The cytologic features of chlamydial cervicitis

Chlamydial cervicitis is a common and important infection. Diagnostic cytologic criteria have been proposed, but not generally accepted. To better evaluate the cytologic changes, cervical cultures for Chlamydia trachomatis and duplicate cervical smears for Papanicolaou staining and immunofluorescence staining for chlamydial organisms were taken from 496 patients. A total of 61 (12.3%) of the patients had a positive culture for C. trachomatis. By immunofluorescence, the organisms were present as very small extracellular elementary bodies in mucus or as similar bodies in leukocytes; inclusions within epithelial cells were seen in only two cases. The organisms did not stain with the Papanicolaou stain. Chlamydial infection correlated with the degree of inflammation, with the presence of histiocytes and lymphocytes, especially large "transformed" lymphocytes, and with the presence of unidentified short bacteria, which stained red with the Papanicolaou stain. These features predict which patients should be tested more definitively for the presence of chlamydial organisms. However, we found no cytologic criteria that can reliably permit its diagnosis.     

Sensitivity and specificity of the Papanicolaou-stained cervical smear in the diagnosis of Chlamydia trachomatis infection

Two hundred young women had simultaneously prepared cultures for Chlamydia trachomatis and cervical smears; they also completed a questionnaire. Twelve of the chlamydial cultures were positive. There was poor correlation between the culture results and the cytologic morphology or symptoms. On initial blind reading, only 10% of the smears cytologically interpreted as positive were actually positive by culture. Under the most favorable (non-blind) interpretation, only 23% of the smears cytologically interpreted as positive for chlamydial infection were also culture positive. Because of the high incidence of false positives, we conclude that routine cytologic examination of Papanicolaou-stained smears is not an acceptable method for the diagnosis of chlamydial infections of the cervix. Immunoperoxidase staining of duplicate smears did not appear to be a successful replacement for culture.     

Oncofetal antigens as tumor markers in the cytologic diagnosis of effusions

To test the value of oncofetal antigens in the cytologic diagnosis of effusions, immunoperoxidase staining with antisera to carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), pregnancy-specific beta 1-glycoprotein (SP1) and placental alkaline phosphatase (PLAP) was carried out on pleural and peritoneal fluids from 72 cases. Sections of formalin-fixed, paraffin-embedded cell blocks were used in most cases; cytocentrifuge preparations were used in some. Reactions were negative with all antisera in 23 of 24 nonmalignant effusions as well as in all 7 cases of malignant mesothelioma and 4 cases of malignant lymphoma. In 24 of 36 confirmed carcinomatous effusions, staining was positive with one or more antisera, including anti-CEA positivity in 23 of the 24 cases. In 5 of the 24 cases with positive staining, a confident diagnosis of malignancy had not been made on routine cytologic preparations. Immunoperoxidase staining for CEA appears to be of supportive value in the cytologic diagnosis of malignancy in effusions.     

Evaluation of proposed cytomorphologic criteria for the diagnosis of Chlamydia trachomatis in Papanicolaou smears

To evaluate the proposed cytomorphologic criteria for the cervical cytologic diagnosis of Chlamydia trachomatis infection, a study was made of 171 endocervical smears. All cytomorphologic elements that could be ascribed to Chlamydia trachomatis infection were correlated with the diagnostic confirmation of this microorganism by monoclonal antibody (MAb) staining. The presence of Chlamydia trachomatis was detected in 21 samples (12.28%) by MAb staining. Comparing the cytomorphologic results with the MAb results, the sensitivities and specificities of the Papanicolaou smear diagnoses were 19% and 86% using the cytologic criteria proposed by Gupta and coworkers, 38% and 87% using the criteria proposed by Kiviat and coworkers and 23% and 91% using the criteria proposed by Shiina. In view of (1) its low sensitivity, (2) the subjective elements and individual variations in the proposed cytologic criteria, (3) the similarity with Trichomonas vaginalis-produced exudates and (4) the implications of a misdiagnosis of a sexually transmitted disease, it is concluded that cervical cytology is not useful for ascertaining the presence of Chlamydia trachomatis.     

Determination of estrogen receptors in cytologic imprints of breast cancers using immunocytochemical methods.

