Pathobiology of salivary glands. IV. Histogenetic concepts and cycling cells in human parotid and submandibular glands cultured in floating collagen gels.

Localization of cells with proliferative capacity in human major salivary glands lacks extensive study. Minced fragments of human parotid (n = 3) and submandibular (n = 3) glands embedded in a floating collagen gel matrix and cultured for up to 28 days allowed maintenance of the three-dimensional relationship of the various cell types in these glands. Immunocytochemistry and electron microscopy of a time-dependent series of cultured gland fragments showed gradual cytologic modification of acinar cells so that acini became duct-like but also established that even after 28 days of culture certain cellular features allowed continued identification of acinar cells. Serial section immunostaining for amylase, cytokeratins, and proliferating cell nuclear antigen (a specific marker for cycling cells) revealed that acinar, intercalated duct, and excretory duct (both basal and luminal) cells are all capable of entering the cell cycle. At day 5 of culture, the number of cycling cells increased 16-fold in the parotid gland and 9-fold in the submandibular gland over that in the respective in situ gland. In this in vitro system, which perhaps simulates regenerative processes in human salivary glands, none of the samples showed cycling cells localized only to segments of intercalated duct or the basal cells of excretory duct as suggested by current histogenetic concepts.

Role of electron microscopy in head and neck pathology in adults and children.

Diagnostic electron microscopic studies specific to the head and neck region are limited in both children and adults. Even information about the workload created by specimen acquisition from this region in the electron microscopy laboratory is lacking. To address these deficiencies, various specimens gathered from adults (N = 6607) and children (N = 1556), over a 5- and 10-year period, respectively, and blocked in plastic resin were surveyed. Head and neck samples accounted for 16.4% of these samples from adults and 13.5% from children, representing sizable contributions to workload from an anatomic region amounting to less than 10% of body mass. In adults, diagnostic problems posed by metastatic neoplasms account for a considerable proportion of the cases submitted for electron microscopy; in the pediatric age group, undifferentiated primary tumors of the head and neck region frequently require ultrastructural study. In both circumstances, spindle cell, look-alike, neuroendocrine, and small cell tumors can frequently be rapidly assessed and differentiated by ultrastructural details. Problem cases are inevitable given the complexity of this region and the number of different tissues and organs in close proximity. It is advantageous to anticipate the possibility of encountering difficult differential diagnostic problems by sampling lesions of the head and neck for glutaraldehyde fixation more frequently.

An ultrastructural morphometric study of follicular center lymphocytes: III. The control of lymphocyte nuclear size in reactive hyperplasia and non-Hodgkin's lymphoma.

Since lymphocyte nuclear size and shape play such a key role in the classification of non-Hodgkin's lymphoma (NHL), the mechanisms and nuclear compartments responsible for the control of nuclear size are of fundamental importance to pathologists. To assess the role of condensed chromatin and the interchromatinic region of the nucleus in determining the size of this organelle, morphometric image analysis of total nuclear area and the area occupied by these two compartments was performed on electron micrographs of nuclear profiles from lymphocytes in lymph node biopsies of four cases of reactive lymphoid hyperplasia and ten examples of NHL. Using bivariate linear regression analysis, it was clear that, whether in normal mantle zone and follicular center lymphocytes or in the neoplastic lymphocytes of various NHLs, there is an extremely high correlation (R = 0.96) between the size of the interchromatinic region and the area of nuclear profiles. In contrast, there is a poorer correlation (R = 0.63) between the area occupied by condensed chromatin and nuclear area. The results demonstrate for the first time that even in NHL, where some nuclear features clearly vary from the normal counterpart, the control of nuclear size continues to be largely dependent on a particular subcompartment of the nucleus, the interchromatinic region. How this fact influences the categorization of subtypes of NHL for therapeutic and prognostic purposes will be an essential avenue for further investigation.

Ultrastructural morphometric study of follicular center lymphocytes: II. Analyses of cleaved-cell populations do not support the Lukes-Collins' concept.

