The Reduction of the Cell Nuclear Size in the Cell Body Space During the Differentiation Might Be Cell Lineage Specific (A Retrospective Morphological Note).

The cell body space occupied by the nucleus decreased during the cell differentiation of the granulocytic cell lineage in CML (Chronic Myeloid Leukemia) patients. In contrary, in patients suffering from CLL (Chronic Lymphocytic Leukemia), the cell body space occupied by the nucleus during the cell differentiation of the lymphocytic lineage did not decrease despite the reduction of the cell size. Thus, the cell body space occupied by the cell nucleus during the differentiation was characteristic for each of these cell lineages.

RNA Concentration and Content in the Nucleoli and Cytoplasmic Rim in Differentiating Lymphocytes of Patients Suffering from B Chronic Lymphocytic Leukaemia - a Cytochemical Note.

Nucleolar RNA optical density (concentration) measurements at the single cell level indicated that differentiation of lymphocytes is accompanied by a slightly decreased nucleolar RNA concentration in contrast to the cytoplasmic rim around the nucleus. On the other hand, the nucleolar size was markedly reduced and the cytoplasmic rim surrounding the nucleus was reduced only weakly. Concerning the calculated rough estimate of the RNA content, the differentiation induced its larger decrease in the nucleoli than in the cytoplasmic rim. These observations indicated that the nucleolar RNA concentration and RNA content together with the nucleolar morphology are more sensitive markers of the differentiation process than the RNA concentration and content in the cytoplasm. Thus, the nucleolar RNA transfer to the cytoplasm in advanced differentiation steps might still be going on regardless of the decreasing or inhibited nucleolar biosynthetic activity. In addition, the presence of ring-shaped nucleoli and micronucleoli characteristic of mature and terminal lymphocytes in some lymphocytic less differentiated steps, i.e., lymphoblasts and prolymphocytes, might indicate the premature differentiation state of such cells.

The Morphology of Cell Differentiation, Terminal Differentiation and Ageing Seems To Reflect the Same Process: a Short Note.

Based on simple microscopic cell morphology in blood and bone marrow smear preparations, it seems to be likely that the cell differentiation and terminal differentiation in human blood cells, and particularly in erythroid or granulocytic lineages, simultaneously reflect ageing of the lineage progenitors and terminal differentiation steps. The terminal differentiation stages of both these lineages actually appear as senescent cells. Abnormal ageing of progenitor cells may represent one of the "dysplastic" phenomena of the premature terminal differentiation state. Such state is characterized by heterochromatin condensation and nucleolar morphology similar to that in fully differentiated terminal cells of granulocytic or erythroid lineages. It should also be mentioned that in some known erythropoietic disorders, less differentiated erythroblasts may lose nuclei similarly as "normal" fully terminally differentiated cells of the erythroid cell lineage. It seems to be clear that cells in both abnormal less differentiated and terminally differentiated stages of erythroid or granulocytic lineages lose the ability to multiply similarly as senescent cells. On the other hand, the background of cell ageing and differentiation is very complicated and requires a different approach than the simple microscopic morphology at the single cell level. However, the morphology and clinical cytology at the single cell level might still contribute with complementary data to more sophisticated complex studies of that topic. In addition, the morphological approach facilitates the study of the main components of single cells in various states, including the differentiation steps or ageing.

The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

Dominant Nucleolus in the Progenitor Cell Using Human Bone Marrow Erythroid and Granulocytic Cell Lineages as a Model. A Morphological and Cytochemical Note.

Progenitor cells of the human erythroid and granulocytic cell lineages are characterized by the presence of several nucleoli. One of these nucleoli is larger and possesses more fibrillar centres than others. Such nucleolus is apparently dominant in respect of both size and main nucleolar function such as nucleolar-ribosomal RNA transcription. Such nucleolus is also visible in specimens using conventional visualization procedures, in contrast to smaller nucleoli. In the terminal differentiation nucleated stages of the erythroid and granulocytic development, dominant nucleoli apparently disappeared, since these cells mostly contained very small nucleoli of a similar size with one fibrillar centre. Thus, the easily visible dominant nucleoli appear to be useful markers of the progenitor cell state, such as proliferation, and differentiation potential.

