Structure of layer IV in the somatosensory neocortex of the rat: description and comparison with the mouse.
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Animals↗
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Three-dimensional counting: an accurate and direct method to estimate numbers of cells in sectioned material.
We introduce a way to count and measure cells in an optically defined volume of tissue called a counting box. This method--direct three-dimensional counting (3DC)--eliminates the need for correction factors, such as that introduced by Abercrombie (Anat. Rec. 94:239-247,'46), to determine the number of cells per unit volume (NV). Problems caused by irregular cell shape and cell size, nonrandom orientation, and splitting of cells by the knife during sectioning are overcome. Furthermore, 3DC is insensitive to large variations in section thickness. The innovative feature of 3DC is the definition of a counting box with top and bottom sides located inside the section a precise distance away from the cut surfaces of the tissue. The positions of the top and bottom sides of the counting box are delimited by using a digital length gauge and a Z-axis control unit. Sections of tissue between 8 and 100 micron thick are examined with a high numerical aperture objective in combination with video-enhanced differential interference contrast optics (DIC). Cells are marked on a television screen while the microscopist scans systematically from the top to the bottom of the counting box. Cells that are located completely inside the box and cells that only cross through its top, right, or back sides are counted. All cells that cross the planes that define the bottom, left, and front sides of the counting box are not counted. Direct 3DC provides an accurate, simple, and reliable way to count cells, nuclei, nucleoli, or other objects in sectioned material.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus.
This paper is the third in a series to quantify differences in the composition of subcellular organelles and three-dimensional structure of dendritic spines that could contribute to their specific biological properties. Proximal apical dendritic spines of the CA3 pyramidal cells receiving synaptic input from mossy fiber (MF) boutons in the adult rat hippocampus were evaluated in three sets of serial electron micrographs. These CA3 spines are unusual in that they have from 1 to 16 branches emerging from a single dendritic origin. The branched spines usually contain subcellular organelles that are rarely found in adult spines of other brain regions including ribosomes, multivesicular bodies (MVB), mitochondria, and microtubules. MVBs occur most often in the spine heads that also contain smooth endoplasmic reticulum, and ribosomes occur most often in spines that have spinules, which are small nonsynaptic protuberances emerging from the spine head. Most of the branched spines are surrounded by a single MF bouton, which establishes synapses with multiple spine heads. The postsynaptic densities (PSDs) occupy about 10-15% of the spine head membrane, a value that is consistent with spines from other brain regions, with spines of different geometries, and with immature spines. Individual MF boutons usually synapse with several different branched spines, all of which originate from the same parent dendrite. Larger branched spines and MF boutons are more likely to synapse with multiple MF boutons and spines, respectively, than smaller spines and boutons. Complete three-dimensional reconstructions of representative spines with 1, 6, or 12 heads were measured to obtain the volumes, total surface areas, and PSD surface areas. Overall, these dimensions were larger for the complete branched spines than for unbranched or branched spines in other brain regions. However, individual branches were of comparable size to the large mushroom spines in hippocampal area CA1 and in the visual cortex, though the CA3 branches were more irregular in shape. The diameters of each spine branch were measured along the cytoplasmic path from the PSD to the origin with the dendrite, and the lengths of branch segments over which the diameters remained approximately uniform were computed for subsequent use in biophysical models. No constrictions in the segments of the branched spines were thin enough to reduce charge transfer along their lengths.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Ultrastructural and immunocytochemical observations on the superior olivary complex of the mustached bat.
