Microscopic notes on the perinucleolar chromatin region in immature and mature human B-leukemia lymphocytes.

Perinucleolar region was studied in lymphocytes of patients suffering from chronic B lymphocytic leukemia to provide more information on the perinucleolar-condensed chromatin - heterochromatin - during the maturation of these cells. The perinucleolar heterochromatin of lymphocytes in smear preparations was visualized using a simple, but sensitive cytochemical method for the demonstration of DNA. The perinucleolar heterochromatin was also easily visible as unstained perinucleolar regions in specimens stained for RNA. In addition, the perinucleolar heterochromatin of lymphocytes was distinct and apparent in the transmission electron microscope using conventional as well as cytochemical methods for visualization of chromatin structures. Despite the great variability, the maturation of leukemic lymphocytes was accompanied by an increased width of the perinucleolar heterochromatin shell. It seems to be also interesting that the perinucleolar region of both immature as well as mature leukemia lymphocytes contains heterochromatin bodies about 2µm in diameter. They appeared to be a regular component of the perinucleolar heterochromatin shell and were apparently different from other nuclear bodies present at the nucleolus. In contrary to other known nuclear bodies, perinucleolar heterochromatin bodies in leukemia lymphocytes consisted only of conglomerates of DNA containing chromatin fibrils and did not contain other structural components including RNA. The presence of perinucleolar heterochromatin bodies in the perinucleolar region of leukemia lymphocytes is not contradictory with the present knowledge on that nuclear territory. They might be associated with presumed special perinucleolar DNA loci, which according some previous studies were more expressed in malignant cells.

A short cytochemical note on the nucleolar and cytoplasmic RNA concentration in differentiating cells represented by human erythroblasts.

The present study was undertaken to provide more information on the nucleolar and cytoplasmic RNA concentration in differentiating cells of the erythroid lineage. These cells represent a convenient model to study cell differentiation since all stages are morphologically well characterised. The bone marrow of patients suffering from the chronic phase of chronic myeloid leukaemia without a large increase in the granulocyte to erythroid ratio provided erythroblasts for computer-assisted image density measurements of RNA in nucleoli and cytoplasm at the single cell level. The measurements indicated a significant decrease of the nucleolar and cytoplasmic RNA concentration only in advanced stages of erythroblast differentiation (polychromatic and orthochromatic erythroblasts). The ratio of the nucleolar to cytoplasmic RNA concentration was otherwise very stable and did not change during differentiation, being similar in the early and advanced stages of erythroblastic development. In contrast, the nucleolar size significantly decreased even during the early stages of erythroid development (basophilic erythroblasts). This marked decrease in the nucleolar diameter in differentiating erythroblasts and the less marked decrease in the nucleolar RNA concentration suggest that the amount of RNA in the nucleolus is closely associated with nucleolar size rather than on its concentration within the nucleolar body.

The RNA content of nucleolar bodies is related to their size - a cytochemical study on human monocytes and lymphocytes in blood smears and blood cytospins.

The present study was undertaken to provide more information on the relationship between the nucleolar size and RNA density. Mature monocytes circulating in human peripheral blood appeared to be very convenient for such study because they contain multiple nucleoli of various sizes in one and the same nucleus. In addition, nucleoli without perinucleolar chromatin represented by nucleolar bodies are easy to be visualized by a simple cytochemical procedure for RNA demonstration. The diameter and density of NoBs in specimens stained for RNA were determined by computer-assisted measurements of individual cells. According to the results, the nucleolar RNA content was apparently related to the nucleolar size because the RNA density of small and large NoBs was practically the same. In addition, the diameter measurements also indicated that one or two of several NoBs in one nucleus were dominant - larger - than the others. It should also be mentioned that the diameter and RNA density of NoBs were studied in monocytes in peripheral blood smears regardless of their limited number. Additional study on lymphocytes indicated that the preparation procedure of smears modified both diameter and density of the measured parameters less than the preparation of cytospins as demonstrated by lower variability of the resulting values. Thus, the observed differences between smears and cytospins also indicated that the specimen preparation procedures should always be considered during evaluation of the nucleolar size or staining intensity of nucleoli or cytoplasm due to the presence of RNA.

