Morphometric ultrastructural analysis of nuclear components in T-cell acute lymphoblastic leukaemias

The paper deals with the morphometric ultrastructural analysis of the nuclear components performed in five cases of T-cell acute lymphoblastic leukaemia. All these cases constituted a homogeneous group in clinical and immunological evaluation. The cases analysed did not differ in cell nucleus and euchromatin volume as well as in their number of nucleoli per one cell profile.     

Evaluation of human bladder tumors by ultrastructural morphometric techniques

To estimate the malignancy of the human urinary bladder tumors, various cellular components of normal and neoplastic bladder epithelial cells were examined using morphometric techniques at the electron microscopic level. Nuclear to cytoplasmic ratios were found to be 1:5 in normal epithelial cells, 1:4 in superficial grade 1 (G1) tumor cells, 1:3 in superficial grade 2 (G2) tumor cells and 1:2 in invasive grade 3 (G3) tumor cells. Nuclear volumes were largest in G3 tumor cells, but no differences were seen in cell volumes between normal epithelial and tumor cells. The volumes and surface areas of the rough-surfaced endoplasmic reticulum (rER) and lysosomes per cell decreased progressively from G1 to G3 tumor cells. The volume density of tonofilaments was increased in G1, but decreased in G2 and G3 tumor cells. The cisternal thickness of the rER and Golgi complex was also increased in G1 tumor cells. From these results, it was concluded that morphometric analysis may be useful in evaluating the degree of differentiation and invasive potential of human bladder tumor cells in the low and intermediate risk groups.     

Cytomorphometric analysis of small cell neoplasms of the lung from specimens obtained via bronchoscopy.

Primary neoplasms of the lung composed of small cells consist of undifferentiated small cell carcinoma (UCS), carcinoid tumors (CT) and some non-small cell carcinomas (NonSC) that lack cytoplasmic differentiation. The cytologic identification of tumors from specimens obtained via bronchoscopy sometimes presents difficulties due to overlapping patterns. In order to define additional differentiating criteria, we evaluated Papanicolaou-stained smears from 30 histologically proven cases, 10 from each of the three groups, with statistical analysis of computer-assisted morphologic measures. We identified significant differences between NonSC versus USC and NonSC versus CT with regard to nuclear and cytoplasmic dimensions (P less than .0001) but not between USC and CT. The nuclear:cytoplasmic ratios were similar in all three groups (P = .39). From this preliminary study we conclude that morphometric analysis can distinguish NonSC from USC and CT but not USC from CT.     

Comparative ultrastructural morphometrical studies in acute leukemia.

Four hundred paramyeloblasts (from meyloblastic, promyelocytic, monoblastic and lymphoblastic types), isolated from peripheral blood of untreated leukemia patients, were studied by planimetric ultrastructural morphometry. The data of 19 parameters for these four paramyeloblastic types were compared with statistical methods. More central "scattered" heterochromatin was found from this morphometric investigation, i.e. early prophases in the lymphoblastic type of acute leukemia (these cases are more sensitive to therapy). The absolute mean values of the areas of whole cells, areas of the nucleus and nucleolus, areas of the heterochromatin and other indices show that different cell clones will undergo leukaemic transformation.     

Morphometric study of hepatic ultrastructure in alcoholic hepatitis. Veterans Administration Cooperative Study Group on Alcoholic Hepatitis

We undertook a morphometric analysis of hepatocellular organelles in an attempt to correlate their changes with the clinical stages of patients with alcoholic hepatitis. Although hepatic ultrastructural alterations did not correlate with disease severity, we found significant differences between patient and control groups in the measured parameters of non-organelle cytoplasm, mitochondria, SER, RER, glycogen, and lipid.     

Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

Bimodal variations in subcellular structures of rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

To clarify 24-hr variations in rat thyroid follicular cells under physiological conditions, their subcellular structures were examined at six evenly spaced times during 24 hr by using a morphometric technique. The volume, surface, and numerical densities of subcellular structures varied distinctly over each 24-hr period, with a bimodal pattern. The cellular and nuclear volumes varied also bimodally over 24 hr. A decrease in the surface density of the apical plasmalemma at 1200 and 0000 hr coincided with an increase in volume density of cytoplasmic granules representing colloid droplets and dense bodies. Most granules (colloid droplets) appearing at these times were reduced in electron density. At other times, especially at 1600 and 0400 hr, morphometric parameters of rough endoplasmic reticulum (rER), Golgi complex, and subapical vesicles were prominently increased, although values for rER did not peak at 1600 hr. At these times, the volume densities of cytoplasmic granules, most of which were heterogeneous and of homogeneous electron density, were decreased. These findings coincided with immediate and subsequent reactions of follicular cells after injection of thyroid-stimulating hormone (TSH). From the evidence, it seems likely that variations in follicular cells over a 24-hr period reflect variations in blood TSH concentration. The total membrane areas of membrane components in follicular cells were calculated from the morphometric measurements. These areas fluctuated unimodally during 24 hr over a 65% range. This suggests that the membranes in follicular cells are subjected to cyclic degradation and regeneration during each 24-hr period.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-IgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron- dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-IgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation. These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.     

