Working Group: Cytopathology II

Abstracts· 369

Working Group: Cytopathology II

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Mm·1 (Kl·67) IMMUNOSTAINING OF BREAST CANCER CELLS IN CYTOLOGIC SMEARS P. DALQUEN*. B. BASCHIERA (a.G.)*. R. CHAFf.ARD (a.G.)*. H. DIETERICH (a.G.)**. O.E. FEICHTER*. K. KRAMER (a.O.)**. J. TORHORST (a.G.)*. *Institute for Pathology. University of Basel. **Frauenklinik Rheinfelden

INTERPHASE CYTOGENETICS AS A NEW DIAGNOSTIC TOOL IN CYTOPATHOLOGY

AIMS: Recent studies have shown that "neoadjuvant" chemotherapy downstagi~g large primary breast cancers prior to surgery may allow more often breast conservation. Since proliferation index correlates with the biological behaviour of breast cancer and since it has been demonstrated that tumor responsiveness to cytotoxic drugs may depend on proliferation, the assessment of proliferating capacity of a carcinoma may contribute to the selection of high risk patients who could be candidates for adjuvant chemotherapy. The purpose of this study was to test the reliability of immunocytochemical evaluation of the proliferation capacity on cytologic specimens. METHOD: The monoclonal antibody MIB·l was employed to 83 smears prepared from frozen tissue (FTS) and to 51 fine-needle aspirates (FNA) from 119 breast cancer patients. MlB-l staining indexes were compared with various tumor parameters assessed on histologic material. RESULTS: MIB-l positive cell fraction established on cytologic smears was significantly different in ductal and lobular carcinomas (p=O.024) and was significantly correlated with mitotic activity (p
Interphase or molecular cytogenetics allows the detection of karyotype changes in non-dividing cells. The various applications of this technique are based on recent developments in non-radioactive in situ hybridization. The possibility to examine intact interphase cells enables one to assess cytomorphology together with genetic characteristics. Thus, gains and losses as well as structural changes of chromosomes or genes can be detected at a single-cell basis.

M. WERNER and A. GEORGII Pathologisches Institut der Medizinischen Hochschule Hannover

Enzymatic visualization of hybridized probes, e.g. by peroxidase or alkaline phosphatase. is a stable reaction, and morphological details can be evaluated by conventional light microscopy. The use of fluorescing dyes, e.g. fluorescein or rhodamin, is necessary to detect specific chromosomal translocations. Comparative genomic hybridization can demonstrate non-ballanced changes of the genome but requires homogenized cells. Molecular cytogenetics is well established for the diagnosis and detection of residual disease in chronic and acute leukemias. In these disorders, cells affected by clinically significant structural aberrations such as t(9;22), t(15;17) or inv(16) can be quantified by using triple color fluorescence. This technique also promises to be suitable in the classification of non-Hodgkin's lymphomas or the differential diagnosis of solid tumors, especially those of soft tissue origin.

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PROGNOSTIc!! SIGNrFICAlfCB OF CHROMATIN FEATURES IN BREAST CARCINOMAS

Detection of human papilloma virus In cytologic specimens by hybrid

K.D. KUNZE, V. DIMMER (a.G.), G. HAROSKE (a.G.), W. MEYER (a.G.), F. THEISSIG (a.G.) Institute of pathology, Technical University Dresden

capture-technlque

Aims: The prognostic significance of chromatin features was studied on 187 invasive ductal breast carcinomas (mean follow up 63 months) and compared with other clinicopathological factors (lymph node status, histopathological grading, tumor size). Methods: The studies were performed with two different image cytometry workstations. Image cytometric features included 54 parameters of nuclear size, shape and chromatin structure. Multivariate, discriminant analysis and the Cox regression proportional model were used for statistical evaluation. Results: Combining clinicopathological and cytometric features a correct prediction of prognosis could be made in 83 t. By statistical evaluation only three out of 70 features were found to contribute significantly to the discrimination of subgroups: the number of coarse chromatin particles, lymph node involvement and tumor size. For prediction of a 5 or 7 year survival the significance of the chromatin particle number is equivalent to the significance of the nodal stage. Independent measurements with two different image cytometric systems indicated a high reproducibility of chromatin features. Conclusion: 'The image cytometric stUdies on breast carcinomas substantiate the prognostic significance and reproducibility of chromatin features.