To the density and distribution of heterochromatin in differentiating, maturing and apoptotic cells represented by granulocytic, lymphocytic and erythrocytic precursors.

The present study was undertaken to provide more information on the density and distribution of heterochromatin in early and advanced stages of the granulocytic, lymphocytic and erythroid development. Heterochromatin was visualized using a simple cytochemical method for the demonstration of DNA followed by computer-assisted densitometry of the digitized images. The largest heterochromatin density in early proliferating stages of all studied blood cell lineages was noted in the perinucleolar region and centrally located chromocentres. In contrast, the heterochromatin density at the nuclear membrane was significantly lower. In advanced nonproliferating stages or apoptotic cells the heterochromatin density increased and was similar in all nuclear regions, i.e. in the perinucleolar regions, chromocentres, and at the nuclear membrane. Thus, such observations indicated that the heterochromatin condensation in the perinucleolar region and chromocentres, i.e. in "gene-rich nuclear regions", of differentiating and maturing progenitors of blood cells preceded that at the nuclear periphery.

A karyometric note on nucleoli in human early granulocytic precursors.

The diameter of nucleoli was measured in human bone marrow early granulocytic precursors after visualization by a simple cytochemical method for demonstration of RNA. Such method facilitated to clearly see nucleolar bodies without perinucleolar chromatin, including those of micronucleoli. The bone marrow of patients suffering from chronic myeloid leukaemia (untreated with cytostatics) provided a satisfactory number of both myeloblasts and promyelocytes for nucleolar measurements because of prevailing granulopoiesis. The direct nucleolar measurement was carried out on digitized and processed images on the screen at magnification 4,300x. It seems to be likely that the nucleolar size is directly related to the number of nucleoli per cell. The largest nucleoli were present in both myeloblasts and promyelocytes that possessed a single nucleolus. In contrast, the nucleolar diameter was significantly smaller in cells with multiple nucleoli. However, in cells with small multiple nucleoli, one of them was always larger and dominant with a large number of AgNORs. Such large nucleoli are possibly visible in specimens stained with panoptic procedures or methods staining nuclear chromatin or DNA. It should also be mentioned that both myeloblasts and promyelocytes mostly possessed two nucleoli with the mean diameter close to 1.5 microm. The incidence of early granulocytic precursors classified according to the nucleolar number and size strongly suggested that the various nucleolar number and nucleolar size in these cells might be related to the different stage of the cell cycle and might also explain their heterogeneity.

Intranucleolar translocation of AgNORs in early granulocytic precursors in chronic myeloid leukaemia and K 562 cells.

The present study was undertaken to provide missing information on the distribution of AgNORs in large nucleoli of human leukaemic early granulocytic precursors in vivo as well as in vitro. In vivo, the distribution of AgNORs was studied in early granulocytic precursors of patients suffering from chronic myeloid leukaemia who were both untreated and treated with imatinib mesylate. AgNORs were visualized by silver reaction under conditions which facilitated to see their distribution by light microscopy. In vitro, the distribution of AgNORs was studied in proliferating and ageing K 562 cells which originated from chronic myeloid leukaemia. In vitro, the ageing of K 562 cells produced intranucleolar translocation of AgNORs to the nucleolar periphery. Such translocation was also observed in some leukaemic early granulocytic precursors in vivo, e.g. in bone marrow myeloblasts and promyelocytes of leukaemic patients. As was expected, the intranucleolar translocation of AgNORs in early granulocytic precursors was more frequent in patients treated with the cytostatic therapy--imanitib mesylate. The abovementioned findings suggest that myeloblasts and promyelocytes with AgNORs translocated to the periphery of large nucleoli might be in the ageing state, similarly as blastic cells of leukaemic myeloid origin (K 562 cells) in ageing cultures. Thus, the translocation of AgNORs might be a useful marker of premature ageing in the future and might contribute to the evaluation of the single cell state under various experimental as well as clinical conditions. However, more clinically oriented studies are required in this direction.

