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Current methods of detecting micrometastases in breast cancer fail in a large proportion of patients. Therefore an improved method for detection of metastases in blood samples could be of great clinical interest both for prognosis and selection of patients for adjuvant systemic therapy. We have developed a new non-invasive method which associates immuno-magnetic separation and filtration cytometry. The sensitivity of our procedure was evaluated in a model system using a mixture from a human breast cancer cell line (MCF-7) and a normal human blood sample. The identification of tumoral cells was achieved by measuring DNA content in comparison with standard cells. The lowest concentration of MCF-7 detected was 1 tumoral cell in 500,000 white blood cells. In addition, filtration cytometry provides a visual control of nuclei permitting the elimination of all doubtful cases and an automatic count of tumoral cells directly per ml of blood, which may be an independent predictor of early relapse. This new method may avoid unnecessary axillary lymph node dissection in patients with negative nodes. Our procedure seems suitable for the detection of rare circulating cells in routine laboratory testing and could be used in other applications.

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An automated system, TRAKCELL, was developed for the quantitation of cells in culture. It enabled cell counting, classification according to morphological cell characteristics and measurement of cell proliferation and differentiation. The system was tested on the toxic effect of ascorbic acid on rat brain catecholaminergic neurons in primary culture. In parallel, the effects of nerve growth factor, dexamethasone and forskolin on cell differentiation were studied using rat pheochromocytoma PC12 cells. The results show that the system permits rapid and reproducible measurements of cell density and of the morphological changes observed following various drug treatments.

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A fully automated image analyzing system was developed for the quantitative study of cells in culture. It was able to count cells, to classify cells according to their morphological characteristics and to follow cell culture development. A specific procedure was designed to process Hoffman modulation contrast images. It detects local gray level differences while using conditional dilation techniques. We were able to successfully detect aggregated unstained cells, presently a technical limit in image segmentation. Living cells can be studied in a noninvasive and nondestructive way with this system. An improved automatic focusing algorithm was developed which ensured an accurate prediction of the optimal focus position. A strictly defined sampling procedure was applied to estimate unbiasedly cell density and obtain precisely cell contours. The evaluation of the system was carried out on Chinese hamster ovary (CHO-NTR) cell cultures treated with a newly developed neurotensin agonist JMV449. Chinese hamster ovary cell division was found to be retarded 20 hours after the JMV449 treatment, while the morphology of CHO-NTR cells has already undergone significant changes 12 hours after the treatment. This image analyzing system provides the possibility to follow cell culture development (e.g., cell density evolution, cell morphological changes) under various experimental conditions.

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This paper presents a new method for the quantitative analysis of the movement characteristics of spermatozoa. The method is based on (i) measuring the displacement of a spermatozoon observed using a microscope and (ii) automatically compensating for the displacement of the spermatozoon in order to keep it in the center of the microscopic field. The displacement of the spermatozoon is measured by on-line processing of digitized images of the microscopic field, acquired by an image analysis system. The motility characteristics of the same spermatozoon can be evaluated over prolonged periods of time (typically 10 min). The movement characteristics of 19 spermatozoa from five fertile donors were studied. At constant temperature, intradonor variations of the motility parameters were found. No statistically significant differences were found in the mean values of the motility characteristics for three of four donors. At increasing temperature, the mean curvilinear velocity, averaged on the 19 spermatozoa from the five donors, regularly increased between 23 and 30 degrees C and remained constant up to 38 degrees C.

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In dissociated mouse brain cell cultures we frequently observed an association between myelin basic protein (MBP) positive oligodendrocytes and proliferating astrocytes. When MBP was added in a purified form to the culture medium, it greatly stimulated the proliferation of astrocytes, while other proteins tested did not. This finding allows us to speculate that the gliosis observed in demyelinating diseases or/and in central nervous system (CNS) injury would be due to the mitogenic effect exerted by MBP or its fragments when there is myelin breakdown.

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A software package of general interest, and adapted to the analysis of histological sections has been written. The different programs of this package have been classified into four groups: macroanalysis, selection of fields, analysis, and data processing. The macroanalysis allows the acquisition of topographical information that will be used during the selection of fields and the analysis. Mapping programs have been added to the classical data processing of the measured parameters (statistical calculations, edition of values, histograms, graphs, .. .). They allow the generation of parametric images representing the distribution of particular elements in the section, or the topographical variations of calculated parameters. The recombination of information obtained at different magnifications, and the correlation of information of different types is thus possible and of great interest in the study of complex histological structures.

