suspensions in 20 cases of AML and in 30 cases of ALL. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 98% (49 of 50) of acute leukemias. The results matched in 100% (20 of 20) of AML. Five percent (1 of 20) of AMLs expressed CD10. Two of the AMLs with monocytoid differentiation were interpreted as negative for CD10 by flow cytometry, although these had nonspecific dim immunofluorescence for multiple markers, including CD10, and these cases were negative by immunohistochemistry. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 97% (29 of 30) of ALL. Eighty-four percent (21 of 25) of B-precursor ALL and 40% (2/5) of T-lineage ALL expressed CD10 by immunohistochemistry. In 1 case of B-precursor ALL, CD10 was dimly positive in 24% of the blasts by flow cytometry but negative by immunohistochemistry. We conclude that immunohistochemical staining of paraffin-embedded tissue, either B5- or formalin-fixed, is an effective method for the detection of CD10 in acute leukemia. This technique is useful in distinguishing AML from ALL.     

Identification of monoclonal antibodies to pancreatic islet cells by immunoperoxidase staining

Immunoperoxidase staining and enzyme-linked immunosorbent assay (ELISA) were used to identify monoclonal antibodies that reacted with pancreatic islet cells. All monoclonal antibodies produced against isolated human or rat pancreatic islets including one mouse autoantibody reacted with pancreatic islets in formalin-fixed pancreas sections, but not with rat kidney or thyroid. Reactivity was also found with suspensions of normal rat islet cells and rat insulinoma cells using a 3-stage immunoperoxidase procedure and an ELISA technique. Differences were observed in staining intensity between the various antigenic substrates tested suggesting variable cross-reactivity and/or number of epitopes. The sensitivity of the immunoperoxidase technique proved to be favourable for identification of monoclonal antibodies that recognize cellular constituents such as islet cell antigens present in low concentrations.     

Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis

In corneal scraping smears from 13 patients with clinically suspected herpes simplex keratitis (HSK), HSK is demonstrated by means of peroxidase-antiperoxidase (PAP) technique with antisera to herpes simplex virus (HSV) in Papanicolaou-destained cellular samples. The staining for HSV antigen was present in seven cases of corneal scraping smears with superficial keratitis (dendritic and geographic ulcers) while six cases of stromal keratitis (deep keratitis) failed to show HSV antigen except in one case. Specific antigen for HSV was predominantly present in the cytoplasm rather than in the nucleus. Immunoreactions were negative with HSV antisera in patients with other infections and in those in a normal control group. Using the PAP technique, detection of HSV antigen in corneal scraping smears was of great value in the diagnosis of HSK, especially in cases of superficial keratitis.     

Elimination of iron pigments and background staining which mask immunoperoxidase reactions

When immunohistochemical stainings are applied to demonstration of antigens in histopathological specimens, the ferrous pigments which may be present in the tissues usually mask the final precipitates of the reaction. These pigments can be eliminated with oxalic acid or sodium dithionite after the immunohistochemical staining. These treatments also help in the bleaching of unspecific background stain.     

Factor VIII-related antigen staining by immunoperoxidase technic in smaller laboratories: a potential problem

A case of metastatic squamous cell carcinoma to bone was studied using immunoperoxidase methods for Factor VIII-related antigen, producing positive staining in the tumor cells. Electron microscopy proved the squamous nature of the tumor, and, subsequently, a pulmonary primary tumor was found. Testing of additional tumor types revealed positive staining for Factor VIII in squamous cell carcinomas, tumors with squamous differentiation, and in renal cell carcinoma. Problems of antibody purity and specificity always must be considered, but in the smaller laboratory where personnel and expertise for such quality control do not exist, there is a hazard of nonspecific reaction and misdiagnosis.     

Impact of cell culture sensitivity and virus concentration on rapid detection of herpes simplex virus by cytopathic effects and immunoperoxidase staining.

Tremendous interest has been generated in the commercial kits now available that incorporate herpes simplex virus isolation in cell culture with immunoperoxidase staining for viral antigen detection. Most studies comparing commercial kits with conventional cell culture techniques have found the kits to be less sensitive. However, different cell cultures were used for the two methods. In this study, mink lung, rabbit kidney, MRC-5, and Vero cells were compared for reisolation of herpes simplex virus from clinical specimens in which viral infectivity titers were concurrently determined. When specimens contained high titers of infectious virus, the cell system used made little difference and all specimens were detected by immunoperoxidase staining at 48 h postinoculation. However, when specimens contained low concentrations of virus, the differences in sensitivity between cell systems became apparent in rapidity of detection and overall isolation rate. Mink lung and rabbit kidney cells were both more sensitive than MRC-5 cells; Vero cells were significantly less sensitive than the other cells tested. The application of immunoperoxidase staining shortened the time to virus detection and lessened, but did not eliminate, the differences between the cell systems. Cytopathic effects alone in the most sensitive cell system equaled or exceeded immunoperoxidase staining applied in less-sensitive cell cultures.     

Silver staining method for nucleolar organizer regions in cervical smears

The distribution of nucleolar organizer regions (NORs) was studied in Papanicolaou preparations of cervical smears in order to distinguish benign from preneoplastic lesions. Destained smears (six defined as normal, six as inflammatory with squamous metaplasia, six as CIN I, six as CIN II, and five as CIN III) were submitted to the Ag-NOR method after staining with Orange G and EA36. Ag-NOR count was performed in previously outlined fields on the smears. Statistically significant differences (P < .05) were found between the normal smears, inflammatory smears with squamous metaplasia, and each grade of CIN. We conclude that the Ag-NOR technique could be useful to evaluate cervical smears of doubtful interpretation, using previous demarcation of the abnormal fields/cells. Diagn. Cytopathol. 16:497–499, 1997. © 1997 Wiley-Liss, Inc.     

Improved detection of medically important fungi by immunoperoxidase staining with polyclonal antibodies

This study was performed to identify pathological fungi of eight species [Aspergillus fumigatus, Candida albicans, Torulopsis (Candida) glabrata, Cryptococcus neoformans, Fusarium anthophilum, Rhizopus oryzae, Sporothrix schenckii and Trichosporon beigelii] in formalin-fixed, paraffin-embedded tissue sections by indirect immunoperoxidase staining. Mature albino rabbits were immunized with formalin-killed organisms. Antibodies were prepared by precipitation. Immunoperoxidase staining was applied to the paraffin-embedded tissue sections of experimentally infected mice and human autopsy and surgical specimens. Although the cell walls of each fungus stained clearly, many cross-reactivities appeared. However, it was possible to obtain specificity for the eight species by absorption and dilution of the antisera.