Morphometric discrimination between syndromic (Gorlin) and nonsyndromic keratocysts.

OBJECTIVE: To search for morphologic nuclear features in the epithelial lining of odontogenic keratocysts to differentiate simple from Gorlin syndrome cysts. STUDY DESIGN: Five cases of syndrome-associated keratocysts and five of simple ones were studied. Thirty nuclei from the epithelial basal layer for each case were analyzed by the shape analytical morphometry (SAM) software system to quantitatively evaluate nuclear dimensions (area, perimeter, diameter), contour irregularities and nuclear shape asymmetries. Results were subjected to Student's t test and cluster analysis. RESULTS: Values of nuclear dimensions were very close in both groups of keratocysts, without any significant statistical differences. The variables related to nuclear profile irregularities, as well as those describing nuclear asymmetry, showed significantly higher values (P < .001) in syndromic cysts. Cluster analysis produced two different clusters by using variables related to nuclear contour irregularities. CONCLUSION: Preliminary results indicate the existence of nuclear morphologic differences between simple and syndromic cysts.     

Comparative morphometric analysis of intraepidermal and dermal lymphocytes in early mycosis fungoides and some inflammatory dermatoses

OBJECTIVE: Early stage mycosis fungoides (MF) may be indistinguishable from some inflammatory dermatoses (IDs) histopathologically. Various morphologic features of lymphocytes in epidermis and dermis may help to differentiate early-stage MF from other IDs. STUDY DESIGN: We studied 25 cases of early-stage MF and 30 cases of various IDs with prominent exocytosis. The following nuclear variables were measured on dermal and epidermal lymphocytes in MF and IDs (control) groups: nuclear area and perimeter, area convex hull, perimeter convex hull, diameter equivalent circle, minimum feret, maximum feret and form factor. All measurements were done using routinely prepared and hematoxylin-eosin-stained slides. RESULTS: There were statistically significant differences (p < 0.01) for all of the morphometric variables between early-stage MF and IDs. CONCLUSION: Our results revealed that the nuclear contour irregularities of intraepidermal lymphocytes are higher than those of dermal lymphocytes in MF. Their nuclear geometry is also different. Comparison of the nuclear features of intraepidermal with dermal lymphocytes may provide additional support in the diagnosis of early-stage MF.     

Analytical morphometry of sagittal fronto-facial profiles of 2nd-6th month human foetus

A prominent problem in the morphological study of developing biological structures is shape parametrization with the aim to evaluate transformations in a non subjective way. We carried out an analytical morphometrical study on a series of human embryos, by means of the S.A.M. (Shape Analytical Morphometry) software package. After standardization and normalization of the fronto-facial sagittal profiles, the analytical procedures applied were: 1) Fourier analysis: each profile was considered as an irregular but periodic function obtained by the sum of sinusoids of increasing order. 2) Shape Asymmetry Evaluation: couples of profiles are compared by means of "Janus" procedure; by a parabolic fitting we obtained parameters able to evaluate allometric and isometric differences between the two profiles. The preliminary results of the applied procedures indicated that rate and direction of allometric and isometric growth are not constant during the time.     

A comparative mathematical evaluation of contour irregularity using form factor and PERBAS, a new analytical shape factor

Contour irregularity was mathematically evaluated using two different analytical shape factors: (1) "form factor" (FF), a conventional type of shape analysis formula defined by the formula 4 pi A/P2, and (2) the "PErimeter Ratio Before and After Smoothing" (PERBAS). PERBAS values are obtained by first "smoothing" a contour that contains one or more concavities; surface projections are connected using straight lines and include only tangent lines that do not interest the nucleus, resulting in a convex wrap. Perimeters before and after smoothing were obtained using a MOP-3 computerized image analyzer. Using a series of computer-generated images, it was determined that FF was more sensitive than PERBAS to simple deformations of shape (i.e., having no contour concavities) whereas PERBAS was more specific for surface "roughness" (i.e., contour having at least one concavity). As an application to cancer diagnosis, 6 cases of Burkitt's lymphoma (BL) and 14 cases of Burkitt's-like lymphoma (BLL) were compared for nuclear contour roughness using both shape factors. The BLL group could be distinguished from the BL group on the basis of mean PERBAS values (P = .029), but not on the basis of mean FF values (P = .093). The BL group had a mean PERBAS value of 1.051 +/- 0.023 and a mean FF value of 0.790 +/- 0.068. The BLL group had a mean PERBAS value of 1.093 +/- 0.054 and a mean FF value of 0.723 +/- 0.094. Mean FF and PERBAS values, plotted in a bivariate graphic display, established morphometric shape domains. BL cases occupied a significantly smaller shape domain than did the BLL cases, indicating a greater heterogeneity among nuclear shapes in the latter group. Shape discrimination, in general, was greatly increased when both shape factors were used; this has a great potential for determining cancer diagnosis and patient prognosis.     

