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)     

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 CINI and 96.9% of the CINII 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.     

Quantitative electronic analysis of chromosome autoradiographs using a television image analysing computer

A quantitative method for analysis of chromosome autoradiographs, using a TV image analysing computer (Leitz Classimat) is described. The principle of the method is based on an integrated measurement of silver grain areas, selected by grey value discrimination. The areas are shown to be representative for the amount of radioactive substance present, and are suitable for comparison of different grain densities over chromosomes.     

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.     

Automation of routine clinical chromosome analysis. I. Karyotyping by machine

An automated karyotyping system suitable for widespread use in clinical laboratories is described. The software is implemented on a general-purpose, commercially available image analyzer (Magiscan 2) using TV input from a conventional research microscope with minimal modification. The analysis is automatic, but operator interaction is used to resolve difficulties. Extensive experience with a routine clinical workload shows that the system is robust and easy to use and that its use results in a substantially increased laboratory throughput.     

Clinical performance of a system for semiautomated chromosome analysis.

Until recently equipment for automated chromosome analysis has not been used for routine purposes in clinical cytogenetic laboratories. During a 3 1/2-year period the chromosome laboratory of Rigshospitalet has tested the Magiscan chromosome system under routine conditions and performed the first evaluation of its clinical performance. The system consists of an image processor with a light pen for manual interaction connected to a hard-copy printer and a microscope with a TV camera and a motorized scanning stage for eight slides. Automated metaphase finding takes place without operator assistance. An operator is involved in the analysis after the metaphases are located. Using two of these complete systems, we have performed a total of 4,691 chromosome analyses comprising a count of 10 metaphases, of which three were "eyeball" karyotyped and one was "machine" karyotyped. Presently, two-thirds of our prenatal analyses (amniotic-cell cultures) are carried out with these two machines. A third Magiscan system without scanning stage is used as a "karyotyping-only" system to produce hard-copy karyograms in those cases in which metaphases are manually located and counted in the microscope. Since the end of 1984, 4,773 additional machine karyograms have been produced with this system. With a complete system, a prenatal analysis can be carried out in an average of 35 min. The average time for a machine karyotype is 7 min. Since 1984 the productivity of the laboratory has increased 17%-20% without enlarging the staff.     

Performance of a TV image analysis system as a microdensitometer

The performance of a TV image analysis system combined with an automated microscope (the Leitz TAS plus) as a microdensitometer and morphometric device was investigated. There was a strict linear correlation (r greater than .99) between the physically defined optical transmission values and the resulting electronic signals from the plumbicon TV camera for the whole area displayed on the monitor. The shading of the optoelectronic system had a coefficient of variation (CV) of 1.42% for measurements in the center of the displayed area, but a CV of 3.55% for measurements over the whole monitor area. Densitometric measurements remained stable 15 minutes after putting the microscope lamp into operation (T less than 0.075%, remeasuring every two minutes). The geometric distortion, measured as different ferret diameters of ideally round latex particles, ranged from +/- 0.5% to +/- 1.0% deviation over the entire displayed area. These results indicate that densitometric and morphometric measurements with this equipment are sufficiently precise and reproducible when performed in the center of the area displayed on the monitor.     

Multidimensional slit-scan detection of bladder cancer. Preliminary clinical results

A multidimensional slit-scan flow system was developed for the automated recognition of abnormal cells derived from cancer of the uterine cervix and its precursors. It provides great sensitivity in both its ability to recognize cellular abnormality and to deal with the myriad potential causes of false alarms in an automated flow system. While its initial application was the automated recognition of the spectrum of neoplasia in gynecologic cytology samples, a preliminary study was carried out using specimens obtained from the urinary bladder. Cellular material was collected by bladder irrigation and stained with the fluorochrome acridine orange. One hundred fifty-three bladder irrigation specimens, including 115 abnormal specimens containing cells derived from neoplastic lesions of the bladder epithelium, were analyzed. For the purposes of this study, abnormal specimens from the urinary bladder included specimens containing cells derived from the following lesions of the urothelium: dysplasia (atypical hyperplasia), carcinoma-in-situ, and transitional cell carcinoma, grades 1-3. Approximately 50,000 cells were analyzed for most specimens. Of the 38 presumed normal specimens (specimens containing only normal urothelial components), four were instrument classified abnormal. For the 69 specimens containing cells derived from transitional cell carcinoma, grade 1, 1-2, 2, 66 were correctly classified as abnormal while three were classified as normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acriflavine-Feulgen stilbene staining: a procedure for automated cervical cytology with a television based system (LEYTAS).

