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.     

Diagnosis of herpes simplex virus in routine smears by an immunoperoxidase technique

Routine Papanicolaou-stained cervicovaginal smears from 59 patients were cytologically screened for herpetic infection. Forty-one of the smears were positive for herpes, 2 were suspicious and 16 were negative. All 59 slides were then destained and restained by a commercial immunoperoxidase kit for the detection of herpes simplex virus (HSV). The immunoperoxidase stain was positive in 23 of the 41 cytologically positive slides. One of the 2 cytologically suspicious slides was also immunoperoxidase positive, as was 1 of the 16 cytologically negative slides. This study indicates that immunoperoxidase staining is very specific but not quite as sensitive as routine Papanicolaou-stained smears in the detection of HSV. The immunoperoxidase method is thus recommended for the confirmation of HSV cases rather than for the routine diagnosis of HSV infection.     

Detection of Chlamydia trachomatis in Papanicolaou-stained cervical smears by an indirect immunoperoxidase method

A two-step (indirect) immunoperoxidase method directed against Chlamydia trachomatis was developed. The method was then used to evaluate the specificity of cytologic changes suggestive of C. trachomatis in Papanicolaou smears of cervical specimens from women who were culture-negative for the organism. Positive immunoperoxidase staining was detected in 9 of 21 cases (43%) tested. Technical problems, especially background staining, precluded interpretation in the remainder of the cases. Cervical cytology, as demonstrated by immunoperoxidase staining, may, in some instances, be more sensitive than the culture. However, because the etiology of cytologic changes not specifically identified by immunoperoxidase staining may be due to other organisms or factors, immunoperoxidase procedures, as described, should not replace culture for confirmation of cytologic findings suggestive of C. trachomatis.     

Application of the immunoperoxidase technique to cell block preparations from fine needle aspirates

Immunocytochemistry on fine needle aspiration (FNA) material has been mainly performed on cytologic preparations; there have been few reports on the use of FNA cell blocks. This study compared the intensity scores of immunoperoxidase staining on FNA cell block preparations from 21 breast, 12 thyroid and 10 lymph node aspirates with the scores on the corresponding surgically excised specimens. FNA materials for cell blocks were fixed in formalin and embedded in agar. Ten commercially available antibodies forming three panels were studied using standard peroxidase-antiperoxidase and avidin-biotin complex techniques. In general, the staining results on the FNA cell block sections agreed with those on the surgical specimens; in addition, there were fewer aberrant positive staining results and much less background staining in the cell block sections. These phenomena were most striking with the cytokeratin antibodies. It is concluded that immunoperoxidase staining on FNA cell block preparations is reliable; the advantages of the use of cell block sections as opposed to smears are discussed.     

Fine needle aspiration biopsy in the diagnosis of tuberculosis

Bacteriologic data and aspiration smears obtained by fine needle aspiration (FNA) biopsy for a series of 39 cases of tuberculosis were reviewed. Based on the morphologic features of the aspiration smears, the cases were divided into two groups: 18 cases in which distinct epithelioid granulomas were present and 21 in which no granulomas were found but large amounts of necrotic debris with variable numbers of polymorphonuclear cells, histiocytes and lymphocytes were present. Material from the FNA biopsy specimen was submitted for culture and fluorescence studies in 34 cases (15 with and 19 without granulomas). In the first group, auramine-rhodamine staining of smears was positive in 4 of 15 cases and Mycobacterium tuberculosis was isolated in 12 of 15 cases. In the second group, auramine-rhodamine staining was positive in 9 of 19 cases and culture was positive for M. tuberculosis in 16 of 19 cases. The results indicate that studying FNA smears by light microscopy and bacteriologic culture is an effective way of diagnosing tuberculosis.     

Herpes simplex virus in postradiation cervical smears. A morphologic and immunocytochemical study.