Nucleoli in human early erythroblasts (K2, K1, K1/2 cells).

Human early erythroid precursors classified according to the nuclear size were studied to provide information on nucleoli in these cells using simple cytochemical procedures for demonstration of RNA and proteins of silver-stained nucleolar organizers. K2 cells with nuclear diameter larger than 13 microm and K1 cells with nuclear diameter larger than 9 microm corresponding to proerythroblasts and macroblasts (large basophilic erythroblasts) mostly possessed large irregularly shaped nucleoli with multiple fibrillar centres representing "active nucleoli". K1/2 cells with nuclear diameter smaller than 9 microm corresponding to small basophilic erythroblasts were usually characterized by the presence of micronucleoli representing "inactive nucleolar types". On the other hand, a few K1/2 cells contained large nucleoli with multiple fibrillar centres similar to those present in K2 cells and thus appeared as "microproerythroblasts". The nucleolar asynchrony expressed by the presence of large irregularly shaped nucleoli with multiple nucleoli (active nucleoli) and ring-shaped nucleoli (resting nucleoli) in one and the same nucleus of K2 or K1 cells was not exceptional and might reflect a larger resistance of these cells to negative factors influencing the erythropoiesis. The intranucleolar translocation of silver-stained nucleolus organized regions was noted in K2 cells and might indicate the premature aging of these cells without further differentiation. More studies, however, are required in this direction.

A note on the decreased number and loss of fibrillar centres in nucleoli of apoptotic HL-60 leukaemic granulocytic precursors produced by 5-aminolaevulinic acid-based photodynamic treatment.

The nuclear and nucleolar ultrastructure was studied by means of conventional transmission electron microscopy to provide more and complementary information on nucleolar changes accompanying the apoptotic process in leukaemic granulocytic precursors (HL-60 cells) produced by PDT without previous terminal differentiation. PDT induced the apoptotic process using BL irradiation and ALA as a precursor of the photosensitizer protoporphyrin IX. PDT produced marked changes of the nucleolar ultrastructure in apoptotic cells, such as reduction of the number and loss of fibrillar centres surrounding dense fibrillar components. Such nucleolar changes are known to reflect an alteration of nucleolar biosynthetic activities, which are believed to be located at the periphery of fibrillar centres. Some electron micrographs also indicated that fibrillar centres apparently migrated out from nucleolar bodies.

Nucleolar abnormalities--a defect of the nucleolar preribosome assembly--in ringed sideroblasts in refractory anaemia with ringed sideroblasts (RARS) of myelodysplastic syndrome (MDS). An electron microscopic study.

Ringed sideroblasts were studied by means of transmission electron microscopy in patients suffering from refractory anaemia with ringed sideroblasts (RARS) of myelodysplastic syndrome (MDS) to provide more information on the structural organization of nucleoli in these abnormal erythroblasts. For control of the electron microscopic observations nucleoli in erythroblasts were also visualized by two widely used cytochemical procedures for the demonstration of RNA and AgNOR proteins. In contrast to previously described ultrastructure of nucleoli in "normal" erythroblasts, nucleoli of ringed erythroblasts in RARS of MDS were frequently characterized by a reduced incidence or lack of dense ribonucleic acid (RNA) containing granular components. Since the dense RNA containing granular components represent preribosomes, such sideroblasts in RARS of MDS exhibit a further nucleolar abnormality, which reflects a severe alteration of the nucleolar ribosome assembly in these abnormal cells. On the other hand, the alteration of the preribosome assembly was not noted in early developmental stages of ringed sideroblasts such as proerythroblasts. In addition, nucleoli in advanced or terminal stages of few ringed sideroblasts also did not exhibit such nucleolar abnormality and thus confirm a great structural and functional variability of these cells. The defect of RNA containing structures in nucleoli of advanced and terminal stages of erythroblasts are in a hormony with the light microscopic cytochemistry, which demonstrated a significantly smaller incidence of micronucleoli in specimens stained for RNA than in those stained for AgNOR (silver stained nucleolus organizer region) proteins.