Postfach 10 03 46

HEINE M, FLENKER H

27503 Brememaven

The hybrid capture method is an easy to uSe technique for the evaluation

of DNA sequences in HPV infections. The nonradioactive test kit is designed to detect 14 different types of HPV divided into two groups: the "low risk" group ( HPV 6/11/42143/44 ) and the "high risk" group ( HPV 16/18131/33135/45151/52156 ) for the genesis of cervical cancer. The

sensibility of this RNA-DNA technique is higher than in Southern blOtting. An outline of the method: after denaturation of DNA in infected cells of the cervical smear, a specific RNA probe is added thus forming RNADNA hybrids. These are detected by an immunological reaction with monoclonal antibodies. The test is recommended for follow-up after a cytologic diagnosis of HPV associated mild or moderate dysplasia , ( group III 0 in the MUNICH classification ). Practical value of the test, corresponding cytologic diagnoses as well as different therapeutiC consequences will be discussed.

370 . Abstracts

225 DETECTION OF EWING'S SARCOMA CYTOLOGIC SMEARS BY PCR

227 IN

ARcmvAL

T. SCHLOIT (a.G.), H. NAGEL (a.G.), I. RUSCHENBURG (a.G.), S. REIMER (a.G.), M. DROESE Zentrum Pathologie, Abteilung Zytopathologie, Universitat Gl>ttingen Aims: Ewing's sarcoma (ES) and related subtypes of primitive neuroectodennal tumor share a reciprocal chromosomal t(II;22) (q24;qI2) .translocation. Concerning morphologic similarities between ES and other small round cell tumors the detection of this unique genetic feature by means of reverse transcriptase polymerase chain reaction (RTPCR) serves as important ancilliary method of differential diagnosis in pathology. We optimized the underlying methodical conditions and tested cytodiagnostic reliability ofRT-PCR to establish a sensitive assay on archival ES smears. Methods: Archival fine-needle aspiration (FNA) biopsies isolated from patients with concordant cytologic and histologic diagnosis of ES were collected. Using pediatric material aspirated from osseous tumor lesions the t(11;22)-specific RNA sequences were amplified by RT-PCR and the nucleotide sequences of the PCR products were analysed by automatic DNA sequencer. Results: Cytologic Ewing's sarcoma smears from 16 cases were prepared and tested with p-actin specific primers for presence of RNA by RTPCR. Samples from 12 tumor patients contained RNA which was transcribed into cDNA and subsequently amplified with ES-specific oligonucleotides. RT-PCR detected t(11;22)-specific RNA in cytologic smears obtained from seven patients. For reaffinnation of PCR results, the ES-PCR fragments were sequenced using an automatic DNA sequencing system. Conclusions: In this study, we have shown that conventionally stained cytologic smears are suited for molecular genetic analysis which serves as a helpful tool in differential diagnosis of small round cell tumors.