A sample preparation and staining procedure for automated cytology with a TV based system (LEYTAS) is described. It consists of a centrifugation technique and automated acriflavine-Feulgen stilbene staining of cervical specimens. The advantages of using both the fluorescence and the absorption image of acriflavine-Feulgen stilbene stained cervical cells for a television based system are discussed.     

False alarms in a slit-scan flow system: causes and occurrence rates. Implications and potential solutions.

A slit-scan technique was developed as a basis for an automated prescreening system for gynecologic cytology. A flow system based on this technique was fabricated and tested and results indicated that false alarms (misclassification of objects or events from normal specimens as abnormal) are the greatest remaining obstacle to development of an automated prescreening instrument. A dual view correlation system was fabricated to provide exact image-contour correlation in flow and permit precise determination of causes and occurrence rates of false alarms. This paper presents data from correlation analyses of 23 normal cytologic specimens. Major causes of false alarms and their implications to automated prescreening are discussed. A technique that would eliminate the majority of false alarms in flow is presented.     

Development and validation of a computerized cytomorphometric method to assess the maturation of vaginal epithelial cells

BACKGROUND: After menopause, declining levels of estrogens may cause vaginal discomfort, or so-called "vaginal atrophy." Evaluation of therapies for vaginal atrophy may be performed using the so-called "maturation index." The maturation index is expressed as the percentage of (para)-basal, intermediate, and superficial epithelial cells in a vaginal smear. Manual assessment of the maturation index is subject to inter- and intraobserver variations. In this study, assessment of the maturation of cells in vaginal smears using automated image analysis was investigated. MATERIALS AND METHODS: Automated assessment, using a commercially available image analysis system, was performed on hematoxylin-eosin-stained cytospin specimens. A training set was constructed by an experienced cytotechnologist, based upon visual classification of stored grey value images. From this, two discriminant functions (DFs) were calculated capable of classifying cells in one of the three types. These cell classifiers were capable of classifying 97% of the cells correctly. Data from automated assessment were compared with those of classical manual counting. Specimens of 13 mature and 6 atrophic vaginal specimens were assessed in duplicate, both manually and by image analysis, using the DFs. RESULTS: No significant interobserver effect was found for image analysis, whereas a significant effect was found for manual counting. Both methods were able to distinguish between matured and atrophic specimens. CONCLUSIONS: It was concluded that for assessment of vaginal maturation, the use of automated image analysis systems is recommended. Besides increased reproducibility, image analysis systems yield additional data describing the size and shape of the cytoplasm and nucleus of cells, which might increase discriminating power.     

一种目视激光显微镜

本文介绍一种目视激光显微镜。该装置采用白炽灯和激光做光源。通过调压器衬底亮度可以调整到零。由于激光的高亮度和强相干性,与普通显微镜相比,该显微镜具有景深长,分辨率高,层次丰富的特点。使用该显微镜时能实现镜象的假色彩编码,且镜象具有立体感。文中报导了该显微镜的原理和使用效果。     

Bowenoid papulosis. Classification as a low-grade in situ carcinoma of the epidermis on the basis of histomorphologic and DNA ploidy studies

The nature of bowenoid papulosis was investigated by a comparative investigation of 12 biopsy specimens of this lesion, 19 biopsy specimens of Bowen's disease, 14 biopsy specimens of squamous cell carcinoma and 10 biopsy specimens of seborrheic keratosis. In addition to conventional histomorphologic and cytomorphologic studies, nuclear DNA measurements on single cells isolated from tissue blocks were performed using a TV image analysis system combined with an automatic microscope. Two parameters, the "5c exceeding rate" (5cER) and the "2c deviation index" (2cDI), were computed from the single-cell DNA values to arrive at a "DNA diagnosis" and a "DNA malignancy grade" (DNA-MG). All specimens of bowenoid papulosis and Bowen's disease were morphologically diagnosed as in situ carcinomas of the epidermis; a DNA diagnosis of malignant was rendered in all of these specimens due to the detection of aneuploid nuclei (5cER greater than or equal to 1). DNA diagnoses of malignant were also rendered on all specimens of squamous cell carcinoma (100% sensitivity) while DNA diagnoses of benign were rendered in all specimens of seborrheic keratosis (100% specificity). The mean DNA-MG for bowenoid papulosis (0.69) was significantly lower than that for Bowen's disease (1.04) and squamous cell carcinoma (1.15). The mean morphologic (Broder's) grade of malignancy was also lower for bowenoid papulosis than for Bowen's disease and squamous cell carcinoma. HPV 16 DNA was detected in 10 of 12 specimens of bowenoid papulosis. Thus, the results of DNA image cytometry and morphologic investigation suggest that bowenoid papulosis is a low-grade carcinoma in situ of the epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

An automated microscope for cytologic research a preliminary evaluation.