This study investigates the functional organization of the superior olivary complex of the mustached bat with classical transmission electron microscopy and postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) and glycine antisera in semithin serial sections. The ultrastructure and distribution of terminal types in the lateral superior olive (LSO) and the medial nucleus of the trapezoid body (MNTB) closely resemble that of other mammals; the organization within the medial superior olive (MSO) differs significantly. The differences concern the relative proportion of putatively inhibitory boutons, which appear as symmetrical synapses with flattened vesicles on MSO somata. In the bat, inhibitory boutons comprised 75-100% of perisomatic boutons, a value identical to that observed in the LSO. These terminals most likely arise from the MNTB. In other species, putatively inhibitory terminals form a much smaller proportion of perisomatic boutons in MSO. This difference suggests that in the bat MSO excitatory input to cell somata is considerably reduced and outweighed by inhibitory input. This suggestion is corroborated by immunocytochemical data. Glycine-immunoreactive puncta encrust somata of LSO and MSO cells to a similar degree and in rather homogeneous patterns throughout these nuclei. Putatively GABAergic terminals are located mainly on distal dendrites of MSO and LSO cells. Regional variations in the density of GABA-immunoreactive puncta in LSO suggest that different tonotopic zones are under differential modulatory influence. Both the LSO and MSO of the mustached bat contain significant amounts of putatively inhibitory projection cells. Coexistence of both antigens was commonly observed in subsets of cells. Quantitative analyses of labeling patterns and comparisons with other mammals suggest that the mix of neurotransmitters in projection cells of LSO and MSO is phylogenetically flexible, and thus the details of the functions of ascending pathways are species specific. In contrast to other mammals, the bat MSO forms parallel output pathways with excitatory and inhibitory components. Data are discussed in relation to specialized physiological response features.
Animals↗
Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin-embedded tissues.
DNA content determinations were carried out by flow cytometry on nuclear suspensions prepared from the same paraffin-embedded tissue block for each of eight surgically resected human carcinomas at section thicknesses of 5,10,20,30,40,50, and 100 millimicrons. Flow cytometric DNA determinations were also obtained on fresh tissue specimens in four of the eight carcinomas. As section thickness decreased below 50 millimicrons, there was a progressive increase in the histogram baseline noise at low DNA values and a decrease in the relative peak height of aneuploid DNA. The former was attributed to an increase of nuclear fragments in thinner sections, and the latter to the greater probability of transection of the larger aneuploid cells within a specimen. Both artifacts were minimized at section thickness of 50 millimicrons or greater.
Breast Neoplasms↗
Flow cytometry using paraffin-embedded tissue: five years on.
The use of paraffin-embedded tissue for flow cytometry is reviewed. A number of technical modifications of the original 1983 method have been described, aimed at improving the accuracy of DNA measurements by minimizing cell debris or reducing coefficients of variation, and at simplifying sample preparation. Over 100 clinical studies have now been reported, mainly assessing the effect of DNA index on prognosis, and those published up until mid-1988 are summarized in an appendix. More recently there have been developments in the use of monoclonal antibodies to measure oncogene products or proliferation markers in addition to DNA content. Detailed clinical evaluation and standardization of these more sophisticated methods is still some way ahead, but as was the case with DNA index, the use of archival material from patients whose outcome is already known should speed this process.
Animals↗
Towards a quantitative grading of bladder tumors.
The histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated. Three hundred thirty-three images were digitized from 111 bladder tissue sections (4 microns thick, Feulgen stained), using the ICAS microscope-camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity. The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as "in between grade 1 and grade 3" and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population. The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields-of-view are chosen for analysis.
Epithelium↗
Quantitative analysis of chromosome in situ hybridization signal in paraffin-embedded tissue sections.
Interphase cytogenetic analysis using chromosome-specific probes is increasingly being used to detect chromosomal aberrations on paraffin-embedded tissue sections. However, quantitative analysis of the hybridization signal is confounded by the nuclear slicing that occurs during sectioning. To determine the sensitivity and accuracy of chromosome in situ hybridization for detecting numerical chromosomal aberrations on paraffin-embedded sections, in situ hybridization was performed on sections derived from mixtures of cell populations with known frequencies of numerical chromosomal aberrations and the Chromosome Index (CI) was calculated (i.e., total number of signal spots/number of nuclei counted) as a quantitative measure of chromosome copy number. The presence of 25% or more monosomic or tetrasomic cells in a given population was easily detected as a change in CI (P < 0.05). Lower degrees of polysomy could be detected as a small percentage of nuclear fragments with > 2 signal spots. The CI was not significantly influenced by a change in section thickness from 4 to 8 microM, by an increase in cell size from 478 to 986 microM3, or by the choice of detection method (fluorescence vs. conventional bright-field microscopy). Comparative analysis of touch preparations and tissue sections from the corresponding breast tumors showed that CI accurately reflects the average copy number of chromosomes in intact nuclei and may actually be superior to in situ hybridization on whole nuclei for the detection of numerical chromosomal changes in defined histologic areas. This method is thus a sensitive and accurate means of studying genetic changes in premalignant and malignant tissue, and of assessing the genetic changes associated with specific phenotypes.