Immunocytological detection of micro metastatic tumor cells in the peritoneal cavity and the bone marrow of gastrointestinal cancer patients H. Juhl, H. Kalthoff, A. Schott, U. KrUger, D. Henne-Bruns, B. Kremer Chirurgische Universitiitsklinik Kiel Aimes: Several immunocytological studies have shown that dissiminated tumor cells are frequently detectable in the bone marrow of gastrointestinal cancer. However, bone metastases are a rare event in gastrointestinal cancer while metastatic diseases occur usually within the abdominal cavity. Methods: We investigated peritoneal lavage samples of patients with gastric (n=46), colorectal (n=66) and pancreatic cancer (n=47) for isolated tumor cells using a panel of five different monoclonal antibodies against tumor-associated antigens and compared the detection rates with findings in bone marrow probes which were investigated in parallel. The survival rate (median follow up of 3 years) was detennined in correlation with the detection rates in bone marrow and peritoneal cavity samples. Results: The finding of tumor cells in peritoneal cavity samples increased in parallel to the tumor stage in gastric, colorectal and pancreatic cancer from 20%-30% (Stage I) to 60-70% (Stage IV), in bone marrow samples only in pancreatic cancer patients. The 3-year survival rate of 3S gastric, 46 colorectal and 15 pancreatic cancer patients who were surgically treated by RO-tumor resection including a radical lymphadenectomy, showed a statistically significant difference between patients with and without immunocytological positive peritoneal cavity samples. No correlation was seen with bone marrow samples. Conclusion: Using immunocytology, disseminated tumor cells can be detected in bone marrow and peritoneal cavity with high frequency. Especially the finding of tumor cells in the peritoneal cavity serves at a good indicator for cancer relapse and might be used as a guideline for further adjuvant therapy.

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MOLECULAR DNA PROBES DETECTING REARRANGEMENTS OF THE HMGI-C GENE AN ADDmONAL TOOL FOR TIlE CYTOLOGY OF BENIGN MESENCHYMAL TUMQRS

STANDARDS FOR QUALITY ASSURANCE IN DIAGNOSTIC CYTOLOGY FROM THE VIEWPOINT OF MEDICAL ORGANIZATIONS F.W.Kolkmann,Vorsitzender der Qualitiitssicherungsgremien der Bundesarztekammer, Gemeinschaftspraxis flir Pathologie, Niirtingen

J. Bullerdiek (aG.i, S. Wanschura(aG.)', S. Bartnitzke (aG.)', u. BODIt'

, Institut fUr Humangenetik, Universitlit Bremen Institut fUr Pathologie, Zentralkrankenhaus-Nord, Bremen

2

Aims: A huge variety of aberrations involving the chromosomal region 12q13-1Sare a nonrandom cytogenetic abnormality in frequent benign tumors main1y of mesenchymal origin e.g. uterine leiomyomas, pleomorphic adenomas of the salivary· gland, lipomas, or hamartomas of the lung. In addition to the frequent tumors mentioned above these abnormalities have also been found in rare mesenchymal tumors, e.g. hemangiopericytomas or aggressive angiomyxomas. Recently, we have identified the molecular basis of these aberrations of chromosomal region 12q14-1S ie. a rearrangment of the HMGI-C. Interestingly, this type of rearrangement has exclusively been found in benign tumors as a rule of mesenchymal origin. Method: Using appropriate DNA probes about 80% of these rearrangements can be detected by fluorescence in situ hybridization (FISH) which can also be applied on cytologic preparations. It is based on the use of a cosmid pool containing DNA probes from the 3' and the S'end of the third exon of the gene. By the rearrangements the 3'end becomes translocated to another recipient site resulting in the appearance of a double and a split signal in the nuclei Results: According to our experience based on ISO tumors or corresponding cell lines the test is informative in about 60-70% of all lipomas, 80% of the pulmonary chondroid hamartomas, and 30-40% of the uterine leiomyomas and allows for the identification of a particular subset of salivary gland pleomorphic adenomas. It will become an additional tool for the cytological diagnosis of soft tissue tumors also including e.g. hemangiopericytomas or enchondromas. The molecular basis of the technique and examples will be demonstrated.

Medical quality assurance is a joint obligation of the responsible scientific societies and medical organizations. The scientific societies must develop the necessary quality assurance projects and measures and test and evaluate them in pilot studies, before they can be applied routinely. The medical organizations help when it comes to applying quality assurance measures in routine work; they settle financial matters and take care of the legal aspects. In gynecologic exfoliative cytology quality assurance is already regulated by the guidelines of the medical organizations. For the much more highly differentiated, wide ranging field of extragenital cytology no routinely applied quality assurance measures exist as yet, apart from some pilot studies. In nongynecologic cytology it is also important to guarantee quality in morphology, in processing and in the results obtained, and the quality must be verified by external and internal quality assurance measures. Whereas morphologic quality and the quality of results can be investigated by such measures as external multi-institutional tests and reference centers, assuring processing quality is an internal task, which can be supported by external measures.