Immunostaining of estrogen receptors (ERs) was carried out on imprints of 62 breast carcinomas using monoclonal antibodies and a sensitive immunoperoxidase technique (the Abbott ER-ICA kit). The results were compared to those obtained by the conventional biochemical analysis of cytosol proteins and to the degree of tumor differentiation. The cytologic specimens were insufficient for analysis in 6 cases; of the remaining 56 cases, 37 (66%) showed a positive ER reaction. In 51 cases with both types of ER analysis, the immunocytochemical staining of the imprints correlated strongly with the biochemical analysis in 44 cases and weakly in 3. Four cases were negative immunocytochemically and positive biochemically. Among the ductal carcinomas, well-differentiated tumors had higher percentages of ER-positive cells than did poorly differentiated tumors. These results show that the immunoperoxidase method is a highly specific and sensitive technique for the evaluation of ER content; it may be applicable to small samples of tumor tissue and may provide additional information for identifying hormonally responsive breast tumors.     

Prevalence of Chlamydia trachomatis and oncogenic human papillomavirus types in cytologic atypia of the uterine cervix

A history of having substantial Chlamydia trachomatis exposure as detected by serum antibodies is a cofactor of human papillomavirus (HPV) mediated cervical carcinogenesis. In this study, we examined the concurrent C. trachomatis infections in cytologic atypia of the uterine cervix in order to evaluate the impact of C. trachomatis infection in patients with high risk for cervical intraepithelial neoplasia. Cervical scrapes form 707 patients were subjected to PCR amplification with primer sets for HPV and C. trachomatis. Based on negative beta-globin results, 10 specimens were not eligible for further analysis. Oncogenic HPV types were detected in 278 specimens (39.8%). C. trachomatis was found only in six specimens (0.9%). In conclusion, concurrent C. trachomatis infection was uncommon and hence it was an improbable risk factor in cytologic atypia.     

A proposed mouse model for acute epididymitis provoked by genital serovar E, Chlamydia trachomatis

This study was conducted to determine the efficacy of the male mouse as a model for epididymitis caused by human genital serovar E, Chlamydia trachomatis. C. trachomatis was reisolated from all tissues removed on Days 3, 5, and 7 post inoculation (pi). Although some infected epididymides removed on Days 10, 14, and 21 pi were positive, control tissues remained negative. Histopathology of tissues showed a heavy, mixed inflammatory infiltrate consisting of polymorphonuclear cells and lymphocytes. Serum antibody to C. trachomatis was detected in the infected mice only (titer greater than or equal to 1:32). Chlamydial inclusions and individual elementary bodies were confirmed by immunofluorescent and immunoperoxidase staining up to Day 7 pi. These data show that the male mouse is susceptible to C. trachomatis infection and is appropriate for studies dealing with the effect of C. trachomatis on male fertility.     

A gastrointestinal-specific monoclonal antibody that may be of clinical value in cytologic material

A new monoclonal antibody (MAb), 29-10, produced by immunization of mice with cells from the SW 1116 colorectal carcinoma cell line, detected an antigen present in cytologic touch imprints of surgically resected normal and neoplastic gastrointestinal (GI) tissue, including specimens from the stomach and the colon. These imprints were fixed in 95% ethanol and stained with the avidin-biotin immunoperoxidase technique. In tested cases, 22 (100%) of 22 imprints from GI adenocarcinomas and from normal GI tissue, as well as 13 (56.6%) of 23 imprints from colonic polyps, stained positively while no staining was demonstrable in imprints from other tissues. In histologic sections, only 4 (23%) of 17 colonic adenocarcinomas and 3 (11.5%) of 26 polyps stained positively. The staining ability of MAb 29-10 was compared to that of MAb 19-9, another colorectal antibody, and was found to be markedly superior for binding of the antigen in cytologic preparations. This tissue-specific antibody may be useful in identifying malignant cells of metastatic carcinoma as to their GI tract origin.     