Aneuploidy↗
Flow cytometric immunphenotyping of epithelial cancer cells in effusions--technical considerations and pitfalls.
BACKGROUND: Data regarding the role of flow cytometry (FCM) in the characterization of malignant effusions are limited to date. In the present study, we optimized the conditions for FCM immunphenotyping of effusions using a four-color analysis and investigated aspects related to the advantages and limitations of this method in this setting. METHODS: FCM analysis optimization for the study of epithelial cells was undertaken using five carcinoma cell lines, and subsequently applied to malignant pleural and peritoneal effusions using antibodies against epithelial and mesothelial markers (Ber-EP4 and EMA), CD138, and integrin subunits. FCM of frozen versus fresh specimens and the performance of FCM compared to immunhistochemistry were evaluated. RESULTS: FCM optimization was achieved and applied to clinical specimens, with resulting detection of epithelial markers and adhesion molecules on cancer cells. Frozen clinical specimens and cell lines showed reduced CD138 expression compared to fresh specimens, with conservation of the remaining epitopes. FCM generally showed comparable performance to immunhistochemistry. CONCLUSIONS: FCM is an effective method for characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and is comparable to immunhistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. The majority of epitopes are conserved in frozen cells, but a minority may be lost, suggesting that the thorough testing of each antibody in both conditions is mandatory.
Antibodies↗
Utility of additional slides from residual Preservcyt material in difficult ThinPrep gynecologic specimens: a prospective study of 58 cases.
ThinPrep purportedly increases the sensitivity of cervicovaginal cytology for detecting abnormal squamous and glandular cells. The value of additional slides from residual Preservcyt material to characterize difficult lesions is unknown. Fifty-eight cases were studied to determine the utility of additional slides for diagnosis and to assess cellular uniformity. In 32 (55%), repeat slides helped make a definitive diagnosis, including 18 atypical squamous cells of uncertain significance (ASCUS) reclassified as low-grade squamous intraepithelial lesion (LGSIL) (13), high-grade squamous intraepithelial lesion (HGSIL) (4), or endometrial adenocarcinoma (1); 5 LGSIL reclassified as HGSIL; 3 atypical glandular cells of uncertain significance (AGUS) reclassified as LGSIL (1) or HGSIL (2); 2 LGSIL?HGSIL classified as LGSIL; and 4 cases confirmed as LGSIL (2) or HGSIL (2). Results were compared to follow-up clinical information, including subsequent cervicovaginal samples and biopsies. The number of abnormal cells was similar between slides in most cases. We conclude that, while ThinPreps prepared from the same vial have similar numbers of abnormal cells, additional slides can be helpful for diagnosis in select cases.
Female↗
Reflex high-risk human papilloma virus DNA test is useful in the triage of women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion.