Emperipolesis--a peculiar feature of megakaryocytes as evaluated in chronic myeloproliferative diseases by morphometry and ultrastructure

A combined morphometric and ultrastructural study was performed on so called emperipolesis or internal wandering of myeloid cells in the cytoplasm of large mature megakaryocytes. Measurements were made on material from a total of 115 patients comprising a normal control group and 5 groups with subtypes of chronic myeloproliferative diseases, including primary (essential, idiopathic) thrombocythemia (PTH). A significant increase in this peculiar phenomenon was noted in myeloproliferative disorders and especially in PTH where the frequency of emperipolesis showed a positive correlation with the number of anuclear cytoplasmic fragments sectioned, with the circular deviation of the shapes of megakaryocytes and with the extent to which the peripheral thrombocyte count was elevated. Electron microscopy in selected cases displayed the integrity of the plasma membranes of engulfed hematopoietic cells within the dilated cavities of the megakaryocytic demarcation membrane system (DMS) and no evidence of phagocytosis. Moreover there was a close relationship between engulfed myeloid cells and the presumptive sites of platelet shedding which had their openings from the cisternal lumina of the DMS. Our results demonstrate that emperipolesis of hematopoietic cells within megakaryocytes should not be regarded as a special nosological feature, but as an indication of enforced thrombocytogenetic activity which is expressed particularly in PTH.     

The pineal gland of the gerbil,Meriones unguiculatus

Summary By means of morphometric analytical procedures, a diurnal rhythm in the cellular volume of gerbil pinealocytes was determined. This rhythm has been attributed primarily to a change in the cytoplasmic volume of the pinealocytes which is low during the daylight hours and increases to reach a peak during the middle of the dark period. At the ultrastructural level, six cytoplasmic components of the pinealocytes were found to exhibit a rhythm: free cytoplasm, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER) and ribosomes, secretory vesicles, microtubules, and mitochondria. The presumptive secretory vesicles and the microtubules reached a peak in volume one hour before lights-off. It is suggested that lights-on and lights-off both signal a decrease in size and/or number of the secretory vesicles. The SER and RER/ribosomes reached their peak volume one hour after lights-off which is interpreted as indicating a peak in indoleamine synthesis and protein synthesis, respectively. The volume of free cytoplasm exhibits two peaks; one occurs one hour before lights-off while the second peak occurs in the middle of the dark phase. It is suggested that, although part of the secretory product of the pinealocyte may be present in dense-cored vesicles, other locations could include the free cytoplasm and clear secretory vesicles.Supported by NSF grant #PCM 77-05734     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes. A morphometric study of so-called "dark cells" and their putative role in epidermal carcinogenesis

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered "clear cells" dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

γ-Aminobutyric Acid Agonist-Induced Alterations in the Ultrastructure of Cultured Cerebellar Granule Cells Is Restricted to Early Development

The effect of 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) on the ultrastructural composition of cultured cerebellar granule cells was investigated during development by quantitative electron microscopy (morphometric analysis). Granule cells were exposed to THIP (150 microM) for 6 h after 7 and 14 days, respectively, in culture. THIP treatment of 7-day-old cultures led to a statistically significant increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus, vesicles, and coated vesicles, whereas no significant increase in the cytoplasmic density of these organelles was observed in 14-day-old cultures exposed to THIP for 6 h. These findings show that the effect of THIP on the ultrastructural composition of cultured cerebellar granule cells is restricted to early development.     

Quantitative electron microscopy of endocrine cells in oxyntic mucosa of normal human stomach

Summary An ultrastructural morphometric study of the endocrine cells of the oxyntic mucosa of the stomach in gastric biopsies collected from five male and five female healthy volunteers aged 19–31 was performed. No sex-related differences were disclosed. Endocrine cells accounted for 1.2±0.4% of the epithelial volume and 0.9±0.4% of the mucosal volume, i.e., including the lamina propria. After classification of the specific endocrine cell types according to the ultrastructural morphology of secretory granules, the volume densities of ECL, P and D cells (30±9%, 24±7%, and 22±4% of the entire endocrine cell mass, respectively) were higher than those of other endocrine cell types. In particular, EC cells contributed less than 10% and X cells represented a very low proportion of the total cells. Non-granulated profiles of cells which in all other respects appeared to be endocrine were also found with a volume density of 8±4%. D cells were distinguished by the high fraction of cytoplasm occupied by secretory granules (31±5%). Subdivision of the whole mucosa into four horizontal segments revealed the endocrine cells to be mostly distributed in the three lower, with virtually no endocrine cells in the superficial segment. The quantitative ultrastructural analysis of the endocrine cell population of the normal human oxyntic mucosa provided by this study may allow a better evaluation of physiological and pharmacological variations of the endocrine cell population.     