Analytical morphometry of seminiferous tubules in maturation arrest: Fourier analysis of the internal contours of tubules

Shape of seminiferous tubuli in maturation arrest, is investigated by harmonic Fourier analysis and upper degree polynomials as attempt to find morphometric parameters useful in morphological diagnosis. Inner luminal contour is considered as a closed curve in a reference system and scattered in a series of points whose coordinates are automatically calculated. Then a software named S.A.M. (Shape Analytical Morphometry) is utilized to find the Kth-order equation and calculate the best-fit curve. Moreover an ellypse is drawn with the same barycenter and axes of original curve. These three curves (real curve, best-fit curve and ellypse) are considered as periodic functions and submitted to Fourier analysis to evaluate the coefficients of the series and the spectrum of harmonics. Among 20 contributors, the subsets of greatest amplitude are selected for comparison and classification in terms of fundamental shape and its perturbations.     

An image analysis morphometric method for the study of myelinated nerve fibers from mouse trigeminal root

For the morphometric light microscopic study of myelinated fibers in mouse trigeminal root, it was necessary to write: (1) an entirely automatic analysis program for the myelinated axons inside the myelin sheath, based on the detection of the myelin sheaths, and (2) an interactive analysis program for the myelinated fibers outside the myelin sheath, due to the high density of compactness of the myelinated fibers based on an indirect fiber individualization by reconstructing them from their axons. In the latter, a semiautomatic correction method (drawing the profile contours with a light pen) allowed compensation for the failures of the automatic method, except for the smallest fibers, which represented 8% of the total. Using these programs, 95% of the axons could be measured and 92% of the myelinated fibers whose axons were analyzed could be measured. The area-equivalent diameter was independent of the detection method; it is a correct-size measurement parameter for axons and fibers that is unrelated to their shape. The projected diameter, an estimation of the perimeter obtained by measurement of the profile projections, depended upon the detection method because the profile contour was influenced by the detection method; it thus takes into account the profile shape. For myelinated fibers, whose analysis program used two detection methods (automatic and semiautomatic), there was an average difference of 16% between the projected diameters obtained with these two methods, whereas the equivalent diameter value was the same. The fiber circularity factor could not be precisely estimated because of the detection error; the axon circularity factor was more reliable since the axon detection was completely automatic.     

Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity

Although sperm head shape and relative dimensions are considered reliable indicators of sperm quality, their quantification is most often operator-driven, e.g., subjective. Artificial insemination semen doses from 35 mature stud boars of known fertility and belonging to three breeds and two hybrid breeds (Duroc, Large White, Landrace, respectively, Yorker and Risco) were used in this study. Sperm samples were extended to 100x10(6) cells per mL and 10microL of the sperm suspension used to made smears which, stained, were examined using phase contrast microscopy interfaced with an automated sperm morphology analyzer (ASMA, ISAS). Each sperm head was measured for four primary parameters [area (A) microm(2), perimeter (P) mum, length (L) microm, width (W) microm], and four derived parameters of head shape [(L/W), (4piA/P(2)), ((L-W)/(L+W)), (piLW/4A)]. Definition of head size was statistically performed. The threshold for each class was established on the basis of the area values, considering the 25th percentile as small and the 75th percentile as large spermatozoa. In a second step, sperm head shape was determined as normal, elliptic, abnormal (rugose) contour, long or irregular and percentiles set as above to define spermatozoa with normal values for each shape parameter. Significant differences were found among breeds in the size of morphologically normal spermatozoa, which were significantly larger and more elliptic (P<0.001) in the Duroc breed. Sperm chromatin integrity was studied using the SCSA-assay, with significant differences observed in the degree of fragmentation intensity (DFI) although this value was consistently low in all animals studied. The hereby-validated ASMA was able to determine significant differences in sperm shape and dimensions among breeds, which were not accompanied by deviations in chromatin structure neither within nor between fertile AI-boars.     

Temperature measurement on neurological pulse generators during MR scans

Background

Electromagnetic stimulation of the nervous system has the advantage of reduced discomfort in activating nerves. For brain structures stimulation, it has become a clinically accepted modality. Coil designs usually consider factors such as optimization of induced power, focussing, field shape etc. In this study we are attempting to find the effect of the coil contour shape on the electrical field distribution for magnetic stimulation.

Method and results

We use the maximum of the induced electric field stimulation in the region of interest as the optimization criterion. This choice required the application of the calculus of variation, with the contour perimeter taken as a pre-set condition. Four types of coils are studied and compared: circular, square, triangular and an 'optimally' shaped contour. The latter yields higher values of the induced electrical field in depths up to about 30 mm, but for depths around 100 mm, the circular shape has a slight advantage. The validity of the model results was checked by experimental measurements in a tank with saline solution, where differences of about 12% were found. In view the accuracy limitations of the computational and measurement methods used, such differences are considered acceptable.

