High DNA content and prognosis in lymph node positive breast cancer. A case control study by the University of Leiden and ECOG. (Eastern Cooperative Oncology Group).

To investigate whether breast cancer cells with unusually high nuclear DNA content are associated with an adverse outcome, Eastern Cooperative Oncology Group investigators selected breast cancer trial patients who suffered an early death (ED) within two years after diagnosis to compare with other trial patients who had a survival of at least 7.5 years. Paraffin blocks of primary breast cancers were obtained from 93 evaluable patients who had been enrolled in two surgical adjuvant trials for lymph node positive (LN+) disease (T1-3N1M0). Single cell monolayer preparations from these blocks were stained with acriflavine-Feulgen and analyzed by image analysis for DNA content with the automated Leiden Television Analysis System (LEY-TAS). Standard prognostic variables (estrogen receptor (ER) status, number of lymph nodes with metastases, and size of the cancer) were compared with three DNA content characteristics: DNA ploidy status, number of nuclei with > 5C DNA content, and percent of nuclei with > 5 C. Estimates of the odds ratio in multivariate comparisons showed that ER negativity was associated with ED (p = 0.0005) and an odds ratio estimate using negative/positive of 4.87. The number of positive lymph nodes associated with ED had a p-value of 0.0005 and an odds ratio estimate of 4.63 when comparing the > 3 nodes group to the 1-3 nodes group. In contrast, the strongest association for any of the DNA content characteristics with ED had a p-value of 0.017 and an odds ratio estimate of 2.76. This power of association disappeared when stratified on ER status.(ABSTRACT TRUNCATED AT 250 WORDS)

Laboratory test of an automated cell analysis system for cervical screening.

An automated cell analysis system (Autoplan-MIAC) for the early detection of precancerous lesions of the cervix was tested under semi-routine conditions in a clinical cytology laboratory. A set of 1500 specimens, highly enriched with abnormal cases, was analysed. Cervical scrapings were collected in suspension and processed by cytocentrifugation for microscopy. Two slides were prepared from each sample: one for staining according to Papanicolaou for the visual reference diagnosis and one for Feulgen staining for automated analysis. The specimens were evaluated in two ways: the first one, which is referred to as the automated machine classification system (AMC), classifies the specimens according to the number and ratio of selected objects (alarms) and is a fully automated system. The second system classifies the specimens after visual evaluation of the stored alarms as they are displayed on a TV monitor, and is designated the interactive machine classification system (IMC). The AMC results showed a false positive rate of 16.5% when the cut-off threshold was selected so that all 117 positively diagnosed specimens were classified 'positive' by the system. In that case 87.4% of the CIN I and 96.9% of the CIN II cases were AMC-positive. The IMC results showed a false positive rate of 2.5%, when 86.3% of the CIN I cases, 96.9% of the CIN II cases and all CIN III and invasive carcinoma cases were positively classified.

The prognostic value of DNA content measured by image cytometry in soft tissue sarcomas.

Nuclear DNA content in soft tissue sarcoma was determined by image cytometry using archival, paraffin embedded material. In a retrospective study 138 specimens of 81 patients have been analysed. The ploidy level was correlated to clinical outcome regarding tumor volume and histological grading, the most important prognostic parameters. Ploidy has a significant prognostic value and correlates well with histological grading (p = 0.01). Tumour volume was found to be an independent prognostic factor (p = greater than 0.1) [chi 2 test]. The DNA content of the primary tumour and of multiple local recurrences remained similar.

Interobserver variability in the cytological diagnosis of 1500 Papanicolaou stained cervical monolayer specimens.

Cervical specimens from 1500 patients were prepared by means of a centrifugation procedure to obtain monolayer specimens suitable for automated screening using a machine. After staining according to Papanicolaou, each specimen was diagnosed by four independent cytologists from two different institutes. Within each institute, noncorresponding screening results were discussed to arrive at a conclusion diagnosis. After discussion of the discrepancies between the two centers, the conclusion diagnoses were combined to one final cytological diagnosis for each specimen. This final diagnosis is to be used as a reference diagnosis to evaluate machine classification as obtained by the AUTOPLAN/MIAC system. This system is presently being tested both in Leiden and in Frankfurt for its accuracy of detecting abnormal lesions in cervical specimens. The used diagnostic procedure resulted in a negative reference diagnosis for 1217 of the 1500 specimens; 170 specimens were diagnosed CIN I or II (mild or moderate dysplasia) and 113 specimens had a positive reference diagnosis (CIN III or invasive carcinoma). Based on these three diagnostic classes, the agreement between the four independent cytologists and the reference diagnosis varied between 93.60% and 96.60%, whereas 95.33% of all 6000 diagnoses correlated with the reference diagnosis.