Abstracts· 371

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A PREPARATION ON A PROl=ICIENCY TESTING ON QUALITY ASSURANCE IN CLINICAL CYTOLOGY

MODERN MICROSCOPE TECHNOLOGY FOR QUALITY ASSURANCE IN CERVICAL CYTOLOGY SCREENING

R. Goer lGhen Inslilul liir Palhol_ogln, klinik"' .. Gollil.

U. SCHENCK, W. PLANDING (a. G.)

The problem is Ihat many special branches of clinical medicine practise the cytology al though it is a pathomorphological method. Until today measures of Qualityassurance for the non-gynecological clinical cytology don't exist however only the arrangement of development, exists. Therefore, it was the aim of study to find the possibilities and groundworks for a facultative certification, for the non-gynecological clinical cytology. A group of cytologists prepared 6 collections with a panel of 33 slides out the whole group of the special organ and fluid cytology. 7 pathologists with experience in the cytology checked these collections with classing by points. The result was that most of the slides represented the extra-gynecological cytology of organs and fluids. The tester only needs just 3 - 4 hours and found that 28 - 29 slides wer" typical. The outcome showed the possibility to create a proficiency testing or a certification in non-gynecological cytology.

230 QUALITY ASSURANCE IN CERVICAL CYTOLOGY AUSTRIA G. Breltenecker Instltut fUr Pathologie der Universltiit Wien, Abt. Gynakopathologie und Zytologie

Labor flir Klinische Zytologie, Institut fUr Pathologie, TU Miinchen Zytologisches Institut der Bayerischen Krebsgesellschaft The applicatiOn of new microscopical techniques for quality assurance in gynecological cytology may relate to quality of structure, process quality and product quality. For structural quality we use for the students of the Munich Cytology School the HOME-Microscope which allows training via monitor images that are projected to the eyepiece of the microscope. Teaching units which are associated with certain slide coordinates are displayed to give object related information or check the student's knowledge with multiple choice questions. Continuous registration of slide coordinates and the objective magnification (every 20 milliseconds) is now in use at all cytotechnology v.urking places for continuous process control in the Bavarian Cancer Society Cytology Lab. In a series of 8653 slides screened the average screening time was 3,5 minutes per slide added by an average two minutes of mostly clerical work. A daily workload protiie demonstrating the number of slides per hour can be generated to check whether the workload limit of a maximum of ten slides per hour, as set by the German Medical Association lire adhered to. Average screening time per case does not vary much with the time of day or the day of the week. Similar systems as developed in our lab are now also commercially offered by different companies either for scanning control or to preset the scanpath with motorized stages. Product quality assurance can now be done with automated screening devices which can be applied presently only after visual diagnosis has been performed. Thlecytology/pathology including microscope remote control is presumed to play an important role when standards and speed of image transfer allow easy use. "supported by EC: Grant Nr. Soc. 94 202 355 05F021 P2261

232 IN

According to International trends In Austria folloWing regulations for quality assurance In cervical cytology are to be established by the Board of Pathologists In the Austrian Medical Association and the social securitles_ 1.) Only pathologists with an additional speciality degree for cytodlagnostics (2-years additional training In cytopathology) or speCialists for laboratory medicine and gynecologists (With 3-years additional training. one of them In histopathology) get a contract for performing gynecological cytology. 2.) Only Medical TechniCians With a 3-year school and diploma and additional special training In cytology are allowed to screen. 3.) Doctors and MTA have to attend continuing education (100 hours within 3 years). 4.) The Investigations have to be performed Within the laboratory (no homework. no sending to other Institutions). 5.) EqUipment has to fullfill requirements of the state of the art 6.) Automatic pre-screening scanners have to be approved by the Austrian Medical AsSOCiation. 7.) A standarized nomenclature and classification system has to be used to provide uniform recording (similar to the Munich Schema). 8.) 10 % of the smears have to be rescreened. 9.) Workload limitations: Maximal workload Is 80 smears per day and 16.000 per year/MTA Maximal workload of a laboratory (one pathologist and 5 MTA) Is 80.000 smears per year. 10.) Annual statistics including statements on quality of the smears 11.) Storing of smears and reports for at least 10 years. 12.1 External controls are possible In certain cases.