Ultrastructural morphometric analysis was carried out on six cases of lymph node biopsies with reactive hyperplasia to establish the frequency and depth of invaginations in nuclear profiles situated in the mantle zones and follicular centers. The frequency distribution of the depth of invaginations was similar in nuclear profiles whether in the small lymphocytes of mantle zones or the small, partially transformed (centrocytes) and fully transformed (centroblasts) lymphocytes of follicular centers. Invaginated and cleaved lymphocytes were not confined to the partially transformed (centrocytic) lymphocytes of follicular centers, and nuclear profiles with invaginations bore no resemblance to those depicted in the Lukes-Collins model. A considerable proportion of mantle zone lymphocyte nuclear profiles had invaginations (ranging from 7.5% to 53.6%) and there was no difference between the frequency of deep indentations or clefts in mantle zone lymphocytes (8.1 +/- 5.4%) and the small unstimulated (9.3 +/- 5.3%) and partially transformed (8.4 +/- 1.4%) lymphocytes in follicular centers. Computer modeling of stylized nuclei with conical indentations indicated that all lymphocytic nuclei likely have multiple invaginations or groove-like creases.

Ultrastructural morphometric study of follicular center lymphocytes: I. Nuclear characteristics and the Lukes-Collins' concept.

A combined ultrastructural and morphometric image analysis study was carried out on the nuclear profiles of follicular center and mantle zone lymphocytes of six cases of reactive hyperplasia in human lymph node biopsies. For accuracy of morphological observations and sampling at low magnifications, sections were mounted on formvar-covered slot grids. Measurements of nuclear profile features of small (untransformed) lymphocytes in mantle zones served as the standard for a supposed unimodal population in each case. Analysis of nuclear profile area values indicated that during lymphocyte transformation in follicular centers nuclei had a gradual and progressive increase in size and that the sampled nuclear profiles in both the mantle zone and follicular center were unimodal. Lymphocyte nuclear shape (contour index) was a more complex, and likely biologically independent, feature than nuclear area in both the mantle zone and follicular center. Nuclear profile contour indexes of mantle zone lymphocytes were more irregular than suspected and in some cases had mean values greater than those of follicular center lymphocytes. Furthermore, the frequency distribution of nuclear contour index was not normally distributed in either the follicular center or mantle zone due to the presence of a small proportion of highly irregularly shaped nuclear profiles in both sites. The results indicated that some premises of existing concepts of follicular center cells and the process of lymphocyte transformation in follicular centers were incorrect and should not be directly extrapolated to the nuclear profile characteristics in non-Hodgkin's lymphoma.

Quantitative ultrastructural study of nuclei from exfoliated benign and malignant mesothelial cells and metastatic adenocarcinoma cells.

Various approaches, including morphometric image analysis, are currently being used to improve the distinction between diffuse mesothelioma and metastatic adenocarcinoma of the serous membranes. Since exfoliated cells of malignant mesotheliomas were thought to have nuclear profile contours with greater irregularity than the similar profiles in metastatic adenocarcinoma cells in pleural effusions, this and other nuclear parameters were measured in ultrastructurally examined preparations from three cases of reactive mesothelial hyperplasia, seven examples of diffuse mesothelioma and three cases of metastatic adenocarcinoma (with primaries in the ovary, esophagus and prostate). Contrary to the subjective impression, the nuclei in metastatic adenocarcinomas actually had a mean nuclear contour index greater than that found in diffuse mesotheliomas; statistically, the difference was not significant. Likewise, such other nuclear parameters as nuclear area, condensed chromatin area and contour index, percentage of condensed chromatin and number of condensed chromatin clumps per nuclear profile did not discriminate between malignant mesotheliomas and adenocarcinomas metastatic to pleural surfaces. These morphometric results quantitate the similarities in nuclear size, nuclear shape and condensed chromatin arrangement in these two types of tumor and explain why the cytopathologist has such great difficulty in distinguishing between exfoliated mesothelioma and adenocarcinoma cells in most cases.

Nuclear morphologic and morphometric analyses of nodular poorly differentiated lymphocytic lymphoma: assessment of small cleaved nuclei.