Chlamydia trachomatis infections in asymptomatic women. Results of a study employing different staining techniques

A total of 300 cervical smears randomly collected from asymptomatic women in a mass-screening program for the detection of cervical carcinoma was investigated for Chlamydia trachomatis infection by the use of Papanicolaou and immunofluorescence staining. Features of chlamydial infection detected in 18 cases by Papanicolaou-stained smears were confirmed in 11 cases with immunofluorescence; not a single case that was negative in the Papanicolaou-stained smears was positive by immunofluorescence. The presence of Chlamydia in the Papanicolaou-stained smears in ten cases, including two cases that were negative by immunofluorescence, was also proven by either immunoperoxidase staining or in situ hybridization. On the other hand, either immunoperoxidase or in situ hybridization gave false-negative results in two of the ten cases. Therefore, the combined use of different techniques demonstrated that false-negative results occurred with all techniques, except with Papanicolaou-stained smears, whose sensitivity is apparently the highest.     

Detection of sexually transmitted diseases by urethral cytology, the ignored male counterpart of cervical cytology

The detection of sexually transmitted diseases by urethral cytology was investigated in 270 men examined by urethral swabbing smears. Each sample was used to prepare a wet mount smear and smears for staining by the Papanicolaou, Gram and methylene blue techniques. A fifth smear was used for direct staining with fluorescein-conjugated monoclonal antibodies for the detection of Chlamydia trachomatis. The smears were examined for cytoplasmic and nuclear changes as well as for pathogenic organisms and inflammatory changes. Infections with Chlamydia trachomatis, Neisseria gonorrhoeae and human papillomavirus (HPV) produced distinctive cytologic patterns similar to those seen in cervicovaginal smears from women. The patterns in candidiasis, trichomoniasis and herpes simplex virus infection were not as diagnostic. Particularly noteworthy were the nuclear alterations, which appeared to be proplastic in HPV infection but retroplastic in Chlamydia infection. The results of this study indicate that urethral cytology would be an invaluable addition in diagnosing sexually transmitted diseases in men, particularly in the case of Chlamydia and HPV infections. The monomorphic structure of urethral columnar epithelium, as compared to the cervical epithelium, seems to result in a clearer and more constant response to pathogenic infections, as seen in the resulting smears.     

The role of immunoperoxidase staining in diagnostic cytology

A survey was made of the immune staining characteristics of 60 malignant neoplasms. Cytologically positive smears from each case were tested against a panel of six antibodies (alpha-antichymotrypsin, carcinoembryonic antigen, cytokeratin, desmin, vimentin and S-100 protein). The smears were decolorized and stained with polyclonal sera using the standard avidin-biotin immunoperoxidase procedure. In selected cases, the application of Diatex compound for partition of smears was necessary to obtain optimal results. Most staining reactions reflected the histogenesis of the neoplasms. However, more than one of five reactions was nonconclusive due to background staining, scanty cellularity or poor cytoplasmic preservation; furthermore, the 238 reactions scored as positive or negative included 33 unexpected positives and 15 unexpected negatives. In a series of 20 additional cases, selective immunoperoxidase staining was used in an attempt to solve specific diagnostic problems; the results in 13 of 15 cases with conclusive staining agreed with the cytologic impression. It is concluded that standard immunoperoxidase techniques can contribute to the solution of certain diagnostic problems in cytology; however, the results should be interpreted with caution and with full knowledge of the limitations of the technique.     

Immunocytochemical detection of cyclin, a proliferation-associated protein, in cytologic preparations.

Cyclin is a nuclear protein associated with DNA-polymerase delta, whose expression correlates with cell proliferation in vitro. To assess the value of cyclin staining in diagnostic cytology, an anticyclin monoclonal antibody was used to survey cyclin expression in cytologic preparations obtained as "bench top" aspirates from surgically resected specimens. The tissues assayed included carcinomas and normal and benign proliferative tissues of renal, mammary, prostatic and colonic origin. Staining was performed via the avidin-biotin-complex immunoperoxidase method. The staining of tumor cells was nuclear, with sparing of the nucleoli; the results were variable in different areas of a given tumor and varied significantly between tumors of the same histopathologic type. Benign proliferative tissues also showed staining. Nonproliferative tissues, such as renal tubules adjacent to a renal cell adenocarcinoma, were largely, but not entirely, nonstaining. The percentage of cyclin-positive nuclei was sometimes much higher than the typical percentages of tumor cells found in S phase. This observation was confirmed in two cases in which cyclin staining was much greater than the percentage of S-phase cells detected by flow cytometry. This suggests either stabilization of the protein beyond S phase in cells and/or dysregulation of cyclin expression in malignant cells. The viability of unfixed surgically resected tissue may also have affected the detection of cyclin, a problem that should not exist with clinically aspirated tissue fixed immediately after aspiration. These preliminary observations suggest that the selective use of cyclin staining may facilitate cytologic diagnoses. Furthermore, the wide range of cyclin expression within tumors of one histologic type suggests that cyclin expression may serve as a new parameter for investigating tumor behavior and prognosis.     

