[Fat pulmonary embolism after liposuction]. / Embolie graisseuse pulmonaire après liposuccion.

A 24-year-old woman undergoes buttock's liposuction as an outpatient procedure. As she went back home, progressive dyspnea, respiratory distress and collapse developed. At hospital admission, she was dyspneic with thoracic oppression, tachycardia and anguish. Chest X-ray and thoracic CT scan suggested a pulmonary localisation of fat emboli. Symptomatic treatment allowed complete recovery. This report discusses diagnosis of fat emboli after liposuction as well as epidemiology and physiopathology.

Classification of lymphoproliferative disorders by spectral imaging of the nucleus.

Spectral nuclear morphometry was used for the classification of lymphocytes in lymphoproliferative disorders. May-Grunwald-Giemsa-stained blood specimens were taken from thirty patients with infectious mononucleosis, non-Hodgkin lymphoma or chronic lymphocytic leukemia, and from ten healthy individuals. Blood specimens were analyzed by spectral imaging. Seventeen distinct spectra were collected into a spectral library and a distinct pseudo color was assigned to each one of them. The library was used to scan all the cells in the database and to create a spectrally classified image of each cell. The spectral map, per cell, reveals distinct spectral-response regions in each cellular compartment, via the distinct region colors. Computational analysis of the spectral maps allows for the objective quantification of a set of parameters, or features, representing the cell. The features used in this work include the area and perimeter of the nucleus, circularity, edginess and the spectral pattern. The analysis pursued showed that each class of cells is associated with a set of unique parameters. We conclude that spectral analysis combined with feature analysis provides significant information in the analysis of lymphoproliferative disorders and may serve as an additional tool for the histopathological evaluation of disease.

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells.

OBJECTIVE: The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS: Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS: Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS: Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.

Spectral imaging of MC540 during murine and human colon carcinoma cell differentiation.

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147-153, 2001)

Nuclear transport of photosensitizers during photosensitization and oxidative stress.

The nuclear transport pathways of the photosensitizers meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4) and meso-tetra(4-N-methylpyridyl)porphyrin (TMPyP) during photosensitization and oxidative stress were characterized in CT-26 murine colon carcinoma cells using fluorescence microscopy and multi-pixel spectral imaging. Prior to irradiation, TPPS4 and TMPyP localized mainly in the lysosomes, while irradiation or H2O2 treatment induced a relocalization into the nucleus and nucleoli. Flow cytometry analysis of isolated nuclei from the treated cells showed an increase in nuclear fluorescence accompanying the relocalization. Isolation and separation of the nuclear proteins according to molecular weight was performed using a sephadex G-100 column. The protein fractions exhibiting high fluorescence were separated by high performance liquid chromatography. Five major classes of proteins with a retention time of 1, 7, 11, 12 and 15 min were obtained. Each photosensitizer was associated with a distinct class of proteins. While TPPS4 fluorescence was detected in the protein fraction with a retention time of 11 min, TMPyP fluorescence was associated with a protein fraction having a retention time of 7 min. We conclude that although oxidative stress triggers entry into the nucleus of both TPPS4 and TMPyP, each sensitizer uses a distinct transport mechanism based on its chemical properties.

Potential use of spectral image analysis for the quantitative evaluation of estrogen receptors in breast cancer.

Evaluation of estrogen receptor (ER) content is an important factor in the choice of therapy and prognosis of breast cancer patients. In this study, we demonstrate a new spectral image analysis technique for objective and quantitative evaluations of stained specimens. The SpectraCube system was used to analyze nuclear antigens in thirteen cases of breast cancer stained by the immunoperoxidase method with hematoxylin counterstain. Spectral imaging segregated the spectrum of diaminobenzidine (DAB) from the background color of hematoxylin and a spectral index was calculated. The spectral index essentially agreed with the pathologist's index (on a scale of 0 to 3) in seven out of the thirteen cases. A substantial number of ER positive pixels was detected in the two cases scored as 0 by the pathologist's index. In a test case scored as 1 by the pathologist's index we detected a significant number of pixels, representing 47% of the nuclei, with DAB-intensity values higher than the cut-off value of 1.2. These data suggest that spectral image analysis is a sensitive method providing intensive information with high reproducibility. Our spectral imaging method is highly flexible, enabling the user to define the spatial resolution of the analyzed specimen by choosing the number of pixels per one nucleus.