A research-oriented system for automated microscopy is described from an operational point of view. The system consists of a microscope, a TV camera, an automatic cell finder and a servo-driven computer controlled stage. The system is interfaced to a NOVA 840 computer having 112,000 words of 16-bit core memory and extensive peripherals. It is capable of performing a wide variety of image processing tasks and is being used to study various aspects of automated microscopy, with applications in, but not limited to, cytology. Results of preliminary performance evaluations are given.     

A microcomputer system for video image analysis and diagnostic microdensitometry

A system for microdensitometry based on a microcomputer, video digitizer and solid-state camera has been developed. Image analysis and densitometry are achieved with convenient control over image editing and calibration. The linear photometric properties of the imaging device enable measurements of high accuracy. The system has proven to give rapid and repeatable performance for determining DNA content distribution from measurements of Feulgen-stained cell nuclei. The results show that a practical image analysis microdensitometer can be designed using a readily available microcomputer. The low cost and simple operation are of benefit for diagnostic applications in which flow cytometry is not possible, the time required for microscope photometry is too great or an automated image analyzer and support staff are not available.     

STESYS2: extended STESYS software for MS Windows

The STESYS2 software is a new version of the IBM PC software supporting interactive stereological measurements. In comparison with the previous STESYS, it is enhanced by a number of useful options, e.g. on-line image input via a TV camera coupled with a microscope operating under MS Windows OS. The main advantage, when compared with other such software packages, is the design of the STESYS2 as a module of the freeware image processing system Image Tool which provides a user-friendly environment including a number of image processing and preprocessing routines. Capabilities of the STESYS2 are illustrated by a practical example: estimation of the surface area of capillaries in the terminal villi of human placenta by the Sandau spatial grid method.     

Quantitative cytology of the positive region in flow sorted vaginal smears.

Fifty-one gynecologic specimens were collected from three women's hospitals and mailed in a prefixed status to our laboratory. The specimens were classified into a negative, a suspicious, a postradiation, and a positive group. After single cell dispersion the samples were stained for DNA and protein, analyzed, and sorted in the dual laser equipped Heidelberg flow analyzer sorter (HEIFAS). Particles with elevated DNA values (beyond 3.5 ploidy) and with intermediate protein values were sorted as the positive fraction directly on microscopic slides. After restaining according to Papanicolaou, they were re-evaluated cytologically and identified as tumor cells, dysplastic cells and false alarms. The latter consist of doublets and aggregates of more than two cells, binucleated cells, sperm aggregates and epithelial cells contaminated with bacteria. The different groups showed significant differences regarding the total rate of aggregates to single cells. In general, false alarms were very frequent in the positive region and impeded the statistical classification of the sample. The reduction of false alarms is a prerequisite for prescreening with flow instrumentation.     

Rapid diagnostic DNA cytometry with an automatic microscope and a TV image-analysis system

A TV-based image-analysis system (Leitz TAS plus) combined with a Leitz automatic microscope was used for rapid DNA cytometry for diagnostic purposes. Malignant or suspicious cells found by the cytopathologists in conventionally stained smears were automatically relocated and measured. A program for automatic detection and measurement of nuclear DNA content was developed. DNA data are processed by an algorithm for the diagnosis and grading of malignancy. This diagnostic procedure is performed in real time and yields highly reproducible results. With this equipment, diagnostic DNA cytometry may be introduced in the routine cytology laboratory. Our mode of application does not replace the cytopathologist, but assists him or her in cases of doubtful diagnosis and in the grading of malignant tumors.     

In vitro quantification by image analysis of inotropic and chronotropic effects of drugs on cultures of cardiac myocytes

Quantitative image analysis is used to measure the inotropic and chronotropic effects of drugs on cultured heart cells maintained at 37°C on the stage of an inverted light microscope, and sequentially superfused with control and treatment media. The beating of the cardiac myocytes is evaluated by simultaneously selecting up to eight areas, including cell edges, from digitized video image. The sizes and positions of these areas are controlled by the operator. To analyze the motion of cell edges in each area, the computer measures the shift of the mass center of pixels' grey levels. Finally, a few parameters are calculated for the eight areas and displayed graphically. In order to assess treatment effects, appropriate statistical tests are performed on the data. Image analysis is an efficient screening test for evaluating the pharmacologic or toxic effects of a substance on isolated or cultured cardiac myocytes from various species.Abbreviations MEM Minimum Essential Medium