To the approximate size of the nuclear region occupied by nucleolar bodies during cell differentiation and maturation using the human leukemic granulocytic lineage as a convenient model.

The present study was undertaken to estimate the approximate size of nuclear regions occupied by nucleolar bodies during the cell differentiation and maturation. The differentiation and maturation of human leukemic granulocytic cells in patients suffering from the chronic phase of the chronic granulocytic leukemia (CML) represented a convenient model for such study because of the large number of cells for the diameter measurements at the single cell level. Early and advanced differentiation or maturation stages of these cells are well defined and nucleolar bodies and nuclear outlines are easily seen by simple cytochemical methods for the visualization of RNA and silver stained proteins in smear preparations. During the cell differentiation and maturation, the estimated size of the nuclear region occupied by nucleolar bodies decreased in both untreated and treated patients with the anti-leukemic therapy. However, the size reduction of nucleolar bodies in differentiated and mature cells was larger than that of the nucleus. In addition, the results also indicated that the nuclear region occupied by nucleolar bodies was characteristic for each differentiation and maturation stage of the granulocytic cell lineage and was not substantially influenced by the anti-leukemic therapy of CML patients.

Morphometric and Densitometric Analysis of Heterochromatin during Cell Differentiation Using the Leukaemic Granulocytic Lineage as a Convenient Model.

Granulocytic early progenitors and terminally differentiated - mature granulocytes with segmented nuclei were studied using computer-assisted diameter and heterochromatin optical image densitometry to provide more information on the nuclear size and heterochromatin condensation state. Bone marrow smears of patients suffering from chronic myeloid leukaemia untreated as well as treated with "specific" anti-leukaemic therapy with imatinib mesylate are a convenient model for such study because they possess a satisfactory number of cells for diameter and optical density measurements. In addition, the identification of developmental stages of granulocytes is very easy and the morphology is not different from that in not-leukaemic persons. As it was expected, the mean diameter of nuclear segments in fully differentiated and mature granulocytes was much smaller than that in non-segmented nuclei of early granulocytic precursors. Therefore, no wonder that the heterochromatin condensation state in nuclear segments of mature granulocytes was much larger than in non-segmented nuclei of granulocytic progenitors. On the other hand, the sum of mean diameters of all nuclear segments per cell was close to the mean nuclear diameter of early granulocytic progenitors. The heterochromatin condensation state in granulocytic progenitors or fully differentiated mature granulocytes exhibited marked stability and did not change after the anti-leukaemic therapy. In addition, Barr bodies of characteristic drumstick appearance bearing inactive X chromosome in interphase nuclei of mature granulocytes in fertile female patients exhibited a heterochromatin condensation state similar to nuclear segments. This heterochromatin condensation state was also stable and constant, and was not apparently influenced by the anti-leukaemic therapy.

Quantitative monitoring of WT1 expression in peripheral blood before and after allogeneic stem cell transplantation for acute myeloid leukemia - a useful tool for early detection of minimal residual disease.

Overexpressed Wilms tumor gene 1 (WT1) has been found in a majority of patients with acute myeloid leukemia (AML). The aim of this study was to confirm the applicability of WT1 expression measurement as a marker of minimal residual disease (MRD). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood (PB) according to European Leukemia Net (ELN) recommendations. The WT1 expression was related to the expression of a reference gene Abelson (ABL) and the results were calculated as a number of WT1 copies related to 104 copies of ABL gene. The upper normal limit of WT1 expression was set at 50 copies of WT1 to 104 copies of ABL. Morphological, flow cytometry and chimerism examinations were evaluated according to standard protocols.A total of 51 AML patients with overexpressed WT1 gene were analyzed. The median follow-up after transplantation was 14 (2-72) months. WT1 expression levels exceeding the upper normal limit were considered as a sign of impending hematological relapse, in accord with morphological, flow cytometry and chimerism data, as well as with the expression of the specific fusion genes. Moreover, in 7 patients the rise of WT1 expression preceded all other standard methods. Patients with high WT1 expression before allogeneic hematopoietic stem cell transplantation (allo-HSCT) had significantly worse outcome than patients with low WT1 level. Examination of WT1 expression in PB of patients with AML is a useful tool for MRD monitoring. Moreover, the WT1 gene expression before stem cell transplantation seems to be of prognostic significance.