From January 1987 to August 1988, cytomorphologic criteria of both herpes simplex virus (HSV) and radiation effects were observed in Papanicolaou smears from 3 of 1,340 patients who had received radiotherapy for squamous cell carcinoma of the cervix. Avidin-biotin immunoperoxidase staining, using a rabbit IgG polyclonal HSV antibody, confirmed the presence of HSV antigen in those three postradiation smears. Both multinucleated molded cells and epithelial cells that lacked cytopathic effects were positive for HSV. Three other postradiation smears from these cases were similarly positive for HSV antigen; the one preradiation smear was negative. In situ hybridization and immunoperoxidase studies on sections from the preradiation biopsies were negative: severely altered neoplastic cells showed no reactivity. The absence of HSV markers in the preradiation specimens suggests that the HSV infections were secondary to the radiotherapy; further studies are needed to prove this association and to assess the possible mechanisms. These cases clearly indicate that the overlapping features of radiation and viral effects (such as multinucleation) may be present simultaneously.     

Value of image-guided needle aspiration cytology in the assessment of pelvic and retroperitoneal masses. A study of 112 cases

OBJECTIVE: To evaluate the diagnostic value of the noninvasive method of image-guided needle aspiration cytology (NAC) in the assessment of radiologically detected pelvic and retroperitoneal space-occupying lesions (excluding the pancreas, kidney and adrenal). STUDY DESIGN: NAC was performed under computed tomographic or ultrasound guidance on 112 patients suspected of having a pelvic or retroperitoneal mass. Cytologic examination was performed on site after staining smears with the Papanicolaou method. In addition, air-dried smears, fixed smears, filter preparations from needle washings and cell blocks were studied. The NAC diagnosis was supported by examining cell blocks; further support was obtained with a tissue biopsy in some cases. Additionally, pertinent immunoperoxidase and/or histochemical studies were done. RESULTS: Eighteen cases were diagnosed as inflammatory lesions, 17 cases consisted of normal cellular elements, and 12 cases showed scanty material and were considered unsatisfactory/inadequate for a diagnosis. Five cases were suspicious for malignancy, and in 39 cases metastatic tumors were diagnosed from a previously known primary. Thirteen cases were diagnosed as lymphoma, and in 8 cases a diagnosis of soft tissue sarcoma was made. There were no false positive diagnoses of malignancy. Cell block preparations and immunohistochemistry were helpful with tumor typing, although lymphoma subtyping and soft tissue tumor typing generally required open biopsy. CONCLUSION: NAC, as the first-line investigation, is not only useful in the diagnosis of space-occupying lesions of the pelvic and retroperitoneal region but can also help in choosing appropriate management. The technique is most useful in diagnosing metastases but is also helpful in excluding malignancy in some cases and in suggesting diagnoses of lymphomas and soft tissue tumors.     

Factors affecting the immunoperoxidase demonstration of intracellular immunoglobulins and J chain from cytocentrifuged cell smears

Immunohistochemical methods were used to study 1) the optimum fixation conditions for the preservation of human J chain and immunoglobulin (Ig) immunoreactivity and 2) the relation of J chain synthesis by plasmablasts and plasma cells to Ig synthesis in cell smears of cultured human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). J chain was demonstrated using the indirect immunoperoxidase method, and intracellular Ig was demonstrated with the unlabeled antibody--enzyme method. In the sequential double staining procedure, J chain was demonstrated using the indirect immunoperoxidase method followed by the demonstration of Ig with the direct immunofluorescence method. Optimum preservation of J chain immunoreactivity was obtained with fixation in neutral buffered formalin at 22 degrees C for 5 min followed by immediate immunoperoxidase staining. False negative results were seen when the slides were stained 2 weeks after fixation. In PWM-stimulated smears, J chain appeared on day three, simultaneously with or after the onset of Ig synthesis. In double stained smears most IgG-positive cells also showed immunoreactivity for J chain from the third day on.     