TELECONSULTATION AND QUALITY CONTROL IN DIAGNOSTIC CYTOLOGY K. KAYSER*, G. NICOLO", H.-J. GABlUS"· .. Abteilung fUr Pathologie, Thoraxklinik, Heidelberg; **Institut fUr Pathologie, Nationales Krebsforschungszentrum, Genua; ***Institut fUr Physiologische Chemie, Maximilians Universitat MUnchen Modem communication techniques permit a contemporary transfer of visual and acoustic data between two or several partners including visual or acoustic interactions from both parts. Teleconsultation in pathology is a subdivision oftelepathology and comprises the consultation of medical experts, control of fixation, staining and quantitative or semiquantitative measurements of histological and cytological slides. It permits the application of techniques which may, in addition, improve the standardization and interpretation of slides, imprints, or smears. A contemporary exchange of information is prerequisite if colleagues are consulted. Contemporary data exchange with individual interaction is only possible if common standard or digitized telephone lines (ISDN) or broad band connections are used. Quality control and standardization of staining or image interpretation with time delay (in anal(lgues to the common mailing of slides) can also be performed using the "world wide web" or internet connections. Regular teleconsultations between the Institute of Pathology of the National Cancer Center in Genua and the Department of Pathology, Thoraxklinik, Heidelberg are performed since 1994. Especially images of smears hard to classify have been transmitted. The transfer of one image lasts for about one minute and prohibits a detailed screening. It is, however, of great benefit for interpretation or classification of abnormal cells or organisms, and can be performed using all kinds of stains including immune- and ligandocytochemistry. Teleconsultation can diminish significantly the error rate in tumor classification and indicate non-sufficient quality of technical procedures. An improvement of the screening facilities cannot be expected.

372 " Abstracts

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CONTRIBUTION OF AUTOMATION TO QUALITY ASSURANCE IN PROGNOSTIC VALIDITY OF DNA·IMAGE CYTOMETRY IN DYSPLASTIC SMEARS OF TIlE UTERINE CERVIX CERVICAL CYTOPATHOLOGY R BOLLMANN·; A. BOCKlNG·· p, SCHWARZMANN (a,G,) • Institute of Pathology, Bonn-Duisdorf Institut fOr Physikalische Elektronik, Universitat Stuttgart •• Institute of Cytopathology, Heinrich-Heine-University, DUsseldorf Aims: We tried to find out the positive and negative predictive values for The development of equipment for cytoautomation began in the the presence or absence of squamous cell carcinomas in smears from the seventies with the primary aim to increase the capacity for the uterine cervix with mild or moderate dysplasias by DNA-image cytometry resently inTroduced programs for the prescreening of PAP-smears, using single-cell-aneuploidy as a marker. At the end of the eighties in the USA cytoautomation was regarded Methods: DNA-image cytometry has been performed on 593 convenas a way out of the difficulties which began with the Wall street tionally Papanicolaou-stained smears with mild or moderate squamous Journal article in which 25% false negative PAP-smear diagnosis in dysplasias (group HID Munich nomenclature II) with the Cydok System routine practice were reported for the conventional prescreening (Hilgers, Konigswinter). At least 100 dysplastic cells were measured per procedure. smear. DNA-single-cell-aneuploidy was assumed if >3 cells with a DNAThe main arguments for the introduction of machine assisted content >5c or >9c could be detected per smear, dependent on the abevaluation of PAP-smears with respect to quality control sence or presence of koilocytes and dyskeratocytes. 1-3 cells >5c19c were improvements consisted of: interpreted as suspicious for aneuploidy. The cytometric results could be - Standardization of all steps beyond the material collection correlated with a histological follow up (within 4 weeks) in 191 cases, and procedure with a cytological follow up (within 2 years) in 306 cases. 40 negative - Evaluation of statistical (population analysis) and subvisual smears served as negative controls, 19 imprints from squamous cell carciinformation nomas, and 15 tumor cell positive smears as positive controls. - Investigation and evaluation of all events and not only 'typical' Results: 40/40 negative smears revealed DNA-euploidy, 33/33 tumor cell events found on a slide positive slides DNA-aneuploidy, 48 % of the dysplastic cases were DNA- Constant and unbiased quality aneuploid, 18 % DNA-euploid, 33 % suspicious for DNA-aneuploidy. In Presently 4 systems (Cytyc, Neopath, NSI, Roche) have reached a 811191 DNA-aneuploid cases the follow up revealed histologically proven premarket state. The systems differ in strategy and requirements carcinoma in situ, in 182/191 cases high grade squamous intraepithelial with respect to preparation. Roche and Cytyc rely on monolayer lesions were found (positive correctness of 42 % resp. 95 %). All of the preparations made in dedicated machines, whereas Neopath and remaining aneuploid cases revealed dysplasias but never normal epithelium NSI work with conventional smears. The error rates with respect to in the follow up. In 108 DNA-euploid cases the follow up revealed only 2 the detection of cancer or precanceros states have been reported cases of carcinoma in situ (negative correctness 98 %). to be below 2 - 5 % false-negative rate and are dependent on the Conclusions: Revealing a negative predictive value of98 % and a positive type of the lesion. • predictive value of 42 % resp, 95 % diagnostic DNA-cytometry seems to One of the challenging problems in the application of machine be able to identity the prospectively malignant cases among mildlmoderate assisted diagnosis will be: both, the machine as well as the human cervical dysplasias, DNA-aneuploidy in smears with mild/moderate dysobserver make mistakes, but they make different mistakes! plasias should result in immediate histologic investigation.