Immunocytochemical detection of estrogen receptors by staining with monoclonal antibodies on cytologic specimens of human breast cancer

A study was undertaken to test the possibility of determining the estrogen receptor (ER) content in human breast cancers by staining with commercial specific monoclonal antibodies (MAbs) on cytologic specimens (touch imprints and fine needle aspirates). The aspirates were suspended in a cell culture medium and cytocentrifuged onto slides to preserve their morphologic characteristics and to allow a proper immunocytochemical staining for ERs. MAb staining for ER was also performed on the respective surgical samples. The staining of cytologic samples for ER showed 100% specificity and 95% sensitivity in comparison to the staining of the histologic samples. Moreover, comparison of the percentage of stained cells in the cytologic specimens to the ER content in the respective surgical specimens, as assayed by the dextran-coated charcoal method, showed the MAb staining of cytologic samples to have 94% specificity and 100% sensitivity. These results support the reliability of MAb staining for ERs in cytologic samples and suggest that it could be the assay of choice in particular clinical settings in the evaluation of primary and recurrent breast cancers.     

Fine needle aspiration cytology of granulomatous diseases of the lung, including nontuberculous Mycobacterium infection

The spectrum of cytomorphologic changes of pulmonary granulomas diagnosed by fine needle aspiration (FNA) biopsy is reported, with a review of the pertinent literature concerning the cytologic diagnosis of granulomatous disease. In our cases, organisms were not seen in the Papanicolaou-stained smears. Recognition of the granulomatous cellular pattern, however, resulted in a thorough search for organisms by special stains, and an etiologic diagnosis was made in each case. These cases emphasize the need for routine staining and culture of FNA material when an initial diagnosis of malignancy is not made. One of the cases appears to be the first report of a nontuberculous Mycobacterium infection diagnosed by fine needle aspiration biopsy.     

Infantile chlamydial conjunctivitis. A comparison of Papanicolaou, Giemsa and immunoperoxidase staining methods

The use of conjunctival smears to diagnose infantile Chlamydia trachomatis infection increased sixteen-fold in our hospital between the years 1979 and 1984. The present study was conducted to compare Papanicolaou and Giemsa staining methods with the avidin-biotin technique of immunostaining utilizing a highly specific antichlamydial monoclonal antibody. On retrospective review of 33 patients, chlamydial infection was diagnosed in 61% of the Papanicolaou-stained and 64% of the Giemsa-stained slides. After the Papanicolaou-stained slides were destained and immunostained with the antichlamydial antibody, round particles corresponding in size to elementary and reticulate bodies were readily seen in 79% of the cases. In comparison with the immunoperoxidase method, the sensitivity and specificity of Papanicolaou staining were 73% and 86%, respectively, while the corresponding figures for Giemsa staining were 77% and 100%, respectively. The study established the applicability of the immunoperoxidase method to this clinical condition, confirmed the accuracy of diagnoses with routine stains and highlighted the increasing incidence of chlamydial conjunctivitis in our hospital population.     

Detection of herpes simplex virus infection using glucose oxidase-antiglucose oxidase immunoenzymatic stain

Herpes simplex virus (HSV) infected cells have been detected in tissue culture and human cell specimens by an immunoenzymatic staining method using the fungal enzyme glucose oxidase. Infected cells from culture or human specimens appear as dark blue, brown, or red, depending on the tetrazolium salt used in the disclosing reaction, with virtually no staining of uninfected cells. The specificity and sensitivity of this method and of the more commonly used immunoperoxidase method are comparable, but the immunoglucose oxidase method avoids the problems of nonspecific staining by the endogenous peroxidase present in mucosecretions and inflammatory cells. Staining time can be reduced up to 40% of that necessary for the unlabeled immunoperoxidase procedure without compromising the quality of staining results.     