Confirmation of genital herpes simplex viral infection by an immunoperoxidase technique

Over the 12-month period from April 1984 to April 1985, 512,000 gynecologic (Papanicolaou) smears were examined in the Provincial Screening Program in British Columbia. During this time, 307 patients were found to have smears that contained cells consistent with, or suggestive of, a herpes simplex viral (HSV) infection. The Papanicolaou-stained smears from these 307 cases were subsequently restained, without prior destaining, using an immunoperoxidase technique specific for type 2 HSV (HSV-2) and cross reactive with HSV-1. Of the 205 smears containing cells considered to be consistent with a herpes infection, 187 were positive using the immunoperoxidase technique. Of the 102 smears showing reactive cell changes though unlikely to be causes by an HSV infection, only 5 were positive using the immunoperoxidase technique. The results show that the immunoperoxidase technique is a rapid and reliable method of confirming a suspected diagnosis of herpetic infection and that it is particularly useful in those patients in whom the Papanicolaou smear findings are equivocal.     

Air-dried smears for optimal diagnostic immunocytochemistry

OBJECTIVE: To exploit formalin-postfixed, air-dried smears for diagnostic immunocytochemistry (ICC). STUDY DESIGN: A series of 144 cases of diagnostic fine needle cytology samples in which air-dried, supplementary smears were available was used to exploit postfixation in the process of antigenic stabilization and rescue for immunocytochemical staining. RESULTS: Postfixation with formalin and with a formalin/ethanol solution gave comparable results as far as recovery and immunocytochemical detection of most monoclonal and polyclonal antibodies. The visualization of the antibody reactions was often superior to that obtained with wet-fixed slides, probably due to the interaction of slow cell dehydration with their consequent optimal flattening observed with formalin postfixation after short rehydration in physiologic saline. CONCLUSION: Although wet fixation of cytopathologic slides in 95% ethanol represents a common standard for ICC, the usage of formalin-postfixed air-dried smears proved reliable and efficient for antigenic rescue and may enter routine usage in cytopathology laboratories. Moreover, in some instances, the visual evaluation of results was easier in the larger, well-flattened cells obtained in air-dried cells.     

Role of CD10 immunochemistry in differentiating hepatocellular carcinoma from metastatic carcinoma of the liver

Background:  Differentiation of hepatocellular carcinoma (HCC) from metastatic carcinoma in liver may be difficult on fine needle aspiration cytology (FNAC), especially when both appear as moderate to poorly differentiated tumours. A panel of immunocytochemical stains is frequently used in case of diagnostic difficulty. Recently, CD10 immunostain with a canalicular staining pattern has been shown to be a specific marker for hepatocytic differentiation.
Objective:  The present study was designed to assess the value of CD10 immunostain in distinguishing HCC from metastatic carcinoma in material obtained by FNAC of liver masses.
Materials and methods:  Formalin-fixed, paraffin-embedded cell blocks of 22 cases (7 cases of HCC and 15 cases of metastatic carcinoma), direct acetone-fixed smears and destained smears of 28 cases (18 cases of HCC and 10 cases of metastatic carcinoma) prepared from FNAC of the liver were immunostained using monoclonal antibody against CD10.
Results:  Seventeen (68%) of twenty-five cases of HCC were positive for CD10 with a canalicular staining pattern. Among them 7 (70%) of 10 cases were well-differentiated HCC and 10 (66%) of 15 cases were moderate to poorly differentiated HCC. Of 25 cases of metastatic carcinoma, four (16%) were positive for CD10 with a cytoplasmic (three cases) and membranous staining (one case) pattern.
Conclusion:  CD10 immunostaining is useful in discriminating HCC and metastatic carcinoma of the liver and is easily applied on cell blocks as well as FNAC smears.     

The value of epithelial membrane antigen in the diagnosis of ovarian tumors.