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Telecytology - A new project between Aurich and Westerstede.

DNA-CYTOMElRY FOR QUALITY CONTROL IN GYNECOLOGIC CYTOLOGY A. BOCKlNG·, R. BOLLMANN··

G. SLauch 1, K. -W. Schweppe (aG)2, R.Dorrer(aG)3, Path. Institut KKH Aurichl, Gyn. Klinik KKH Westerstede 2, Roche Image Analysis Systems - HabsheimFrance 3. Telepathology has been shown to be a suitable solution for frozen sections diagnosis in I'emote areas. Beside a I'outinely used system we introduced an second image transfer system. The system wOI'ks wi th a programmable high resoiuLion camera. In,ages were digi talized on lr'ue colo!' using. Pixel size from 512 - 384 to 2048-1450. Images were transmitted with varying degrees of compression. We invest.igated 1. paralelly frozen sections and imprint cytology of breast lesions and pel vic and oval'y endometriosis, 2. suspect cervical smears and fine needle aspirates and 3. extragenital cytology and blood- and bone marrow smears. Images and slides were reviewed and ISDN diagnosis with definite diagnosis compared, Aim of this study is to desoribe 1. diagnostic accuracy in a) various organs, b) various staining, 2. trainings effect of technicians, who are demonstrating the images, , 3. image quality and compression mode in a) vadous magnification and b) various staining, 4. image tl"ansmission time and real diagnostic time.