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B and T lymphocytes of known origin were used to test a pretopologic (mathematical) classification model that uses chromatin texture as a parameter. One data base was obtained by digitizing the cells with a standard TV camera coupled with a microscope, with the images transmitted to an image-analysis system (ASTI) coupled with a multi-20 computer. A second data base was obtained from the same slides using the TAS coupled with a PDP 11/34. The data bases were analyzed by the pretopologic program, which paramatizes the dispersion and aggregation of 15 possible optical densities (O.D.) and their relation to each other for each cell. Sub-categories were formed according to an algorithm that forms categories of images having the same pretopologic predominating sequences of O.D. values. In the ASTI system, 92% of the B-cell and 94% of the T-cell training samples were classified unambiguously; the test sample was classified as 60% B cells and 40% T cells, which is in close agreement with the spleen distribution as noted in the immunologic literature. The classification algorithm found 13 B-cell subcategories and 12 T-cell subcategories in the respective training sample; classification of the spleen test sample added no new subcategories. The TAS results were in agreement with the ASTI results within the limits of a restricted sample size. The overall B-cell and T-cell classification results were in agreement with those obtained by other authors . The subcategories found are unlikely to be fortuitous since only certain combinations of predominating O.D. values occurred and a large number of digitized nuclei had identical parameters. Nevertheless, the biologic significance of the subcategories cannot be assessed by mathematical methods alone, and these methods must be tested with appropriate biologic models.

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Vascular growth in the median suprasylvian gyrus of the cat has been analysed quantitatively with respect to mitoses, vascular density, vascular diameters, vascular coefficient and preferential vascular orientation. After correction for shrinkage and growth, four maturation periods were identified: (i) Immature period (first postnatal week), when the tissue exhibited numerous vascular mitoses, a low but constant vascular density and vascular coefficient, preferentially radiate vessels and immature (small) vascular diameters. (ii) Premature period (second to fourth week), with few mitoses, a rapidly increasing vascular density, immature (small) vascular diameters, an increase in the number of vessels more than 10 microns in diameter, and a preferential orientation of tangential vessels. (iii) Pre-adult period (fifth to sixth week), without mitoses. The vascular density increased greatly, the vascular diameters reached adult profile, the number of vessels over 10 microns in diameter decreased, the vascular orientations were both tangential and radiate and the vascular coefficient remained slight. (iv) Adult period, when the vascular density and the vascular coefficient were maximal, the vessel diameters were of adult type, and the orientation was tangential. From six weeks, the vessels increased only in length. Vascular maturation proceeded from the depth toward the surface of the cerebral cortex. Layers II-IV were those most highly vascularised, regardless of age. This is discussed in relation to synaptic growth. There was no direct relation between vasculogenesis and myelination. The white matter had a typically radiate vascularisation. A transitional zone between cortex and white matter was identified. It had a loose mesh vascular network and corresponded to the area in which dendrites of inverted pyramidal cells were found. The role of immature vascularisation in the nutrition of the neuropil is discussed.

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Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.

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Neurogenesis was studied in the duck olfactory bulb by injection of tritiated thymidine into the eggs at 53 h and at the 3rd, 5th, 8th, 10th, 12th, 14th and 18th days of incubation. The large neurons appear before the small ones: the mitral cells arise between embryonic day 3 (E3) and E5, the tufted cells between E5 and E8 and the granular cells between E12 and E14. The periglomerular cells could be formed after E18. The order of appearance of this 4 main neurons of the duck olfactory bulb is the same as that in the mouse. All the neurons, except maybe for the periglomerular cells, are principally formed before the hatching of the duckling and the olfactory sense seems to have acquired most of its principal functional aptitudes at this moment.

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Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.

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The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.

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To obtain a reliable and fast quantification in low cell density tissues, we have developed a standardized procedure based on image analysis. The successive steps of quantification, starting with the necessary histological procedure and ending with the desired quantitative parameters, are described. Excepting the histological procedure, we have attempted to automate all the subsequent steps of the experiment, especially the acquisition of the data by an image analyser TAS (Leitz), and the storage and the analysis of these data by a PDP 11-34 computer. The final parameters calculated by such a method are the total number of cells and the cell density within the organ under study, either for all the cells, or for different cellular categories. This procedure has been applied to the quantitative study of cerebellar cells of an adult Staggerer mutant mouse and the results obtained are presented.

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In order to obtain an overall view of neurogenesis in the main central nervous structures of the mouse, we have undertaken a systematic study of the time of neuron origin and of the gradients of neuronal production. The animals were the offspring of pregnant females given a pulse label of 3H-thymidine on selected days after conception. These days were chosen to cover the principal brain development stages: E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5 and E18.5. The offspring were sacrificed during the fourth postnatal week and labeling indices were calculated in the different brain structures. The study of labeling indices as a function of the day of injection and of the brain level studied provided us with information on the major periods of neuronal proliferation in these structures and allowed us to analyse the formation gradients inside a given nervous structure. As a whole, for a majority of nervous structures (olfactory, cortical, septal, hippocampal, preoptic, thalamic, epithalamic, hypothalamic and cerebellar structures), our results are in good agreement with the already published observations. However, we outlined two major phenomena apparently unobserved until now in the mouse: the first is a rostrocaudal gradient of neuronal production in the inferior colliculi, and the second is the long period of neuronal proliferation in the caudate nucleus lasting from E11.5 to E18.5, along a complex inside-out and mediolateral gradient.

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Iron overload has been measured in 100 hepatic biopsies by three different methods: (i) biochemical assay of the liver iron concentration (LIC), (ii) histological grading (HISTO) and (iii) automated image analysis with a Leitz Texture Analysis System by estimating two parameters, (a) the total iron area (TIA) and (b) the first grey level step (FGLS) at which the iron is detected. Image analysis appears as a specific, sensitive, quick, reproducible and valid method.

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