A cytochemical note on nucleoli of granulocytic precursors and granulocytes in patients suffering from the refractory anemia with excess blasts (RAEB) of the myelodysplastic syndrome (MDS).

Nucleoli were studied in the proliferation as well as maturation granulopoietic compartment in patients suffering from refractory anemia with excess blasts (RAEB) of the myelodysplastic syndrome (MDS) by means of simple cytochemical procedures for the demonstration of nucleolar RNA and silver stained proteins of nucleolus organizer regions. Regardless of the procedure used for the nucleolar visualization, early stages of the granulopoietic compartment and particularly myeloblasts of RAEB patients were characterized by reduction of the nucleolar number expressed by the nucleolar coefficient the values of which resembled those described previously in acute myeloid leukemias. The reduced values of the nucleolar coefficient of these cells in silver stained specimens of RAEB patients were accompanied by a decreased number of clusters of silver stained particles representing interphasic silver stained nucleolus organizer regions (AgNORs). The reduction of these clusters was also described previously in leukemic cells. In addition, the differences in the values of the nucleolar coefficient of granulocytic precursors between specimens stained for RNA and those stained with the silver reaction might reflect changing composition and proportions of nucleolar components in the course of the granulocytic development.

A short note on micronucleoli in the course of terminal maturation of human erythroblasts.

The incidence of micronucleoli in the course of terminal differentiation of human erythroblasts was studied by the cytochemical procedures for demonstration of RNA and characteristic proteins of interphase AgNORs. The last dividing stages of the erythroid lineage--polychromatophylic erythroblasts--characterized by the presence of micronucleoli exhibited significantly larger values of the nucleolar coefficient in specimens stained for AgNOR proteins than in those stained for RNA. In addition, both these and terminal non-dividing nucleated stages of the erythroid lineage--orthochromatic erythroblasts--possessed micronucleoli after staining for RNA in a much smaller percentage of cells than after staining for AgNOR proteins. Thus, both these observations indicate that micronucleoli in the course of terminal maturation of erythroblasts apparently lose the nucleolar RNA detectable by the light microscopic cytochemistry. In addition, silver-stained micronucleoli--nucleolar remnants--were also noted in erythroblasts expelling the nucleus.

Incidence of nucleoli in erythroblasts in patients suffering from refractory anemia of myelodysplastic syndrome.

Nucleoli of erythroblasts have been studied in patients suffering from refractory anemia (RA) of myelodysplastic syndrome (MDS) and in control patients without a disturbed erythropoiesis in order to provide information on the incidence of nucleoli and micronucleoli in these cells. Nucleoli in erythroblasts were visualized by a simple cytochemical procedure for the demonstration of RNA which facilitated the visualization not only large nucleoli but also micronucleoli in advanced stages of the erythroblastic maturation. In control patients nucleoli were detected in all stages of erythroblastic development. In patients suffering from RA of MDS, a relatively large population of polychromatic and orthochromatic erythroblasts was characterized by a loss of nucleoli accompanied by the decreased incidence of micronucleoli characteristic of these cells. In contrast to control patients, in patients suffering from RA of MDS the number of nucleoli expressed by the values of the nucleolar coefficient of erythroblasts was smaller, particularly in both the early and terminal stages of erythroblastic development. Thus in patients with RA of MDS both the abnormal loss of nucleoli and decreased number of nucleoli in erythroblasts apparently represent and reflect a further abnormality of disturbed erythropoiesis.