• Institute of Cytopathology, Heinrich-Heine-University, DUsseldorf •• Institute of Pathology, Bonn-Duisdorf DNA-Aneuploidy has often been described as a highly sensitive marker for (prospective) malignancy in dysplastic squamous epithelia of the uterine cervix, This marker is present in about 98 % of squamous cell carcinomas of the uteru.s but has never been detected in benign reactive squamous cells. The detection of DNA-aneuploidy by static cytometry is therefore suitable 1. for an objective confirmation or a subjective diagnosis of tumor cells, 2. for the identification of obligatory precancerous cases among dysplasias and borderline lesions, 3. for the diagnostic evaluation of otherwise atypical cells. We propose as indications for diagnostic DNA-image cytometry cervical smears of the diagnostic groups 11K (for inexperienced cytologists only), III, HID and IVa/b (if a carcinoma could not be found histologically) of the Munich classification II and oflow and high grade squamous intraepithelial lesions (LSIL, HSIL) of the Bethesda II nomenclature. The following aspects in histograms may be taken as evidence of DNA-aneuploidy: 1. abnormal position of stemline (modal value <1.85c >2.15c or <3.7c >4.3c, and p5c without and >9c with evidence of koilocytes or dysceratocytes, and/or 3. coefficient of variation of stemIine fraction> 15 %. Indicators of DNA-polyploidy are combined stemlines >1,85c <2,15c, and >3,7c <4,3c, and single values around 8c. Other histograms are defined as DNA-diploid. Cases revealing unequivocal DNA-aneuploidy have to be looked at as prospectively malignant and should be further investigated by histology. In cases revealing DNA-polyploidy typing of HPV may be appropriate. DNA-diploid cases should be controlled by cytology after 3 months. If no cancer could be detected by histology after cytological diagnosis of tumor cells the identification of DNA-aneuploidy can be taken as argument either to further investigate the histologic specimen by serial sections or to obtain a more representative tissue specimen.

Abstracts·

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G.Richartz (a.G.), A.Meissner (a.G.), A.Kohl (a.G.), QUALITY ASSURANCE IN DlASNOSTIC BNA IMAGECYfOH.-J. Knieriem METRY: PRECISION AND ACCURACY OF MEASUREMENTS Institut fUr Pathologie, Ev. Krankenhaus Bethesda, G. HAROSKE , W. MEYER (aG.), F. THEISSIG (aG.), K.D. KUNZE Duisburg/GERMANY Institute of Pathology, Technical University Dresden Quality Control in Gynecological Cytology Using Quantitative DNA-Image Analysis AIMS: Quality control studies in gynecological cytology gain increasing importance during the 1ast years with special reference to the classification of the different degrees of dysplasia. METHODS: Therefore, we investigated from a total of 39.408 gyn. smears during a period of one year 321 cases with cytological grading of PAP IIW, IIID, IVa, IVb and V, respectively. The mean age of the women was 39,4 years. The original PAP-smears were restained with Feulgen solution and interactive DNA-image cytometry was performed using a Becton- Dickinson CAS 200. RESULTS: In 60 % of all cases DNA-cytometry confirmed the cytological grading, whereas in 36 % DNA analysis demonstrated additional severe dysplasia. Only in 4 % the cytometric results were more favorable. Cytological and cytometrical results were compared with consecutive histological findings. Histology confirmed the cytological findings in 65 %. In 26 % histology showed more pronounced dysplastic changes and in 9 % less severe lesions. Comparing DNA cytometry with histology the confirmation of our findings was more obvious (78 %), whereas in 20 % of our cases DNA analYSis demonstrated a higher degree of dysplasia. Re-eva1uation of histological specimen revealed foci of previously missed severe dysplastic changes. CONCLUSION: Quantitative DNA-image analysis can be a very useful method for quality control in gynecological cytology and also consecutive histology.

Aims: In recent TV-based image cytometers considerable disproportionaIities exist between the IOD values of reference cells, as well as diploid, tetraploil, and octoploid analysis cells compared with their theoretical IOD ratios. most important source of those deviations is the limited spatial resolution of the microscopic objectives, based on the effects of diffraction. These effects as well as the glare of the microscope are based on physical and technical conditions and not to influence per se. Methods: For reducing the effects on the DNA measurements a correc~g method is given, which is applicable to any kind of objective and cell type. De method was tested in Feulgen stained imprints from 43 rat livers and 247 hUman breast cancers with a set of eight different microscope objectives at ~ OPTIMAS-based cytometry workstation Results & Conclusions: The use of highly qualitative optics for reducing tile glare and limiting the optical errors due to diffraction (oil immersion microscope objectives, no phase contrast optics) has to be recoInIrended. A high 0ptical resolution in a Kohler illuminated microscope is to prefer as to keep tile marginal pixel proportion of the nuclear area as small as possible. The varianQ:e of both the reference cells and the analysis cells could be evidently reduced Ity applying the correction methods (cv about 2% and 3%, resp.). All stemline ratios are close to the theoretical ones. Most importantly, the slide-by-slide variations of stem1ine ratios, remaining after the corrections, have been reduced by 50%. The higher the precision of the measurements of both reference and analysis cells, the closer a detectable aneuploid stemline can be located to a euploid one. Statistical approaches for sternline and single cell aneuploidy detection are proposed.