Spectral imaging of red blood cells in experimental anemia of Cyprinus carpio.

In the present work we have studied the effect of experimental anemia induced at both low and optimal temperatures on erythropoiesis in Cyprinus carpio. The results showed that hemoglobin concentration per cell was similar in both temperature conditions, however, red blood cell (RBC) concentration was higher at the optimal temperature. Induced anemia caused an abrupt decrease in RBC concentration, while the hemoglobin concentration per cell remained unchanged. Recovery, as shown by electron microscopy, was characterized by the release of differentiating young and intermediate cells to the peripheral blood. It was revealed that with the progression of differentiation the nucleus/cytoplasm ratio decreases, the chromatin condenses and the shape of the nucleus changes from round to elliptical. Spectral imaging revealed an increase in the optical density of chromatin with the maturation of the cells. The chromatin that was dispersed over the nuclear volume in the young cells becomes highly ordered in the mature cells. Spectral similarity mapping revealed the formation of a novel structure of high symmetry, representing chromatin rearrangement during the process of cellular differentiation.

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen.

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines.

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.

Spectral morphometric characterization of breast carcinoma cells.

The spectral morphometric characteristics of standard haematoxylin and eosin breast carcinoma specimens were evaluated by light microscopy combined with a spectral imaging system. Light intensity at each wavelength in the range of 450-800 nm was recorded for 10(4) pixels from each field and represented as transmitted light spectra. A library of six characteristic spectra served to scan the cells and reconstruct new images depicting the nuclear area occupied by each spectrum. Fifteen cases of infiltrating ductal carcinoma and six cases of lobular carcinoma were examined; nine of the infiltrating ductal carcinoma and three of the lobular carcinoma showed an in situ component. The spectral morphometric analysis revealed a correlation between specific patterns of spectra and different groups of breast carcinoma cells. The most consistent result was that lobular carcinoma cells of in situ and infiltrating components from all patients showed a similar spectral pattern, whereas ductal carcinoma cells displayed spectral variety. Comparison of the in situ and the infiltrating ductal solid, cribriform and comedo carcinoma cells from the same patient revealed a strong similarity of the spectral elements and their relative distribution in the nucleus. The spectrum designated as number 5 in the library incorporated more than 40% of the nuclear area in 74.08% of the infiltrating lobular cells and in 13.64% of the infiltrating ductal carcinoma cells (P < 0.001). Spectrum number 2 appeared in all infiltrating ductal cells examined and in none of the lobular cells. These results indicate that spectrum number 5 is related to infiltrating lobular carcinoma, whereas spectrum number 2 is characteristic for infiltrating ductal carcinoma cells. Spectral similarity mapping of central necrotic regions of comedo type in situ carcinoma revealed nuclear fragmentation into defined segments composed of highly condensed chromatin. We conclude that the spectral morphometric features found for lobular and ductal cell populations may serve future automated histological diagnostics.

Spectrally resolved morphometry of the nucleus in hepatocytes stained by four histological methods.

