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.     

Development and validation of a sensitive immunohistochemical oestrogen receptor assay for use on archival breast cancer tissue

In a preliminary paper (Teasdale et al. 1987) comparing the oestrogen receptor (ER) content of breast cancers by the biochemical dextran coated charcoal (DCC) method and by two histochemical methods, peroxidase immunocytochemistry (ERICA) and immunogold-silver staining (IGSS), it was indicated that ERICA is more sensitive than DCC and that IGSS is as specific as ERICA but less sensitive. This paper describes the comparison of the above three assay methods with two other biochemical methods, iso-electric focusing (IEF) and an enzyme immuno-assay (EIA) on a larger number of cancers. All methods gave statistically comparable results except that IGSS remained less sensitive than the rest. Various modifications to IGSS showed that an immunogold streptavidin enhancement method (IG-SAM) produced sensitivity and specificity equal to that of ERICA. Since IGSS and its modifications are the only methods which can be used on archival paraffin-embedded cancers and IG-SAM gives results highly comparable to ERICA, retrospective studies can be performed on patients whose outcome and response to various treatments are known. Most recent studies have shown that ER positive results can be obtained from 10-year-old paraffin blocks.     

Usefulness of silver intensification of immunostaining for cytologic diagnosis of primary melanoma of the female genital organs

OBJECTIVE: To improve the procedure for diagnosing vaginal melanoma with cytopathologic analysis of HMB-45. STUDY DESIGN: The study examined silver intensification of immunostaining of HMB-45 in nine cases of primary melanoma of the vagina and vulva using archival Papanicolaou-stained smears. RESULTS: All nine samples showed positive staining for HMB-45. Five cases showed intensive staining, two moderate and two weak. The positive staining was black in the cytoplasm of melanoma cells but was detected in neither the background nor normal squamous cells. Though destaining of Papanicolaou stain was not performed before immunostaining, the positivity of immunostaining was easily judged. CONCLUSION: After morphologic observation, immunocytochemical study of HMB-45 is possible even though time has passed since the cytologic specimen was obtained. When there is a suspicion of amelanotic melanoma or scantily pigmented melanoma of the vagina and vulva, cytogenesis with HMB-45 is helpful, especially because it involves little invasion.     

Development and validation of a sensitive immunohistochemical oestrogen receptor assay for use on archival breast cancer tissue

Summary In a preliminary paper (Teasdale et al. 1987) comparing the oestrogen receptor (ER) content of breast cancers by the biochemical dextran coated charcoal (DCC) method and by two histochemical methods, peroxidase immunocytochemistry (ERICA) and immunogold-silver staining (IGSS), it was indicated that ERICA is more sensitive than DCC and that IGSS is as specific as ERICA but less sensitive. This paper describes the comparison of the above three assay methods with two other biochemical methods, iso-electric focusing (IEF) and an enzyme immuno-assay (EIA) on a larger number of cancers. All methods gave statistically comparable results except that IGSS remained less sensitive than the rest. Various modifications to IGSS showed that an immunogold streptavidin enhancement method (IG-SAM) produced sensitivity and specificity equal to that of ERICA. Since IGSS and its modifications are the only methods which can be used on archival paraffin-embedded cancers and IG-SAM gives results highly comparable to ERICA, retrospective studies can be performed on patients whose outcome and response to various treatments are known. Most recent studies have shown that ER positive results can be obtained from 10-year-old paraffin blocks.     

Cytomorphological and immunocytochemical study of non-Hodgkin's lymphoma in pleural effusion and ascitic fluid

INTRODUCTION: Non-Hodgkin's lymphoma (NHL) is often complicated by pleural effusion and ascites. The present study is an attempt to categorize the lymphomatous effusions according to the WHO classification, using archival material. METHODS: May-Grünwald-Giemsa and Papanicolaou-stained smears of 31 lymphomatous effusion specimens were reviewed. Of these, detailed cytological assessment was done on 12 pleural effusions and ten ascitic fluid specimens from 22 patients using the WHO lymphoma classification system. Immunocytochemical studies were performed in 21 specimens. RESULTS: Based on cytomorphological features, the 22 lymphomatous effusion specimens were categorized into lymphoplasmacytoid lymphoma (1), follicle centre cell (FCC) grade-1 (centrocytic) lymphoma (3), FCC grade-2 (centrocytic-centroblastic) lymphoma (3), FCC grade-3 (centroblastic) lymphoma (4), large cell immunoblastic lymphoma (4), lymphoblastic lymphoma (2), anaplastic large cell lymphoma (3) and miscellaneous types (2). Immunocytochemically, the lymphoma cells were T-cell (positive for CD3) and B-cell type (CD20 positive) in five and six cases respectively. CONCLUSION: Cytological examination of pleural effusion and ascitic fluid samples, supported by immunocytochemical studies, may be useful for the classification of lymphomas under the WHO system.     