The silver reaction of nucleolar proteins in the main structural compartments of ring-shaped nucleoli in smear preparations.

The present study was undertaken to provide more information on the conditions which result in preferential silver staining of the main nucleolar structural compartments using silver stainable proteins as their markers at the light microscopic level. For this study the mostly used method in cytology and pathology in which the nucleolar silver-positive structures are "developed" with the colloidal developer (Howell and Black, 1980; Ploton et al., 1986) was selected as silver reaction. Ring-shaped nucleoli of mature human lymphocytes represent a convenient model for such a study because they consist of one large fibrillar center, adjacent nucleolar regions with dense fibrillar components and the nucleolar peripheral shell with dense granular components. All these nucleolar compartments are known to possess characteristic silver stainable proteins. The results demonstrated that proteins of the fibrillar center and possibly adjacent nucleolar regions reacted preferentially with silver after a relatively long fixation with formaldehyde or methanol in unwashed specimens before the silver reaction. In contrast, the preferential staining of proteins in the nucleolar peripheral shell with silver was achieved after the fixation with acidified methanol or ethanol as well as after short fixation with formaldehyde vapors. In addition, the commonly used fixation before the silver reaction are not necessary and may be omitted for the visualization of all silver stainable proteins present in the fibrillar center as well as in the adjacent nucleolar regions and the nucleolar peripheral shell. In addition, similar results were achieved for the simultaneous visualization of proteins in the fibrillar center and nucleolar peripheral shell after fixation with ethanol.

Malignancy: The Number of Nucleoli and Main Nucleolar Types in Lymphoblasts of Children Suffering from Acute Lymphoid Leukemia.

Nucleoli were studied in lymphoblasts of children (untreated with cytostatic therapy) suffering from acute lymphoblastic leukemias (ALL) by means of a simple cytochemical procedure for the demonstration of RNA to provide information on the incidence of the main nucleolar types and the number of nucleoli in these cells. The values of the nucleolar coefficient reflecting the number of nucleoli per lymphoblast ranged between 1.66 and 2.03. The slightly larger values of the nucleolar coefficient in T-ALL were not statistically significant in comparison with those in nonT-ALL. Lymphoblasts in the bone marrow as well as the peripheral blood of both nonT and T-ALL patients mostly contained, "active" (RNA transcribing) large nucleoli with a relatively uniform distribution of RNA. "Inactive" micronucleoli or particularly "resting" ring shaped nucleoli were noted less frequently in these cells. On the other hand, the larger percentage of lymphoblasts determined in specimens stained with the panoptic staining (May-Grünwald-Giemsa) in comparison with that of lymphoblasts with "active nucleoli" in specimens stained for RNA apparently indicates the absence of such nucleoli in some of these cells. These cells might represent ageing, not proliferating, cells the existence of which has been already suggested by previous studies based on a different methodical approach.

A note on nucleoli in granulocytic precursors of the granulopoietic proliferating compartment in patients suffering from the chronic phase of chronic myeloid leukemia treated with two different drugs with different mode of action.

The incidence of main nucleolar types in granulocytic precursors was studied in the granulopoietic proliferating compartment (GPC) of patients suffering from chronic phase of the chronic myeloid leukemia (CML) who were treated by the widely used therapy with two different drugs with different mode of action - hydroxyurea (HU) and interferon alpha (IFN-alpha). In comparison with IFN early stages of GPC, i.e. myeloblasts and promyelocytes in patients treated with HU possessed more frequently micronucleoli which are known to reflect the direct as well as indirect inhibition of the nucleolar biosynthetic activities. On the other hand, the incidence of micronucleoli in these cells of a small percentage of patients treated with IFN also reached the average values of these nucleoli which were noted in patients treated with HU.

The asymmetric distribution of interphasic silver-stained nucleolus organizer regions in human and rat proerythroblasts.