Conclusion

We applied an optimization approach, using the calculus of variation, which allows to obtain a coil contour shape corresponding to a selected criterion. In this case, the optimal contour showed higher intensities for a longer line along the depth-axis. The method allows modifying the induced field structure and focussing the field to a selected zone or line.     

Morphometric distinction of granulomas in tuberculosis and sarcoidosis. Difference in nuclear profiles

Morphometric measurements were carried out on epithelioid-cell nuclei of noncaseating granulomas in paraffin-embedded sections of lymph node biopsy specimens originating from 15 patients with sarcoidosis and from 18 patients with tuberculosis. The results, which were obtained with the help of a computer-assisted tracing device, established highly significant differences in shape and size of epithelioid-cell nuclear profiles in these two diseases. Direct measurements and computations on these results showed that epithelioid-cell nuclear profiles in sarcoidosis have a smaller mean and median perimeter (boundary length), area and long diameter (maximum length of the nuclear profile) and exhibit a more plump, elliptical-like shape than do those in tuberculous granulomas. Epithelioid-cell nuclear profiles of the latter, as a whole, show more irregular contours, resembling those, e.g., of the sole of a shoe or other elongated patterns. Differences in shape of nuclear profiles were best demonstrated by a size-independent form factor (4 pi *area/perimeter2).     

An application of mathematical morphology to analysis of the size and shape of nuclei in tissue sections of non-Hodgkin's lymphoma

Using principles from the theory of mathematical morphology, a semiautomatic analysis of the size and shape of cell nuclei on tissue sections was carried out on a Leitz Texture Analysis System (Leitz-TAS). The four parameters proposed here are more discriminatory than conventional shape evaluation by the form factor (FF), which is based on the ratio of perimeter squared to area. The parameters quantified, respectively, nuclear elongation (ND), narrow (R1) and wide (R2) irregularities, and the distribution of R1 and R2 along the nuclear contour (ID). The properties of these parameters were tested nucleus-by-nucleus on 24 nuclear models. The methodology was then illustrated by a study of lymph node nuclei in non-Hodgkin's lymphoma (NHL). Prior to analysis, 45 lymphomas were classified into five categories of nuclear size and shape according to the International Working Formulation (IWF). Two hundred nuclei were measured on each lymph node section. Statistical interpretation was based upon an analysis of the nuclear surface area on sections and upon the mean values of R1, R2, and ND, the standard deviations of R1 and R2, and the percentage of cleaved nuclei detected by ID. The mean value of R2 discriminated best between the two sets of populations with regular and irregular nuclear contours, respectively. Parameters R1, ND, and ID permitted the distinction of certain NHL cases among populations with irregular nuclei. Nuclear invaginations decreased in depth as the nuclear area became greater. The median surface area was well correlated to the IWF, and the skewness coefficient (third statistical moment of the nuclear surface area distribution) was related to the number of nuclear size or shape subpopulations.     

Megakaryocytic features useful for the diagnosis of myeloproliferative disorders can be obtained by a novel unsupervised software analysis

An unsupervised method for megakaryocyte detection and analysis is proposed, in order to validate supplementary tools which can be of help in supporting the pathologist in the classification of Philadelphia negative chronic myeloproliferative disorders with thrombocytosis. The experiment was conducted on high power magnification photomicrographs taken from hematoxylin-and-eosin 3 micrometer thick sections of formalin fixed, paraffin embedded bone marrow biopsies from patients with reactive thrombocytosis or chronic myeloproliferative disorders. Each megakaryocyte has been isolated in the photos through an image segmentation process, mainly based on mathematical morphology and wavelet analysis. A set of features (e.g. area, perimeter and fractal dimension of the cell and its nucleus, shape complexity via elliptic Fourier transform, and so on) is used to characterize the disorders and discriminate between essential thrombocythemia and idiopathic myelofibrosis. Features related to the general contour of the cell like cytoplasmic area and perimeter are good markers in distinguishing between normal or reactive and pathologic megakaryocytes while nuclear features and global circularity are helpful in the differential diagnosis between ET and prefibrotic IMF. The method proposed should be considered as a fast preprocessing tool for the diagnostic phase and its use can be extended to solve different object recognition problems.     