This study is aimed to investigate the role of reflex high-risk human papilloma virus (HPV) DNA testing as an alternative triage method to colposcopy for women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) on Papanicolaou (Pap) tests. Reflex HPV DNA testing using Hybrid Capture II method was carried out on 88 women with ASC-H diagnosed by Thin Prep Pap test. Correlation with follow-up biopsies was available on 42 of these patients. The reflex HPV DNA test showed an overall positive rate of 67% and negative rate of 33% in 88 patients with ASC-H. Using age 30 as the cut off point, the positive rate had increased to 83.3% (35/42) in patients 30 yr or younger, while the positive rate for patients older than 30 yr had decreased to 52.2% (24/46). Follow-up colposcopic biopsy results were available in 35 of 59 HPV-positive women, which revealed 15 (43%) high-grade squamous intraepithelial lesions (HSIL), 12 low-grade squamous intraepithelial lesions (LSIL), and 8 negative for dysplasia. In 7 HPV-negative patients, the follow-up biopsies showed no evidence of HSIL or LSIL. Correlation between clinical risk factors and the HPV results demonstrated no significant differences in HPV positivity between the high-risk and low-risk patients. The high sensitivity (100%) and negative predictive rate (100%) in detecting HSIL in our study provide strong evidence that, instead of automatic referral to colposcopy, reflex HPV DNA testing may be used as an alternative triage method for women diagnosed with ASC-H on Thin Prep Pap test, especially for women older than 30 yr of age.
Adolescent↗
Tissue imprints in surgical pathology: a rapid intraoperative diagnostic aid.
We studied the intraoperative diagnostic value of imprint cytology in 230 samples obtained from surgical specimens submitted for frozen section diagnosis. A rapid hematoxylin-eosin stain was used. Intraoperative imprint cytology achieved an accuracy rate of 94.3%; for benign lesions the accuracy was 97.5%, and for malignant lesions it was 91%. Overall, the false-negative and suspicious-for-malignancy rates were 1.3% and 4.3%, respectively. No false-positive results were found. The diagnostic yield when intraoperative imprint cytology and frozen section were used together was 99%. It is apparent that imprint cytology is a quick and simple method with wide applicability in the histopathologic diagnosis of lesions from all organs. The value of the method is enhanced when it is used with frozen section diagnosis.
Breast Neoplasms↗
Confocal optical sectioning and three-dimensional reconstruction of carcinoma fragments in Pap smears using sophisticated image data processing.
Carcinoma fragments found in Pap smears contain important diagnostic information not available to the light microscopist because of their thickness and consequent blurring. Optical sectioning by the confocal microscope allows us to reclaim the mitotic figures, glandular architecture, and abnormal chromatin patterns in the restained original smears. The high spatial resolution of the confocal microscope can be further exploited by processing the digital images with the sophisticated Application Visualization System (AVS) on a CONVEX computer. Serial sections in which the fluorescent signals are color coded by this software package and three-dimensional reconstructions of the nuclei and mitotic figures expand our knowledge of these malignant epithelial fragments.
Adenocarcinoma↗
The accuracy and usefulness of frozen-section diagnosis.
A retrospective review of 57 patients, who had received frozen-section diagnoses, was performed both to ascertain the accuracy of diagnosis and to evaluate its usefulness in assessing tumor surgery. Except for 2 cases, each with a parotid gland tumor, the frozen-section diagnoses were consistent with the final diagnoses using permanent paraffin sections. Fifteen cases with either T1 or T2 squamous cell carcinoma of the oral cavity and 2 cases with malignant salivary gland tumor, showing tumor-free surgical margins by frozen-section examination, were followed. A local recurrence of the carcinoma was observed in 1 case during the follow-up period. Prior to neck dissection, lymph node metastases were examined using frozen sections. Ten cases, which were diagnosed as negative for metastasis, showed no subsequent development of cervical node metastasis.
Adult↗
Accuracy of frozen-section diagnosis in salivary gland lesions.
A retrospective study is presented comparing the results of 462 frozen-section analyses of surgically extirpated salivary gland tumors with the permanent-section results. The overall agreement between frozen- and permanent-section analyses was 95.7%. A separate review of the last 47 months of this 32-year study revealed an accuracy rate of 98.8%. Frozen-section diagnosis of salivary gland tumors has been reliable and clinically valuable in our practice.
Biopsy↗
Diverticular bile duct lesion in chronic active hepatitis.