[Visceral leishmaniasis (two case reports)]. / Viscerální leishmanióza (dve kazuistiky).

Two cases of imported visceral leishmaniasis are described. The first patient was a 32-year-old Czech man who developed leishmaniasis 5 months after a holiday in Italy (Bibione). The second patient was a 62-year-old Czech man who developed leishmaniasis 18 months after visiting Croatia (Makarska); the disease began after a course of chemotherapy due to metastasizing testicular tumor. Both patients were successfully treated with amphotericin B lipid complex (Abelcet). Difficulties in establishing the correct diagnosis of visceral leishmaniasis are discussed.

[Common, standardized and recommended approaches in the diagnosis and monitoring of paroxysmal nocturnal haemoglobinuria using flow cytometry]. / Bezné, standardizované a doporucené postupy v diagnostice a monitorování paroxysmální nocní hemoglobinurie prutokovou cytometrií

Paroxysmal nocturnal hemoglobinuria is an acquired clonal disease characterized by proliferation of stem cells, deficient of proteins linked to the membrane via glycophosphatidylinositol (GPI) anchors. PNH cell characterization by flow cytometry was introduced in 1986, since 1996 is considered as method of choice for PNH diagnosis. Flow cytometry PNH analysis is nowadays crucial for disease monitoring in terms of progression, regression, remission or response to therapy and screening for small PNH clones (< 1.0%) in patients with aplastic anemia or myelodysplastic syndrome. Flow cytometry is unfortunately still poorly standardized, there is a variety of different methodological approaches for PNH evaluation and results from external quality assurances schemes reveal heterogeneous results. The aim of this work is to review the applicability of flow cytometry for the diagnosis and monitoring of PNH with respect to our experience and in the context of the recent trends and guidelines for PNH evaluation by flow cytometry.

Heterochromatin density (condensation) during cell differentiation and maturation using the human granulocytic lineage of chronic myeloid leukaemia as a convenient model.

The present study was undertaken to provide complementary data on the heterochromatin condensation in both central and peripheral nuclear regions during the cell differentiation and maturation using computer-assisted density measurements at the single-cell level. The lineage of neutrophilic granulocytes in the bone marrow of patients suffering from chronic myeloid leukaemia was very convenient for such study because the increased number of granulocytes in all developmental stages was satisfactory for heterochromatin density measurements. The morphology of leukaemic and non-leukaemic neutrophilic granulocytes is similar and each differentiation or maturation stage is easily identified. A markedly increasing heterochromatin density--condensation--in the peripheral nuclear region at the nuclear envelope accompanied both the differentiation and maturation of these cells. Thus, peripheral chromosomal territories at the nuclear envelope are important for both the differentiation and maturation process. In contrast, the heterochromatin density of nuclear central regions was already high in early differentiation stages and exhibited a less distinct increase during the differentiation, but was more apparent in late maturation stages representing the terminal differentiation. A limited number of maturing cells with persisting large heterochromatin density in central nuclear regions without markedly increased heterochromatin condensation at the nuclear periphery might represent a further maturation abnormality--asynchrony--during the granulocytic development. From the methodological point of view, both, the cytochemical method for the DNA demonstration and the panoptic May-Grünwald-Giemsa staining, are convenient for computer-assisted chromatin densitometry at the single-cell level.

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.

[Urgent states in hematology: acute promyelocytic leukemia--principles of diagnosis]. / Urgentní stav v hematologii: akutní promyelocytární leukemie--principy diagnostiky.