A novel concept of spectrally resolved morphometry for histological specimens was developed using light microscopy combined with spectrally resolved imaging. The spectroscopic characteristics of rat hepatocytes stained by Haematoxylin and Eosin, Romanowsky-Giemsa, periodic acid-Schiff and Masson's trichrome were assessed. Light intensity in the range 450-850 nm was recorded from 10000 pixels of nuclear domains of each stained cell and represented as light transmittance spectra and optical density. In order to identify spectral shifts caused by stain-macromolecule interactions, we compared the spectra of individual stain components with those of DNA and bovine serum albumin. Chromatin and interchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for each staining method. The spectral fingerprints of Masson's trichrome stain distinguished the nucleolus from the rest of the nuclear chromatin, enabling the demarcation and calculation of the nucleolar area. Spectrally resolved imaging of human hepatocytes stained by Masson's trichrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masson's trichrome stain also distinguished the nucleolar area in human breast carcinoma cells and keratinocytes.

[Rare neurological disorders and hypoglycemia. 3 cases]. / Déficits neurologiques rares et hypoglycémie. 3 cas.

We relate three cases of rare neurological disturbances associated with hypoglycemia (paraplegia, quadroplegia). Clinical symptoms completely disappeared within a few minutes after glucose administration. The pathogenesis of these types of deficit is not fully clarified, and a search of hypoglycemia should always be performed in these circumstances.

Spectrally resolved imaging of Cabot rings and Howell-Jolly bodies.

The spectral characteristics of erythropoietic cellular inclusions stained by May-Grunwald Giemsa (MGG) were determined by spectrally resolved imaging. Multipixel spectra were obtained from Cabot rings and Howell-Jolly (HJ) bodies, displaying a range of wavelengths of transmitted light. The spectral characteristics of these inclusions were compared with those of isolated DNA, histones (type II) and arginine-rich histones (type VI), all stained by MGG. Results of single-cell spectroscopy show that the spectra of Cabot rings and HJ bodies share spectral characteristics with the type II and type VI histones. However, no resemblance was found between Cabot rings and DNA spectra. The spectral analysis of heterochromatin displayed a spectral pattern with characteristics of both DNA and histones, while the euchromatin showed a major contribution of the DNA component.

Spectral morphometric characterization of B-CLL cells versus normal small lymphocytes.

Spectral morphometric characterization of typical chronic lymphocytic leukemia (B-CLL) cells vs normal small lymphocytes stained by May-Grunwald-Giemsa was carried out by multipixel spectral imaging. The light intensity (450-850 nm of 10(4) pixels) from nuclear domains of each stained cell was recorded and represented as light transmittance spectra and optical density. Transmitted light spectra of two nuclear domains were determined, one with low-intensity light transmittance (LIT) and the other with high-intensity light transmittance (HIT). A spectral library was constructed using the four transmitted light spectra representing the HIT and LIT domains of the normal human lymphocytes and the LIT and HIT domains of the CLL cells. The spectral library served to scan CLL lymphocytes from 10 cases of CLL and the lymphocytes of 10 healthy individuals. Each spectrally similar domain in the nuclei of the lymphocytes was assigned an arbitrary color. The morphometric analysis of the spectrally classified nuclei showed specific spectral patterns for B-CLL in 92% of the cells. The specific spectral characteristics of each of the two cell populations were also observed by their optical density light absorbance spectra. We propose that spectral morphometric analysis may serve as an additional diagnostic tool for detection of CLL lymphocytes in a hematological specimen.

Spectral imaging for quantitative histology and cytogenetics.

Evaluation of cell morphology by bright field microscopy is the pillar of histopathological diagnosis. The need for quantitative and objective parameters for diagnosis gave rise to the development of morphometric methods. Morphometry combined with spectral imaging provides multi-pixel information from a specimen, which can be used for further image processing and quantitative analysis. The spectroscopic analysis is based on the ability of a stained histological specimen to absorb, reflect, or emit photons in ways characteristic to its interactions with specific dyes. Spectral information obtained from a histological specimen is stored in a cube whose appellate signifies the two spatial dimensions of a flat sample (x and y) and the third dimension, the spectrum, representing the light intensity for every wavelength. By mathematical analysis of the cube database, it is possible to perform the function of spectral-similarity mapping (SSM) which enables the demarcation of areas occupied by the same type of material. Spectral similarity mapping constructs new images of the specimen, revealing areas with similar stain-macromolecule characteristics and enhancing subcellular features. Spectral imaging combined with SSM reveals nuclear organization and identifies specifically the nucleoli domains. Therefore, differentiation stages as well as apoptotic and necrotic conditions are easily quantified. The commercial SpectraCube system was developed for the application of spectral imaging in biology, recording both transmitted light and fluorescence. The SKY technique utilizes the advantages of the SpectraCube for multi probe FISH and chromosome karyotyping, identifying marker chromosomes, detecting subtle chromosome translocations and clarifying complex karyotypes.