Increase in the number of contacts of endoplasmic reticulum with mitochondria and plasma membrane in yeast cells stimulated to division with He-Ne laser light

We studied the ultrastructure of Torulopsis sphaerica yeast cells irradiated with He-Ne laser (lambda = 632.8 nm, dose--460 J/m2) and then cultured for 6 h in the nutrient with 1% glucose by aeration. The length of membranes of endoplasmic reticulum (ER) and the number of its associations with mitochondria (M) and plasma membrane (PM) were measured on ultrathin sections. A distance of less than 50 nm between heterogeneous membranes was considered as an "association". The cells from irradiated cultures are characterized by the following features: 1) the length of cortical ER membranes in relation to cellular perimeter, and the length of perinuclear ER membranes in relation to nuclear perimeter increase, resp., by 21 and 79%; 2) the number of ER-PM associations per cellular section, and that per unit of PM length increase, resp., by 26 and 41%; 3) the number of ER-M association in relation to the total mitochondrial perimeter, and to perimitochondrial ER increase by 80 and 87%, resp. The latter may be associated with Ca2+ uptake by mitochondria associated with ER, which results in activation of respiration and ATP production.     

The use of intraoperative grid pattern markings in lipoplasty

Intraoperative grid pattern markings have been used in the performance of liposuction. Grid pattern markings include series of longitudinal and transverse lines to delineate various anatomical boundaries and landmarks, including the midline, lateral line, and medial line. The markings are superimposed on the customary preoperative markings and divide broad or circumferential body surfaces into smaller subunits for liposuction. Grid pattern markings are applied to areas such as the anterior thighs, medial thighs, entire abdomen, flanks, back, arms, buttocks, calves, and ankles; they are not applied to smaller, less curved areas. Eighty-two consecutive patients underwent lipoplasty in 562 areas of the body. The revision rate for postliposuction contour irregularities was 4.0 percent (nine of 224 areas) where grid pattern markings were used; one area had an indentation type of contour irregularity and required autologous fat grafting. The revision rate was 1.5 percent (five of 328 areas) where grid pattern markings were not used; two areas in one patient had indentation-type contour irregularities and required autologous fat grafting. All remaining areas requiring revision had protuberant-type contour irregularities and responded to additional liposuction only. The use of grid pattern markings is associated with a low incidence of serious contour-related complications.     

Quantitative imaging of immunocytochemical (PAP) estrogen receptor staining patterns in breast cancer sections

"Receptogram Analysis" has been developed as a pattern-oriented approach for predicting endocrine response in breast cancer based upon quantification of the estrogen receptor immunocytochemical assay (ERICA), using a Quantimet Imaging System. Response prediction was evaluated in 58 stage III and IV patients receiving endocrine therapy (primarily Tamoxifen). The Receptogram is a composite of the univariate distributions of nuclear receptor content, IOD(S), and concentration (MOD), and their bivariate contour plot; where (S) is the calculated nuclear radius in section. MOD distributions were classified into four types based upon peak modality and kurtosis (I-IV), and contour plots were classified into four subtypes (A-D) based upon contour slope. Patients failing therapy were ERICA--or their receptogram revealed co-existent ER+ and ER- tumor cells (type II), highly skewed MOD distributions lacking defined peaks (type IV), or contours with nearly horizontal slope (type C). Response was realized in 9/16 type I patients, with a single positive MOD peak, and in 9/15 type III patients, with discrete, multimodal MOD peaks. In contrast, 0/8 type II, 0/12 type IV, and 0/10 type C patients were responders. Receptogram analysis was superior to cytosol assay (DCC) as a response discriminant: positive predictive value, 53% vs. 33%; negative predictive value, 100% vs. 75%; sensitivity, 100% vs. 83%; specificity, 68% vs. 23%; and accuracy, 78% vs. 41%, respectively. Alternately, patients were assigned to potentially responsive or non-responsive groups based upon thresholded mean receptor parameters: field MOD, mean nuclear MOD (NMOD), and mean NMOD(PF) where PF is the ER+ nuclear fraction. While these parameters correlated with DCC (r = .72, 0.69, and 0.69), they were only marginally better in predictive value.     

Small angle x-ray scattering of the hemoglobin from Biomphalaria glabrata

The hemoglobin from Biomphalaria glabrata is an extracellular respiratory protein of high molecular mass composed by subunits of 360 kDa, each one containing two 180 kDa chains linked by disulfide bridges. In this work, small angle x-ray scattering (SAXS) measurements were performed with the hemoglobin at pH 5.0 and 7.5. Radii of gyration of 98.6 +/- 0.5 and 101.8 +/- 0.2 A and maximum diameters of 300 +/- 10 and 305 +/- 10 A, respectively, were obtained from Guinier plot extrapolation and analytical curve fitting. The pair distance distribution functions p(r) corresponded to globular particles with a somewhat anisotropic shape for both preparations. Computer analysis of the low angle part of the scattering curve led to the determination of the low resolution envelope of the protein, revealing a P(222) symmetry. Shape reconstruction from ab initio calculations using the complete scattering curve furnished a compact prolate three-dimensional (3D) bead model for the protein. Hydrodynamic parameters were obtained from experiments and theoretical calculations using the 3D model. The results of the structural and biochemical studies reported herein indicate that the multisubunit structure of this hemoglobin is compatible with a tetrameric arrangement.