A review of diagnosis of acute promyelocytic leukemia (APL) is presented. There are still many patients with progressive disease with leukocytosis at presentation. These are at greater risk of early death due to bleeding (often intracranial), or, less frequently, due to thrombotic complications. In Czechia, we have, in some instances, noted an unacceptably long time from the first symptoms to diagnosis and to administration of the highly specific differentiation therapy with tretinoin (ATRA) along with anthracycline chemotherapy. This combination is highly efficient--cures are seen in some 70% of patients. Therefore, we present a diagnostic minimum for each and every internist, and even better for every general practician, to get acquainted with. All cases of pancytopenia and consumption coagulopathy should be suspected of APL and referred to a specialized hematologist without any delay. In the following more detailed review of diagnostic measures, much attention is given to APL morphology, which is the first clue leading to diagnosis. The finding of the typical hypergranular FAB M3 morphology and of cells with bundles of Auer rods ("faggot cells"), along with the HLA-DR, CD33+ immunophenotype, is highly (but not absolutely) specific for APL. In cases of the micro-/hypo-granular variant FAB M3v Form, and whenever APL cannot be ruled out with certainty, a test to prove the presence of the PML/RARalpha fusion gene is indicated, using either RT-PCR or, eventually, immunological demonstration of the specific distribution of the PML protein in the cell nucleus. Given that morphology of APL cases, as defined according to WHO criteria (95% of which carry the PML/RARalpha fusion gene), admits extremely divergent morphological pictures ofthe variant forms, we recommend these investigations to be performed in every case of de novo acute myeloid leukemia. A review of the less frequent morphological, as well as genetic variants is given, and the principles of immunophenotypic, cytogenetic and molecular diagnostics are also reviewed.

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.

On the nucleolar size and density in human early granulocytic progenitors, myeloblasts.

Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells) in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.

Prognostic relevance of the FAB morphological criteria in chronic lymphocytic leukemia: correlations with IgVH gene mutational status and other prognostic markers.

Morphological examination is the routine first step in the diagnosis of hematological malignancies, including chronic lymphocytic leukemia (CLL). Atypical cell morphology according to the FAB criteria is known to herald disease progression. Several years ago, it was proposed that FAB morphology at diagnosis had a considerable prognostic impact. However, this proposal has not been widely adopted in practice. Thus we questioned the prognostic value of the morphological examination, which was performed retrospectively in 88 patients out of our 110 institutional registry patients (70 males and 40 females, median age 57 yrs) with CLL at diagnosis. We related the results to the more modern prognostic markers. Atypical FAB morphology was shown to correlate with IgVH gene mutation status, trisomy of chromosome 12 and deletion of 17p detected either by conventional G-banding or by fluorescence in situ hybridization (FISH) analysis. The correlation of FAB morphology with CD38 antigen expression or with the histopathological pattern of bone marrow infiltration was not significant. Overall survival (OS) data were available for 84 morphologically examined patients. The patients with atypical morphology (64 patients) had a significantly shorter OS (103 months) than the 20 patients presenting with typical CLL morphology (237 months; p=0.03). Only the mutation status of IgVH genes correlated more closely with OS (p=0.002). Of note, there was no leukemia-related death within "unmutated" cases who had typical FAB morphology (p=0.14), and vice versa, the mutation status had a significant prognostic impact within the morphologically atypical cases (p=0.01). Thus FAB morphology and the mutation status may yield complementary prognostic information. OS was affected both by the presence of cytogenetic aberrations (p=0.03) - most adversely by deletions of 17p and 11q, and by CD38 expression (p=0.003). We conclude that careful examination of peripheral blood smears according to FAB is a simple, cheap and valuable tool in the first-line assessment of prognosis of CLL patients and should not be overlooked even in 3rd millennium when more sophisticated prognostic markers are at hand. This ought to be confirmed in larger prospective studies with multivariate analysis of data.