Chromatin condensation in erythropoiesis resolved by multipixel spectral imaging: differentiation versus apoptosis.

Chromatin condensation and nuclear organization of May-Grunwald-Giemsa (MGG)-stained normal erythropoietic bone marrow cells and apoptotic red cell precursors were resolved by spectral bio-imaging. Multipixel spectra were obtained from single cells displaying a range of wavelengths of both transmitted and absorbed light. Two groups of spectra, of low- and high-intensity transmitted light, were revealed in the nuclei of each cell. The absorbance spectra served for the reconstruction of "absorbance images" depicting the affinity of MGG stain for the chromatin of proerythroblasts and of basophilic, polychromatic, and orthochromatic normoblasts. The localization of different spectral components in the nuclei was resolved employing two mathematical methods, spectral similarity mapping and principal component analysis. Novel structures of high symmetry revealing windmill-like organization were detected in basophilic, polychromatic, and orthochromatic normoblast cells. Matching structures were detected in apoptotic normoblasts obtained from an agnogenic myeloid metaplasia patient. Apoptosis was associated with a gradual breakdown of the ordered arrays in the nucleus. We propose that DNA cleavage may lead to fragmentation of the symmetrical windmill-like superstructure of the basic nuclear domains.

Subcellular localization of sulfonated tetraphenyl porphines in colon carcinoma cells by spectrally resolved imaging.

Subcellular localization of the dye, 5,10,15,20-tetra(4-sulfonatophenyl)porphine (TPPS4) and the more hydrophobic dye, 5,10,15,20-tetra(1-sulfonatophenyl)porphine (TPPS1), in murine colon carcinoma cells was studied by spectrally resolved imaging (SRI) combined with image processing techniques. Spectrally resolved imaging enabled the acquisition of multipixel fluorescence spectra (> 10(4)) from a single cell. Demarcation of specific localization sites and segregation of the irrelevant fluorescence were based on the pixel spectra and by operating the functions of spectral similarity mapping (SSM), principal component analysis (PCA) and spectral classification. The SRI revealed the fine details of the photochemical process that clarify some aspects of subcellular damage. The SRI depicted the differences between TPPS4 and TPPS1 with respect to their initial localization and their fate at the end of the photochemical effect. The dye TPPS4 was localized initially in lysosomal vesicles, and upon irradiation fluorescence was seen in the nucleus as well as in vesicles. Some of the vesicles were closely related to the nucleus, as resolved by SSM, PCA and spectral classification. Additional light exposure stimulated relocalization of TPPS4 into the nucleus as well as into the nucleolus, which was clearly depicted by SSM and PCA. Spectral classification showed a third, weak residual cytoplasmic array around the nucleus. The dye TPPS1 concentrated in a Golgi-like complex and was resolved in the nuclear envelope and in small vesicles: it was not redistributed into other compartments upon photosensitization. Serum supplementation to the incubation media of colon carcinoma cells treated with TPPS4 or TPPS1 did not change the localization patterns. Pixel spectra of the two dyes in the cells showed spectral shifts and expanded shoulders due to microenvironmental effects. Thus, the chemical nature of the sulfonated phenyl porphines, and not their interaction with serum proteins, was the main determinant of their binding to the lysosomes, nucleus, nucleolus, nuclear envelope or Golgi.