The distribution of SSPs representing AgNORs was studied in human as well as rat proerythroblasts to provide information on the distribution of these nucleolar components in highly immature and proliferating non-neoplastic cells. The distribution of SSPs was asymmetric and most of the cells contained one nucleolus which possessed a larger number of these nucleolar components than the remaining nucleoli. Such nucleolus might be functionally dominant, since the number of nucleolar SSPs is apparently related to the nucleolar biosynthetic activity. On the other hand, when a proerythroblast possessed only one nucleolus, the number of SSPs in such a cell was very similar to the sum of SSPs in a polynucleolar cell. The asymmetric distribution of SSPs characteristic for most proerythroblasts disappeared in the terminal stages of the erythroblastic development. Cells in such stages, as described previously, were characterized by the presence of a limited number of single SSPs.

The Number of Nucleoli and Main Nucleolar Types in Lymphoblasts of Children Suffering from Acute Lymphoid Leukemia.

Nucleoli were studied in lymphoblasts of children (untreated with cytostatic therapy) suffering from acute lymphoblastic leukemias (ALL) by means of a simple cytochemical procedure for the demonstration of RNA to provide information on the incidence of the main nucleolar types and the number of nucleoli in these cells. The values of the nucleolar coefficient reflecting the number of nucleoli per lymphoblast ranged between 1.66 and 2.03. The slightly larger values of the nucleolar coefficient in T-ALL were not statistically significant in comparison with those in nonT-ALL. Lymphoblasts in the bone marrow as well as the peripheral blood of both nonT and T-ALL patients mostly contained, "active" (RNA transcribing) large nucleoli with a relatively uniform distribution of RNA. "Inactive" micronucleoli or particularly "resting" ring shaped nucleoli were noted less frequently in these cells. On the other hand, the larger percentage of lymphoblasts determined in specimens stained with the panoptic staining (May-Grünwald-Giemsa) in comparison with that of lymphoblasts with "active nucleoli" in specimens stained for RNA apparently indicates the absence of such nucleoli in some of these cells. These cells might represent ageing, not proliferating, cells the existence of which has been already suggested by previous studies based on a different methodical approach.

The nucleolar functional asynchrony--imbalance--in the granulopoietic proliferating compartment of patients suffering from chronic myeloid leukemia.

Nucleoli were studied in all stages of the granulopoietic proliferating compartment in the bone marrow of patients suffering from chronic myeloid leukemia to provide an information on the incidence of the nucleolar functional asynchrony (imbalance) in these cells. The nucleolar functional asynchrony is morphologically expressed by the presence of "active" large nucleoli with a relatively uniform distribution of ribonucleic acid (RNA) and "resting" ring shaped nucleoli with RNA only in their peripheral part in one and the same cell. This phenomenon was noted in a small but constant percentage of myeloblasts and decreased in myelocytes regardless of the phase of the disease and therapy. In addition, the nucleolar functional asynchrony was also noted in all stages of the granulopoietic proliferating compartment of control not-leukemic persons.

Preferential silver reaction of nucleolar regions adjacent to fibrillar centers in ring shaped nucleoli of leukemic lymphocytes.

Silver stained proteins (SSPs) characteristic for interphasic nucleolus organizer regions (NORs) associated with fibrillar centers (FCs) and adjacent nucleolar regions of ring shaped nucleoli in leukemic lymphocytes exhibit a different sensitivity to the mild acid extraction including that with HCl. Such extractions permit a preferential visualization of fibrillar centers adjacent regions (FCARs) which are believed to represent sites of the ribosomal RNA (rRNA) transcription. The resistance of SSPs in FCARs to the extraction with HCl seems to be due to their binding to other components present in these regions. The extractibility of SSPs with HCl was influenced by the fixatives used. The largest resistance of SSPs to the extraction with HCl was noted after fixation with glutaraldehyde. In contrast, the largest extractibility of these proteins was observed after fixation with unbuffered formaldehyde.