Automated cell analysis for DNA studies of large cell populations using the LEYTAS image cytometry system.

Image cytometry by means of LEYTAS features analysis of both fresh and archival cellular material. Although not as accurate in ploidy determination as flow cytometry, LEYTAS cytometry incorporates extensive artefact rejection algorithms, thereby allowing detection of low frequency cells. This feature is very useful for the search of rare cells, as e.g. in cervical screening, or for the quantitation of the number of high DNA content cells in the total cell sample. LEYTAS main components are an automated microscope (Autoplan) and a Modular Image Analysis Computer (MIAC), both from Wild Leitz (W-Germany). This paper discusses LEYTAS instrumentation and cell analysis by means of programs especially written for LEYTAS.

Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.

Image cytometry in automated cervical screening.

Severe restrictions with regard to false negative rates have played a major role in the development of the LEYden Television Analysis System (LEYTAS). The present paper describes a test with a continuous series of 1500 cervical samples illustrating the accuracy of LEYTAS in a fully automated screening procedure using cell selection transformations and artefact rejection procedures. Specimen classification with a cut-off at greater than 0.3% alarms (= percentage of automatically selected objects per epithelial cells) and greater than 10 alarms, results in a false negative rate (FNR) of 0.3% (1 case out of 321 cases with severe dysplasia or more serious lesions), a false positive rate (FPR) of 13% (663 negative cases) and a rejection rate of 2.7%. Besides a machine classification, LEYTAS offers a second, machine-interaction classification of those preparations which have been declared positive by the machine. Machine-interaction involves visual evaluation of the stored images of the detected objects (alarms) and reduces the FPR from 13 to 8%. Statistical tests further demonstrate the significance of the screening results. Presently the main drawback for routine use of automated screening with LEYTAS seems to be the time consuming preparation procedure, since instrumentation has now been updated to a new, fast and user-friendly version of LEYTAS.

Automated screening for micrometastases in bone marrow smears.

It is possible to detect micrometastases in primary breast cancer using immunocytochemical staining of bone marrow smears. However, using the light microscope the procedure is time-consuming and laborious because such cells occur rarely (less than 1 in 10,000). Using an image analysis system, the Leytas machine, and a specially prepared reproducible slide it has been possible to automate the technique. A 100% concordance was found between the machine and the light microscope in the identification of slides containing moderate to high numbers of tumour cells in bone marrow, and in those containing no tumour cells. However, in those slides containing low numbers of tumour cells (1-10 tumour cells/10(6) normal bone marrow cells) the sensitivity was decreased to 91%. In the presence of non-specific staining the false positive rate was increased from 0% to 22%. This method represents a potential improvement in the assessment of an important clinical staging procedure.

Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation.

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.

A simple method to select specific tumor areas in paraffin blocks for cytometry using incident fluorescence microscopy.

A simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopathological criteria is feasible using the adjacent section after hematoxylin-eosin staining as a diagnostic guideline. Once the position of the tumor area is determined, a small hollow bore, which is screwed in the objective holder, is pressed in the tissue. The depth is controlled by the microscrew of the microscope. After retraction of the bore, the block is removed, and a thick section is cut. The selected area can be processed for cytometry separately from the remaining tissue. The technique can be used either to enrich the sample to be analyzed with tumor cells or to analyze histopathologically different tumor compartments. Both flow and image cytometry can make use of this selection technique.

Image analysis combined with quantitative cytochemistry. Results and instrumental developments for cancer diagnosis.

This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry.

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine-Feulgen-stained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.

Preparation of monolayer smears from paraffin-embedded tissue for image cytometry.

A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin-embedded tissue this method allows retrospective studies of, for instance, the significance of DNA content with regard to the behavior of a tumor.

Automated recognition of atypical nuclei in breast cancer cytology specimens by iterative image transformations.