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.     

Sensitivity of HPV tests on stained vs. unstained cervical smears

OBJECTIVE: To examine the effect of Pspanicolaou staining of cervical smears on the sensitivity of molecular biologic HPV tests. STUDY DESIGN: Two sensitive HPV tests were used, HPV DNA sequence analysis after polymerase chain reaction (PCR) amplification and the Hybrid Capture II method (HC II) (Digene Diagnostics Inc., Silver Spring, Maryland, USA). Papanicolaou-stained and unstained smears taken simultaneously were examined from 265 women readmitted for examination due to an atypical squamous cells of undetermined significance diagnosis. RESULTS: After an HPV test with the PCR method on unstained slides, 66% of the women were HPV positive, whereas the same women were HPVpositive in 54% when Papanicolaou-stained slides were analyzed. However, this difference was not statistically significant (p > 0.1). With the HC II method, 55% of unstained smears were HPV positive whereas 29% were HPV positive, when Papanicolaou-stained slides were examined. This difference was significant (p < 0.001). The same strong differences in sensitivity were observed when both the PCR and HC II methods were studied on the same Papanicolaou stained glass slides, whereas on unstained slides no significant difference was found. CONCLUSION: The results demonstrate that Papanicolaou staining of a cervical smear significantly decreases the sensitivity of an HPV test performed with the HC II method, whereas the PCR method is less affected. With the Papanicolaou method, the hematoxylin bath is followed by HCl treatment, and strong acid treatment destroys DNA.     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

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.     

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.     

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.     

Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma

athanassiadou p.p., athanassiades p. h., daffaris p., petrakakou e. i., fflerffa ch. i. and kffirkou k. a. (1998) Cytopathology 9, 240–247
Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma
The aim of this study was to add to existing information on the effects of certain tumour markers expressed by breast cancers on tumour malignancy as evidenced by size of primary and occurrence of lymph node invasion. One hundred freshly resected breast cancers were examined by immunocytochemical staining of imprint smears for Cathepsin D and pS2. Oestrogen receptor (ER) and progesterone receptor (PR) were tested for by dextrose-coated charcoal (DCC) assay and the results correlated with tumour size, histology and presence or absence of lymph node metastases at the time of surgery using χ² analysis. A significant positive correlation was demonstrated between Cathepsin D positivity and ER, PR and pS2 positivity. In tumours < 2 cm in diameter at surgery a positive correlation was observed between Cathepsin D positivity and the presence of lymph node metastases. The findings support the hypothesis that Cathepsin D may promote early metastasis, possibly by its proteolytic activity.     

Cytologic diagnosis of estrogen and progesterone receptors in breast imprints

OBJECTIVE: To investigate estrogen receptor (ER) and progesterone receptor (PR) levels in imprint specimens obtained at breast surgery and to compare their correlation with that of standard methods. STUDY DESIGN: Imprint specimens for cytology were obtained from 101 mass-forming lesions in 66 patients, and specimens were frozen in liquid nitrogen for later assay. The imprint specimens were immunocytochemically (ICC) stained by monoclonal antibody to ER or PR; diaminobenzidine-stained cell nuclei in clusters were regarded as positive. Tissue specimens were assayed by the standard method of dextran-coated charcoal assay (DCC) and enzyme immunoassay. RESULTS: Forty-five primary breast cancer lesions, 2 contralateral breast cancer, 49 dissected nodes and 5 benign breast lesions were collected. The correlation between DCC and ICC was 81% (82/101) for ER and 74% (66/101) for PR. That between EIA and ICC was 88% (88/99) for ER and 80% (79/100) for PR, higher than that between DCC and ICC for ER and PR. CONCLUSION: ICC assessment of ER or PR on imprint cytology is a promising clinical test with an acceptable correlation.     