The value of epithelial membrane antigen (EMA) in the diagnosis of ovarian tumors was investigated using an indirect immunoperoxidase staining technique on 91 histologic sections (88 tumors and 3 normal tissues) and 39 ascitic fluid smears (28 from patients with epithelial ovarian tumors and 11 from cases of myoma uteri). The rate of positive EMA staining was highest in malignant tumors (89.2%), second highest in tumors of low malignant potential (33.3%) and lowest in benign tumors (25.0%); normal ovarian tissues were negative for EMA. Of the malignant tumors, all 48 serous cystadenocarcinomas (100%) and 18 of 26 mucinous cystadenocarcinomas (69.2%) stained positively for EMA. In serous cystadenocarcinomas, the EMA staining was mainly localized on the luminal membrane of cells in well-differentiated tumors, but appeared on the entire cell surface and cytoplasm of cells in poorly differentiated tumors. The results of EMA staining on ascitic fluid smears were almost the same as the results for the histologic sections. The intensity and the localization of EMA staining were related to the grade of malignancy in these ovarian tumors. In comparison with staining for other antigens (carcinoembryonic antigen, CA-125 and human keratin protein), EMA was found to be one of the most sensitive markers for the diagnosis of ovarian cancer.     

Species-specific immunocytochemical localization of Mycobacterium tuberculosis complex in fine needle aspirates of tuberculous lymphadenitis using antibody to 38 kDa immunodominant protein antigen

OBJECTIVE: To examine immunocytochemical localization of Mycobacterium tuberculosis (MTB) complex antigen in fine needle aspiration (FNA) smears of tuberculous lymphadenitis (TBLN) using species-specific monoclonal antibody MTSS to 38-kDa immnunodominant protein antigen as a diagnostic adjunct to conventional cytomorphology and its advantage over Ziehl-Neelsen (ZN) microscopy. Study Design FNA smears from 340 cases-174 TBLN; 34 negative controls from nontuberculous, positive controls of 13 known acid-fast bacilli (AFB)-positive sputum smears; 50 blind controls; and 69 other controls (smears from stock cultures of bacterial, atypical mycobacteria and fungal species) were subjected to ZN and immunocytochemical staining using MTSS by the streptavidin-biotin method. RESULTS: Immunocytochemical staining was positive in 59 of 61 (96.7%) archival and 110 of 113 (97.3%) fresh FNA smears; ZN positivity for AFB was observed in 27 of 61 (44.2%) archival and 48 of 113 (42.4%) fresh FNA smears of TBLN. CONCLUSION: The immunostaining using MTSS showed a definite advantage over conventional ZN staining for detection and specific diagnosis of TBLN in FNA smears with 0% false positive results. Immunostaining of cytosmears with species specific antibody to MTB would prove to be a good diagnostic adjunct to morphologic diagnosis.     

Usefulness of estrogen receptor detection using archival Papanicolaou-stained smears.

OBJECTIVE: To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). STUDY DESIGN: Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. RESULTS: In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. CONCLUSION: Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.     

Ferritin immunohistochemical localization in normal and neoplastic colonic mucosa

High levels of ferritin have been detected in serum and tumoral extracts of gastrointestinal neoplasms. However, its histological localization is not well known. An immunoperoxidase technique (PAP) was used for detecting ferritin in 30 colorectal carcinomas, 20 polyps and 8 cases of non-neoplastic mucosae. Ferritin staining was detected in stromal cells (98%) much more than in epithelial cells (21%). Connective cells were positive in 5 cases of normal mucosae (62%), 19 polyps (95%) and all carcinomas (100%). The number of positive cells gradually rose from normal mucosa to carcinoma with an intermediate score in adenomas. However, no relation could be found between the stromal ferritin score and dysplasia in polyps. Likewise, no relation was found between the stromal ferritin score and the differentiation grade, invasion or metastases in carcinomas. The positive epithelial pattern seen in 12 cases (21%) suggests non-specific staining due to passive diffusion from the stroma. Thus, these immunohistochemical findings suggest that in colonic neoplasms, ferritin could be a tumor marker produced mainly by stromal cell reaction more than by the epithelial cells.     

