Signal relay during chemotaxis.

The ability of cells to migrate in response to external cues, a process known as chemotaxis, is a fundamental phenomenon in biology. It is exhibited by a wide variety of cell types in the context of embryogenesis, angiogenesis, inflammation, wound healing and many other complex physiological processes. Here, we discuss the signals that control the directed migration of the social amoebae Dictyostelium discoideum both as single cells and in the context of group migration. This multi-cellular organism has served as an excellent model system to decipher amoeboid-like leukocyte migration and has played a key role in establishing signalling paradigms in the chemotaxis field. We envision that Dictyostelium will continue to bring forward basic knowledge as we seek to understand the mechanisms regulating group cell migration.

Contextual analysis and intermediate cell markers enhance high-resolution cell image analysis for automated cervical smear diagnosis.

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.

Contextual analysis complements single-cell analysis in the diagnosis of breast cancer in fine needle aspirates.

Image analysis techniques were used to characterize individual nuclei of cells and entire clusters of cells in hematoxylin-and-eosin-stained smears of fine needle aspirates of the breast to determine the ability of these techniques to distinguish benign from malignant cases. Analysis of the individual nuclear features showed significant differences in nuclear area, shape (bending energy), texture and integrated darkness between benign and malignant samples. Analysis of the clusters demonstrated that the benign clusters were fewer in number, more cellular (average gray level) and larger than malignant clusters. A statistical classifier was constructed to test the discriminatory accuracy for benign and malignant cases. Good discrimination was found for both the individual nuclei and the clusters when analyzed separately, although a few cases were misclassified by each type of analysis. When combined, the two classifiers achieved a completely accurate classification. This suggests the complementary nature of high-resolution single-cell analysis and the more global cluster analysis techniques.

Evaluation of contextual analysis for computer classification of cervical smears.

A procedure for automated analysis of cervical smears has been implemented in an image cytometry system. Smears are described exclusively in terms of global and contextual information extracted by pattern-recognition algorithms and represented by a vector of proportions of cellular object types. Linear discriminant functions, based on a Fisher criterion, are derived to classify smears with a cross-section of diagnoses into two broad categories, normal and abnormal. Results obtained from 83 smears indicate 78% correct classification. In contrast to most automated systems, good classification results were obtained in normal smears with benign changes caused by inflammation and with postmenopausal atrophia and in abnormals with mild dysplasia. These findings suggest that contextual analysis may be sensitive to subtle changes in cellular morphology and to progressive patterns of dysplasia. When used with standard isolated cell analysis, contextual analysis may provide additional complementary information for automated cervical prescreening.

Assessment of leukocyte alkaline phosphatase by image analysis.

We have shown that it is possible to automate the assessment of leukocyte alkaline phosphatase by using an azo dye cytochemical staining procedure and a commercial, highly sophisticated image analysis instrument originally designed specifically as a differential white cell counter. The data to date indicate that values obtained by this approach are at least as precise and accurate as current manual techniques. Instrumental analysis avoids the subjectivity associated with manual interpretation of staining intensity and should permit meaningful interlaboratory comparisons. The stability of the stained smears upon exposure to immersion oil or Polymount mounting medium proved to be an unexpected bonus. In addition to such functional data on leukocytes as illustrated by this report, these instruments, with appropriate staining methods and software, can also provide clinically useful quantitative data on red cells, as have been described for reticulocytes. We hope to see more clinical applications in the future for these expensive and target-oriented image analysis instruments. They are capable of automatically providing objective quantitative information on a cell by cell basis--providing feature data that cannot be obtained by other means.

Pressure-fixation method of transferring cells from polycarbonate filters to glass slides.

A technique for the preparation of cytology slides is presented by which cells collected on a polycarbonate membrane filter are transferred to a slide by means of simultaneous pressure and fixation. Using cervical samples as a model, the influence of the filtration rate, filter pore size and duration of pressure application on cell recovery was analyzed. The present version of the preparation procedure uses manual techniques that define the operating characteristics of a fully automated procedure.

Cytologic cell collection, suspension and preservation for automated screening procedures.

Since disaggregation for monodispersion is optimal when the specimens are suspended in solutions of low alcohol content, we evaluated a modified Davis' solution as a collection and suspension medium. The preservation of specimens maintained at room temperature (21 C) in this solution, which contained only 10% ethanol instead of the usual 20%, was evaluated both qualitatively and quantitatively. Control slides were made on day 1, and experimental slides were made on days 3, 7, 14, 21 and 28. The slides were evaluated by a cytotechnologist and confirmed by a pathologist for the onset of degenerative changes and loss of diagnostic value. The slides were also quantitatively analyzed by a semiautomatic instrument, the Cyto-Tally, adding substantial numerical value to the inquiry. The fragility of leukocytes was evident by the 14th day. Squamous cell degeneration was seen in the interval between two and four weeks depending on the initial state of the specimen. However, the specimens were of diagnostic value for up to four weeks.

Enrichment of the gynecologic cytologic specimen.

The principal objective of our group has been and continues to be the improvement of the biologic signal-to-noise ratio of the gynecologic cytologic specimen through: the dispersal of clusters and clumps, enrichment of the abnormal component of the dispersed specimen by the method of rate-zonal sedimentation under unit gravity, and monitoring the effects of these manipulations with biophysical non-destructive methods. This report is concerned only with presenting the current status of the effectiveness of our enrichment techniques both qualitatively and quantitatively. Zonal sedimentation creates a broad spatial spectrum of cells by virtue of their different velocities which are directly related to their areas. Certain fractions of this spectrum are extracted, concentrated and deposited on slides for subsequent manual differential analysis, evaluation and documentation. Qualitatively, fractions M1 and M2 show impressive removal of erythrocytes, leukocytes and debris. Quantitatively, enrichment factors vary from 0.1 (depletion) to as high as 26 yielding an average enrichment of about 6, a most rewarding improvement.

Ultrasonic disaggregation of cell clusters.

A technique of cluster disaggregation has been developed and applied to cervical cells in suspension. Preliminary results indicate that ultrasound is capable of cluster disaggregation under controlled energy conditions. Cytologic quality of the resulting preparation is adequate for cell analysis in flow systems.