Immunohistochemical, electron microscopic and morphometric studies of estrogen-induced rat prolactinomas after bromocriptine treatment

To clarify the effects of bromocriptine on prolactinoma cells in vivo, immunohistochemical, ultrastructural and morphometrical analyses were applied to estrogen-induced rat prolactinoma cells 1 h and 6 h after injection of bromocriptine (3 mg/kg of body weight). One h after treatment, serum prolactin levels decreased markedly. Electron microscopy disclosed many secretory granules, slightly distorted rough endoplasmic reticulum, and partially dilated Golgi cisternae in the prolactinoma cells. Morphometric analysis revealed that the volume density of secretory granules increased, while the volume density of cytoplasmic microtubules decreased. These findings suggest that lowered serum prolactin levels in the early phase of bromocriptine treatment may result from an impaired secretion of prolactin due to decreasing numbers of cytoplasmic microtubules. At 6 h after injection, serum prolactin levels were still considerably lower than in controls. The prolactinoma cells at this time were well granulated, with vesiculated rough endoplasmic reticulum and markedly dilated Golgi cisternae. Electron microscopical immunohistochemistry revealed positive reaction products noted on the secretory granules, Golgi cisternae, and endoplasmic reticulum of the untreated rat prolactinoma cells. However, only secretory granules showed the positive reaction products for prolactin 6 h after bromocriptine treatment of the adenoma cells. An increase in the volume density of secretory granules and a decrease in the volume densities of rough endoplasmic reticulum and microtubules was determined by morphometric analysis, suggesting that bromocriptine inhibits protein synthesis as well as bringing about a disturbance of the prolactin secretion.     

A comparative morphometric study of synaptic vesicles and other organelles in mossy fiber endings of the pigeon and the rat

The mossy fiber ending organelles of 4 rats and 4 pigeons were studied using ultrastructural morphometric methods. The number of agranular vesicles per square micrometer of synaptic surface, as well as the number of agranular vesicles per synapse, were found to be greater in the rat than in the pigeon, while no significant differences were found in coated and dense core vesicles. A highly positive correlation was found, in both species, between the synaptic surface and the number of agranular vesicles per unit volume of mossy fiber endings, while no correlation was found between the synaptic surface and numerical densities of coated and dense core vesicles.     

Acute lymphoblastic leukemia: a study of 25 cases by scanning electron microscopy.

Cells from 25 cases of acute lymphoblastic leukemia (ALL) were studied under the scanning electron microscope (SEM). In 24 of the cases, the vast majority of circulating leukaemic cells had few microvilli. Villous cells were rarely encountered and prominent ridge-like profiles and ruffled membranes were not seen. Only six cases were studied by immunological techniques and four of the cases were of the null type while in two the cells bore detectable T-markers. It seems that ALL is almost always associated with the presence of cells with few microvilli in the peripheral circulation, differing in this respect from most cases of CLL. Although circulating leukaemic lymphocytes with few microvilli are sometimes seen in CLL, the most frequent cell type encountered is a more villous lymphocyte. Differences between leukaemic cells from patients with ALL, CLL and non-lymphoblastic leukaemias are discussed. It appears that SEM may help to distinguish lymphoblastic and nonlymphoblastic leukaemic cells in many instances and can be used as a useful adjunct to other modes of microscopy in the diagnosis of acute leukaemia.     

Morphometric differences between cytologically benign and malignant serous effusions

The morphometric differences between benign and malignant serous effusions, as diagnosed by standard cytologic criteria in 95 unselected cases (50 benign and 45 malignant), were studied using the IBAS semi-automated image analysis system, which calculates various parameters from tracings of cellular and nuclear outlines. Fourteen cases were also stained for cytokeratin proteins (with the CAM 5.2 antibody) by the immunoperoxidase technique and reanalyzed for positive cells. Significant differences were found for mean values between cytologically benign and malignant cases for cellular and nuclear areas, perimeters and maximum diameters, but not for two form factors. Some differences were enhanced in the CAM 5.2-stained cases. Real morphometric differences in samples of cells from benign and malignant cases are the basis of cytologic diagnosis. Fully automated diagnostic systems could operate on arbitrary threshold values, but there is considerable overlap in specimen means for all parameters between benign and malignant cases.     

Morphometric ultrastructural evaluation of human peripheral blood T lymphocyte subpopulations isolated on the basis of their affinity for sheep red blood cells

The present investigation were undertaken to compare on the basis of ultrastructural morphometric analysis human T lymphocyte subfractions forming rosettes with sheep red blood cells (SRBC). The following T lymphocyte subfractions were obtained: ARFC (active rosettes), T1RFC (rosettes after 1 h at 4 degrees C) and T2RFC (rosettes after 2 h at 4 degrees C). Analysis of the mean cell and nuclei volumes of lymphocyte subfractions led to the separation of two cell groups differing in volume in each donor: a cell group consisting of large cells (T1RFC) and group of small cells (ARFC and T2RFC).