Liver needle biopsies from patients with non-A, non-B chronic active hepatitis and so-called abnormal bile duct epithelium were studied with a three-dimensional method. Photographs of bile duct structures in serial sections were transferred to acrylic plates. Five bile duct lesions of a not previously described diverticular type were revealed. The diverticuli were of varying shape with a diameter of 30 to 110 microns and a length of 75 to 150 microns budding from small (12 to 25 microns), slightly ectatic bile ducts. The diverticular epithelium was disordered. Some cells appeared as bile duct cells, but most were larger, with rounded nuclei, prominent nucleoli and abundant eosinophilic cytoplasm, sometimes with periodic acid-Schiff-positive, diastase-resistant granules. The lesions were only partly surrounded by a basement membrane. They were all embedded in a tight mononuclear inflammatory infiltrate associated with pronounced periportal piecemeal necrosis. In two cases, a germinal center was adjacent to the epithelium. The pathogenesis of the diverticular bile duct lesion is unknown, but the diverticuli probably represent Hering ducts and groups of periportal liver cells which have escaped the piecemeal necrosis.
Adult↗
The zonal distribution of megamitochondria with crystalline inclusions in nonalcoholic steatohepatitis.
Megamitochondria with crystalline inclusions (MMC) have been previously described in nonalcoholic fatty liver; however, their distribution within hepatic zones is unknown. We sought to determine this distribution from the core liver biopsy specimens of 31 patients: 8 males and 23 females, age range 21 to 72 years. Twenty-nine showed evidence of nonalcoholic steatohepatitis (NASH) on biopsy with steatosis, inflammation, varying degree of fibrosis, ballooned hepatocytes, and Mallory hyaline, and two patients had cryptogenic cirrhosis thought to represent "burned out" NASH. Identified by transmission electron microscopy, the abundance of MMC was compared between low-stage (fibrosis stages 1 and 2) and high-stage (fibrosis stages 3 and 4) groups and between zones with or without difference in fibrosis stage. Regardless of stage, the MMC were distributed equally in all zones and were abundant similarly in low- and high-stage groups. This abundance did not correlate with the degree of oxidative stress (4-hydroxynonenal staining) or with the abundance of ballooned hepatocytes. Consistent with age as a risk factor for more severe disease, the median age for the low-stage group was significantly lower than that of the high-stage group (P =.003). In conclusion, in NASH, the MMC seem to be distributed randomly among zones and without variation in abundance, regardless of the fibrosis stage. The exact function of these structures remains to be defined. In this study, their presence did not seem to correlate with the light microscopic injury pattern represented by ballooned hepatocytes or degree of oxidative stress defined by immunostaining for 4-hydroxynonenal.
Adult↗
Direct comparison of liquid-based and conventional cytology in a South African screening trial.
Our study directly compares the performance of liquid-based (LBC) and conventional cytology for detecting high-grade cervical intraepithelial neoplasia and cancer (CIN 2+) in high-risk, previously unscreened women. As part of a larger randomized clinical trial assessing the efficacy and safety of a "screen and treat" program for cervical cancer prevention, 5,652 South African women, aged 35 to 65 years, were screened using either ThinPrep or conventional Papanicolaou cytology. The cytology method used (i.e., ThinPrep or conventional) was rotated on a 6-month basis for the duration of the study. Directly following collection of the cytology specimen, all women underwent colposcopy with endocervical curettage and biopsy of all colposcopic abnormalities. Assessment of cytology and histology results was blinded and results were compared using histology-confirmed CIN as the "gold standard." The accuracy of LBC and conventional cytology was statistically equivalent, although the sensitivity of conventional cytology was at least 5 percentage points higher at all cutoff levels. For example, at a cytology cutoff level of low-grade squamous intraepithelial lesion, the sensitivity of LBC was 60.3% for CIN2+ vs. 69.1% for conventional cytology and specificity was 94.1% and 94.5%, respectively. LBC specimens were significantly less likely to be "satisfactory-but-limited-by" (6.5% vs. 27.9%) but significantly more likely to be unsatisfactory (2.2% vs. 0.8%). Thus, in this high-risk population, the sensitivity of LBC is no greater than the sensitivity of conventional cytology. Because of the higher unit cost of LBC, low resource settings should carefully consider the potential benefits and drawbacks of LBC before adopting this new technology.
Adult↗