Cytologic and DNA cytometric diagnosis and therapy monitoring of squamous cell carcinoma in situ and malignant melanoma of the cornea and conjunctiva

OBJECTIVE: To investigate the diagnostic accuracy of exfoliative cytology of the cornea and conjunctiva and DNA image cytometry for quality control and monitoring of therapy for malignant neoplasms. STUDY DESIGN: Conjunctival or corneal smears from six cases clinically suspicious for malignant melanomas and eight suspicious for carcinomas in situ were investigated. Smears from 18 cases clinically nonsuspicious for neoplastic diseases served as negative controls. Repeated smears were obtained during and after local mitomycin C (MMC) therapy. RESULTS: In none of 18 nonsuspicious cases, cytology revealed abnormal cells. DNA cytometry showed nonaneuploidy in all of these. All smears from patients with histologically proven malignant melanomas (MM) and squamous cell carcinomas in situ revealed abnormal cells. Image cytometry demonstrated DNA aneuploidy in 66.6% of patients with MM and 80% with carcinoma. Sensitivity of cytology thus was 100% for both MM and carcinoma; specificity also was 100%. DNA measurements after MMC therapy revealed euploid polyploidization of nonneoplastic squamous cells. DNA cytometry provided an objective identification of tumor cell regression. CONCLUSION: Cytologic examination of corneal and conjunctival smears is a noninvasive tool with high diagnostic accuracy for detection of epithelial neoplasms. DNA image cytometry can serve for quality control and for objective monitoring of the effect of local chemotherapy.     

Immunocytochemistry of cerebrospinal fluid

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.     

Detection of amastigotes in cutaneous and mucocutaneous leishmaniasis using the immunoperoxidase method, using polyclonal antibody: sensibility and specificity compared with conventional methods of diagnosis

The indirect immunoperoxidase method was evaluated in 265 biopsies with the purpose of increasing the sensitivity of the diagnostic histopathology of tegumentary lesions caused by subspecies of the Leishmania braziliensis complex. A diagnosis of leishmaniasis was established by parasitological methods (181) or clinical criteria (12) in 193 patients (72.8%). In the latter group of confirmed cases standard histochemistry and immunoperoxidase were compared with direct examination of tissue scraping and culture of lesion aspirates. The detection and localization of amastigotes was more efficient using the immunoperoxidase method (61.3%) than conventional histopathology with hematoxilin and eosin (34.6%) or direct examination of tissue scraping (43.9%). However, culture of lesion aspirates was the most sensitive procedure (89.8%). The efficiency of the immunoperoxidase method was greater in recent lesions, being positive in 75% of cases with less than 3 months evolution, while 55.6%, 37.5%, and 21.1% of cases with lesion evolution of 3-5.9, 6-11, and 12 months or greater, respectively, were positive. The combined use of the direct examination of lesion scraping and immunoperoxidase applied to histological sections of the biopsy from the lesion border allowed an etiologic diagnosis of 72% of confirmed cases. Cross-reactivity was observed with Paracoccidioides braziliensis but not with Mycobacterium leprae, Sporothrix schenckii, or Histoplasma capsulatum.     

Recurrent Hodgkin's disease in the breast. Diagnosis of a case by fine needle aspiration and immunocytochemistry

Recurrent Hodgkin's disease involving the breast in a 17-year-old girl was diagnosed by fine needle aspiration (FNA) biopsy of a solitary mass that developed one year after "curative" radiation. Benign breast disease and breast carcinoma were ruled out upon cytologic examination of the FNA smears, which contained diagnostic Reed-Sternberg cells and the characteristic polymorphic background elements. Follow-up immunoperoxidase staining for Leu-M1 on destained smears confirmed the diagnosis. Definitive therapeutic measures were initiated after the FNA diagnosis.     

THE PAPANICOLAOU-TRAUT METHOD OF CANCER DIAGNOSIS,Its Use as a Routine Pathologic Laboratory Procedure

In a series of 450 examinations by the smear technique in the pathologic laboratory of a small private hospital, the total diagnostic error on all smears was 6.6 per cent. The error on vaginal smears was 4.7 per cent. These figures include false positives, false negatives, and all those smears classified as suspicious even though positive or negative diagnoses were made on subsequent examinations.The Papanicolaou-Traut method of cancer diagnosis can easily be made one of the routine pathologic procedures in the small hospital laboratory. Technicians with little previous cytologic experience can be trained to screen vaginal and other smears accurately after a short training period. This cytologic method is proving to be of value in the early detection of some neoplasms, and if its application is to be extended, the practicing pathologist should add the test to his diagnostic routine.