Comparison of culture, enzyme immunoassay and nucleic acid sandwich hybridization in detecting Chlamydia trachomatis in genital tract infections

Abstract Culture, enzyme immunoassay (Chlamydiazyme™) and nucleic acid sandwich hybridization were compared in detecting Chlamydia trachomatis in uncomplicated genital tract infections. Urethral and cervical specimens were collected from 100 males and 100 females attending a sexually transmitted disease clinic. Chlamydial culture was performed under optimal conditions (duplicate inoculation within the day specimen was collected, culture in vials, monoclonal antibody staining of inclusions, blind passage for negative samples). Here the sensitivity of culture exceeded that of the rapid methods. The sensitivity of a chlamydial antigen detection method (Chlamydiazyme™) was 68% in male and 86% in female specimens, when compared with culture, and the specificity was 100% and 97%, respectively. Acinetobacter calcoace9icus present in clinical specimens did not interfere with Chlamydiazyme™. The sensitivity of the nucleic acid sandwich hybridization was 53% of that of the culture, and specificity 100%. By comparing the three methods it was apparent that the rapid methods did not reveal chlamydial infections not detectable by culture. Thus, if performed carefully, culture is the most sensitive diagnostic method in acute genital infections due to C. trachomatis .     

Estrogen receptor determination in fine needle aspirates of the breast. Correlation with histologic grade and comparison with biochemical analysis

Material obtained by fine needle aspiration (FNA) of 25 surgically removed breast carcinomas was tested for the immunocytochemical localization of estrogen receptor (ER) using the peroxidase-antiperoxidase method and a monoclonal antibody developed against human breast cancer ER. The results were compared to those obtained by the conventional biochemical analysis of cytosol protein. A semiquantitative relationship between the immunoperoxidase stain and the biochemical analysis suggests that cases in which greater than 70% of the cells stain and in which intense staining is present are likely to contain ER in a concentration of greater than 250 fmol/mg of cytosol. Less than 15% stained cells and an absence of intense staining is indicative of a concentration of less than 10 fmol/mg. In only one case was there a significant difference in positivity between the two methods, possibly as a result of a functional heterogeneity of the tumor cell population. Intense staining is strongly suggestive of a tumor of low histologic grade and was never seen in tumors with a high histologic grade or nuclear grade. The immunoperoxidase method of ER detection on material obtained by FNA is a semiquantitative means of selecting patients with breast cancer who are likely to respond to hormonal therapy. The method overcomes many important disadvantages of cytosol analysis and provides clinically significant information regarding the ER content and the degree of tumor differentiation.     

Diagnostic utility of sialosyl-Tn in discriminating carcinomatous cells from benign mesothelium in body cavity effusions

OBJECTIVE: Detecting malignant cells in the setting of reactive mesothelium can be difficult. Several techniques have been tried but without widespread acceptance. Sialosyl-Tn (STn) is an aberrantly glycosylated precursor of the MN blood group antigen frequently expressed in carcinomas and dysplastic epithelium. We investigated the STn monoclonal antibody for its clinical utility as an isolated stain to discriminate benign mesothelium from malignant cells. STUDY DESIGN: Cell block material from 72 cases of body cavity fluids were immunostained for STn using the avidin-biotin complex method without antigen retrieval. Slides were incubated overnight at 4 degrees C in a humidified chamber. RESULTS: Strong immunoreactivity was noted in 31/40 (77%) carcinomatous cases. Only moderate staining was noted in 1 of 28 (4%) benign effusions and weak staining in 5 (18%) additional benign cases. Specificity was 100%, sensitivity 78%, positive predictive value 100% and negative predictive value 76%. No staining was noted in four noncarcinomatous malignant effusions. CONCLUSION: STn may have diagnostic value in this cytologic setting as part of a diagnostic panel but not as an isolated stain.