Desmin detection in FNAB samples of rhabdomyosarcoma: an immunocytochemical study

We performed immunocytochemical (ICC) staining for desmin on 65 fine needle aspiration biopsies from 45 patients with rhabdomyosarcoma, using the avidin-biotin-peroxidase complex method (ABC), and two types of antibodies, D33 and DE-R-11. The material was fixed either in ether-alcohol or in Delaunay's solution. The ABC method was applied to Papanicolaou stained slides, without destaining. We compared the quality of staining on fresh, routinely prepared slides vs archival material and the quality of staining on smears vs cytospins. In 20 cases, D33 and DE-R-II were applied to a pair of slides from the same tumour sample in order to see if there was any difference in their ability to recognize desmin. Desmin was positive in all 18 cases in which ICC staining was performed at the same time diagnoses were given. Among the 27 cases where ICC staining was carried out on archival material, seven were negative. Slides from four of these cases were 20 or more years old and negative reaction could be attributed to heating of slides before coversliping and/or to uneven distribution of desmin immunoreactivity in tumours. The second reason was probably the cause of negative reactions in cases from 1985 and 87. The type of slide preparation had no influence on the quality of staining. However, results were easier to read on cytospins because cells were more evenly distributed. Finally, our results proved that there was no significant difference between D33 and DE-R-11 in their ability to recognize desmin.     

Use of an enzymeimmunoassay (EIA) for quantitation of cytosolic and nuclear estrogen receptor, and correlation with progesterone receptor in human breast cancer

We have adapted a commercially available enzyme immunoassay for ER (ER-EIA) for use with nuclear extracts of breast carcinoma specimens, and have examined the data obtained in relation to the results from cytosolic ER-EIA and radioligand (DCC) assays and from DCC assays for PgR. In a series of 139 carcinoma specimens, we observed a very significant correlation between the cytosolic ER concentration as measured by the EIA and DCC methods, and also between the log10 of the concentration of ER in the cytosolic and nuclear fractions assayed by the ER-EIA method. The correlation between nuclear ER and cytosolic PgR was also highly significant, but not as close as for cytosolic ER. If 130 fmol ER/mg DNA was used as a "cut-off" point to discriminate between specimens "positive" and "negative" for ERN, there was 87% concordance in receptor status between ERN and cytosolic ER, and 79% concordance between ERN and cytosolic PgR. Forty-one percent of specimens were positive or borderline for both ERN and cytosolic PgR, and it is suggested that this receptor combination may be a valuable predictive factor in prognosis and response to hormonal treatment.     

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.     

Immunocytochemical staining of cervical smears for the diagnosis of cervical intraepithelial neoplasia

A total of 233 cervical smears were stained by immunocytochemical methods for epithelial membrane antigen (EMA); the findings were compared with those from Papanicolaou-stained smears from the same women. Squamous epithelial cells from normal cervices did not stain, but cells shed from cervices with cervical intraepithelial neoplasia (CIN) did express the EMA marker. Metaplastic cells from normal and abnormal cervices also frequently stained. The results confirm that this marker detects cervical intraepithelial neoplasia in vitro, but its potential use in an automated screening program may be limited by the staining of the metaplastic cells.     

Retrospective study of Chlamydia trachomatis using the polymerase chain reaction on archival Papanicolaou-stained cytologic smears.

OBJECTIVE: To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique. STUDY DESIGN: A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR. RESULTS: PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted. CONCLUSION: PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.     

Measurement of estrogen receptors in intact cells by flow cytometry

BACKGROUND: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry. RESULTS: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. CONCLUSIONS: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.     

Comparison of biochemical and immunoenzymatic macromethods and a new immunocytochemical micromethod for assaying estrogen receptors in human breast carcinomas

Estrogen receptors (ERs) were assayed in 23 breast carcinomas by: (1) the conventional biochemical assay with dextran-coated charcoal (DCC); (2) the immunoenzymatic assay using a monoclonal antibody (MAb), ER-EIA (Abbott); and (3) an original cytochemical method using another MAb, ER-ICA (Abbott). The first two techniques were performed on biopsy samples, whereas the last was carried out on fine needle aspiration (FNA) samples. The ER contents in aspirates were evaluated by: (1) scaled proportions of colored neoplastic cells; (2) scaled coloration intensity; (3) total grading (= proportion plus intensity); (4) product grading (= proportion times intensity); and (5) a new index (NI) described in this paper. The ER-EIA assay correlated best, with a high statistical significance, with the NI (P less than .001); NI was also the only index that significantly correlated (P less than .05) with the DCC results. The results show that the ER-ICA assay offers the great advantages of being applicable to FNA specimens and of producing rapidly available results. This new technique enriches the panel of MAbs for the diagnosis of adenocarcinomas and offers a new tool for the therapeutic follow-up of breast cancer patients. Our preliminary results suggest that the anti-ER MAbs might be helpful for measuring the hormone dependence of small lesions not assayable by DCC, even under endocrine therapy, thus avoiding false-negative assays.