ne

Reference: Haroske,G. et aL AnaLCell.PathoL 9(1): 1-12, 1995

Working Group: Gastroenteropathology 238

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FEULGEN STANDARDS FOR DNA CYTOMETRY E. K. W. SCHULTE (a.G.) Anatomisches Institut, Universitiit Mainz

HEPATITIS C VIRUS AND AUTOIMMUNE HEPATITIS (AIH): FORTUITOUS ASSOCIATION OR CAUSATIVE RELATIONSHIP? H.P. DIENES ·und P. SCHIRMACHER Institut fUr Pathologie, Universitlit Mainz

Aims: The Feulgen reaction is currently the method of choice for quantitative staining of DNA. However, the Feulgen reaction is complicated, and standards have to be carefully observed. Some of them will be discussed in detail, and a reference protocol is recommended for a quality controlled Feulgen procedure on cytological material. Material: Any cytological specimen (smear, imprint, centrifuged material from serous effusion, aspiration biopsy, cell culture). Fixation: Neutral 4% formaldehyde on air dried material. Postfixation in formaldehyde of wet material (alcohol or spray fixed) is possible. Fixation time I hour. Rinse carefully in water after formaldehyde fixation (otherwise unspecific background staining i). Hydrolysis: 5 M HCl at 220 C. Other protocols can be used if hydrolysis profile of the material is checked. Average hydrolysis time: 45 min. Staining: Commercial Schiff reagent is recommended. Pararosanilin (C.l. 42500) or basic fuchsin of high quality should be used for self-prepared reagent. Thionin (C.1. 52000) may be used if blue nuclear staining is required (cave: shelf-life of reagent i). Staining time 1 hour. Rinse: Freshly prepared (!) sulphite water for at least 10 min. Dehydration: Alcohol 50 - 100%. Measurements: Use interference filter at absorption maximum of reagent (about 550 to 560 nm for Pararosanilin; about 620 nm 630 nm for Thionin). Give bandwidth of filter. Use internal standard (blood cells) wherever possible) for diploid DNA standard. Results: Magenta stained cell nuclei; nucleoli and cytoplasm unstained. Use positive (known standard, e.g. liver imprints or blood smears) and negative (unhydrolyzed) control slides. Avoid: Hot hydrolysis. Microwave heating.

HCV has been implicated in a variety of immune disorders as Sjogren's syndrome, thyroiditis, polyarthritis nodosa, cryoglobulinemia, and AIH. Soon after ils discovery HCV has been causative associated with AIH type I and II (d,efined by autoantibodies ANA, SMA, and anti LKM-l) showed a high incidence of antibodies against the vinis. Furthermore, In many patients with chronic hepatitis C autoantibodies against GOR, a host-coded cellular epitope, can be detected. Another evidence that HCV seems to be able to induce autoantibodies and thus being a candidate agent for the development of AIH comes from the finding that frequences from the viral genome· share homology with cytochrome P450DII6, the target antigen for LKM-I autoantibodies. However, it could be demonstrated that in patients with non-viral genuine AIH a different immunoregulatory mechanism against distinct epitopes working than in patients with autoantibody positive chronic HCV infection. The histopathology of patients with chronic hepatitis Clacking autoantibodies and of those with autoantibodies seems to be identical. It can be concluded that HCV may induce autoantibodies in patients with a certain genetic background. However, the virus itself cannot be regarded as a direct causative agent for subtypes of AIH.