In order to develop an objective grading system for nuclear atypia in breast cancer, an image analysis technique has been applied for the automated recognition of enlarged and hyperchromatic nuclei in cytology specimens. The image segmentation algorithm, based on the 'top hat' image transformation developed in mathematical morphology, is implemented on the LEYTAS automated microscope system. The performance of the segmentation algorithm has been evaluated for fifty malignant and eighty-five benign breast lesions by visual inspection of the displayed 'flagged' objects. The mean number of flagged objects per 1600 image fields for breast cancers was 887 (range 0-7920) of which 87% consisted of single, atypical nuclei. For benign lesions the mean number was 30 (range 0-307) of which 20% were single nuclei. By adaptation of the 'top hat' parameter values, a more extreme subpopulation of atypical nuclei could be discriminated. The large interspecimen variation in the breast cancer results was related to differences in DNA content distribution and mean nuclear area, determined independently with scanning cytophotometry, and to some extent with the histological type.

Combined flow cytometry and image cytometry of the same cytological sample.

Flow cytometry and image cytometry, two measuring techniques in the field of analytical cytology, can be used sequentially on the same cytological sample. Cells stained with a fluorochrome for the determination of for example, DNA or RNA content are first analysed in suspension by flow cytometry. The results of the fluorescence analysis of the individual cells are presented after data processing as frequency histograms of the DNA or RNA content of all the cells of the sample. In these histograms certain cell populations such as those with an increased DNA content are defined and these are then selected for further investigation. This is achieved by sorting cells of interest into centrifugation buckets by means of electrostatic deflection of the droplets containing such cells. Sorted cell populations are then centrifuged on to glass slides and stained according to the acriflavine Feulgen-SITS staining procedure, a quantitative method for DNA and protein. Image cytometry of these stained cells is performed with a computer controlled television based image analysis system (LEYTAS). With this system abnormal cells with elevated DNA content or increased chromatin contrast are automatically detected, thereby eliminating almost all artefacts and normal cells. Subsequently detected objects are stored in grey-value memories after the automated analysis for visual examination by the cytologist. The possibilities of combined flow cytometry and image cytometry are illustrated in typical examples in the field of cervical, bladder and mammary cytology.

LEYTAS analysis of cytological specimens: the results of thousand cervical smears.

This paper describes the application of a television-based system (LEYTAS) in machine analysis of cervical specimens. LEYTAS basically consists of a Leitz microscope, the texture analysis system (TAS, Leitz), a TV camera, a 4-bit grey value memory and a minicomputer (PDP 11/23). A series of 1176 Feulgen stained cervical smears has been analysed in an automated procedure using image transformation for cell selection and artefact rejection. During the analysis of the entire series new artefact rejection procedures have been added to the automated analysis. This addition decreased the percentage of false positives to 15% in the last 277 analysed smears. This percentage concerns machine performance alone. A rapid visual interaction procedure decreases this percentage to 9%. Out of 210 morphologically positive smears (diagnosis Papanicolaou class 3b or more) from the total series, one smear was missed by LEYTAS using the machine classification.

A preparation technique for exfoliated and aspirated cells allowing different staining procedures.

Quantitative cytology requires highly standardized preparation, fixation and staining techniques in order to obtain reproducible morphology (e.g., cell size, cell shape and chromatin distribution). We found centrifugal cytology best suited to this purpose. Therefore, we recently developed an improved bucket for centrifugation that permits sedimentation of cells in a fixative solution (2% polyethylene glycol in 50% ethanol) by using centrifugation at relatively high g forces. The cell quantity, the cell distribution and the flatness of the specimens thus prepared proved to be adequate for automated anlysis using the Leyden Television Analysis System (LEYTAS). Furthermore, different cytochemical and cytomorphologic staining procedures could be performed on different aliquots of the same cytologic sample without any change in the preparation or fixation technique.

Automated analysis of cervical specimens using the T.A.S.

The Leyden Television Analysis System (LEYTAS) is applied to the automated analysis of five positive specimens (2 cases of severe dysplasia, 2 cases of carcinoma in situ and 1 case of invasive carcinoma) and five negative specimens (4 cases of inflammation and 1 normal specimen). Two detection criteria based on size and contrast (so called 'top hat' transformation) are used to detect suspect nuclei. Thresholds and sizes have been used at two levels: 'high level' to detect very large and/or dark nuclei, and 'low level' to detect nuclei, of which absorption and/or size is only slightly increased compared to the nuclei of normal epithelial cells. In all positive specimens analysed with the 'high level' program suspect nuclei are automatically detected, whereas no nuclei are detected in any of the negative specimens. Using the 'low level' program many more suspect nuclei are detected in the positive specimens (up to a number of 294). This program results in some detected nuclei in the negative specimens, but the number remains relatively low (not more than 3 in the investigated cases).