Comparative analytic measurements of nuclear parameters in normal and neoplastic lymphocytes are limited. In the present morphometric study lymphocyte nuclear features in 21 cases of nodular poorly differentiated lymphocytic lymphoma (NPDLL) were assessed with respect to the theoretical aspects of some non-Hodgkin's lymphoma (NHL) classifications. The mean nuclear area of the lymphocytes in NPDLL is generally within the range of the areas of unstimulated (mature) lymphocytes of mantle and follicular regions of lymph nodes with reactive hyperplasia. On this basis, the neoplastic lymphocytes in NPDLL do not reflect, at least cytologically, the antigen-activated, transforming lymphocytes of normal follicular centers. All measured nuclear parameters of small, unstimulated lymphocytes of neoplastic follicles suggest that major proportions of this component are also part of the neoplastic cohort. Sectional nuclear profiles in NPDLL are much more irregular in shape and have a higher percentage of invaginations than normal lymphocytes. However, only 4 to 5 per cent of nuclear profiles in NPDLL are of sufficient depth to be termed clefted. Serial section reconstruction of both normal and neoplastic lymphocytes indicates the degree to which the numbers of invaginated or clefted nuclei are underestimated in the examination of histologic sections. For example, the 4 to 5 per cent of nuclear profiles with clefts in histologic sections of NPDLL actually represent about 25 to 30 per cent of the lymphocyte population. On the basis of computer modeling of stylized nuclei with simple invaginations of varying depths and serial section reconstruction of normal and neoplastic nuclei, it is likely that all lymphocyte nuclei have some form of nuclear membrane invagination and that in poorly differentiated lymphomas these invaginations may be single and multiple discrete indentations or linear, branching grooves. Assessment of the ratio of nuclear invagination depth to nuclear diameter in normal and neoplastic lymphocytes indicates that transforming normal lymphocytes in follicular centers do not undergo a phase of increased nuclear clefting and that this ratio is somewhat greater in lymphocytes in NPDLL than in follicular center lymphocytes. However, the latter effect is not due to increased depth of nuclear invaginations in NPDLL, but rather results from the fact that mean nuclear diameter in this subtype of NHL is considerably smaller than that of normal lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-Hodgkin's lymphoma classification: ultrastructural morphometric studies for the quantification of nuclear compartments in situ.

The condensed chromatin distribution in the nuclei of lymphocytes in non-Hodgkin's lymphoma (NHL) is a key element, along with nuclear size and shape, in the classification of this disease for therapeutic and prognostic purposes. This report describes the ultrastructural comparative quantification of the condensed chromatin and the interchromatinic (nuclear matrix or euchromatin) region in the nuclei of mitogen-stimulated human peripheral T lymphocytes and mouse spleen B lymphocytes, human germinal center lymphocytes, and lymphocytes in ten cases of NHL of a variety of subtypes. The sequential morphologic nuclear changes induced in lymphocytes by mitogens are reflected in human germinal center lymphocyte populations. The common features include the changes in the distribution and volume of condensed chromatin aggregates, as well as the fact that the major increments in nuclear volume during lymphocyte transformation result from increases in the volume of the interchromatinic region. In all subtypes of NHL analyzed morphometrically, subpopulations of lymphocytes were identified in which mean nuclear, condensed chromatin, and interchromatinic volumes were more or less equivalent to those of normal lymphocyte subsets in germinal centers in reactive hyperplasia. However, in NHL the abnormal cytologic characteristics of the nucleus result, at least in part, from a complex interplay of condensed chromatin distribution and amount, and the size of the interchromatinic region. Further complexity is introduced by the fact that in NHL these two nuclear compartments can independently be normal, increased, or reduced in size. Morphometric quantification of lymphocytes in NHL indicates that the interchromatinic (matrix) region of the nucleus is the key element in establishing the nuclear volume of neoplastic lymphocytes. The structural and functional, ribonucleoprotein interchromatinic region of the nucleus was visualized in normal and neoplastic lymphocytes by regressive uranyl-EDTA staining. Quantitative morphometric analysis indicates that the cytologic appearance of neoplastic lymphocytes, even within subtypes of NHL, is heterogeneous and that condensed chromatin quantity and distribution may be more critical than nuclear size in distinguishing between certain subtypes of NHL. Improvements in the classification of NHL will occur only with understanding of the alterations in the biologic mechanisms controlling gross nuclear organization and the morphologic events of the various differentiation pathways available to antigen-stimulated lymphocytes.

Morphometry of normal human lymphoid tissues. Nuclear parameters for comparative studies of lymphoma.

Prior to performing morphometric studies of non-Hodgkin's lymphoma, baseline nuclear parameters of the various compartments of normal human lymphoid tissue are needed. Such information was obtained from traced nuclear profiles from a series of human lymph node biopsy specimens and samples of tonsil and spleen. All tissues showed reactive hyperplasia. In combined samples of adult lymph node specimens, nuclear parameters of mantle and paracortical lymphocytes were similar, but were smaller and more regular than small, unstimulated germinal center lymphocytes. Despite variation in mean nuclear size between various lymph node samples, transformed lymphocytes in germinal centers had a constant relationship to the nuclei of small untransformed lymphocytes in these regions. Our results indicate that there is considerable variation in the nuclear parameters of lymphocytes within a specific region of the normal lymph node of each case, but that such differences are difficult to appreciate in histologic preparations.