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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.

The present study was undertaken to provide more information on the differentiation and maturation of human granulocytes using computer-assisted image RNA densitometry at single-cell level. The bone marrow of patients suffering from chronic phase of chronic myeloid leukemia represents a very convenient model for such measurements because of the satisfactory number of early stages, as well as advanced stages, of the granulocytic cell lineage represented by neutrophils. In contrast to the erythroid cell lineage, similar nucleolar and cytoplasmic RNA density-concentration values were found only in early granulocytic progenitors such as myeloblasts and promyelocytes. In advanced stages of the granulocytic development starting with myelocytes, these cells were characterized by a larger decrease in the cytoplasmic RNA concentration in comparison with that of the nucleoli. Thus, the nucleolar to cytoplasmic RNA concentration ratio in these cells was above 1. On the other hand, it should be pointed out that late differentiation stages of granulocytes, starting with myelocytes, possessed nucleolar bodies (nucleoli without surrounding perinucleolar chromatin) of a markedly reduced size., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

The information on the nuclear size in early granulocytic progenitors is very limited. Numerical data on the nuclear diameter and size in these cells are missing in the literature. Therefore the nuclei of myeloblasts and promyelocytic cells were measured in bone marrow smears of patients suffering from chronic phase of chronic myeloid leukaemia since the general morphology of granulocytic progenitors is very similar to those in non-leukemic persons. Moreover, the increased granulopoiesisinthese patients provided a satisfactory number of early granulocytic progenitors for karyometric measurements. Nuclear diameter in digitised and processed images of these cells was measured directly on the monitor screen at magnification 4300x using a computerised photoprogram. The nuclear mean diameter in both myeloblasts and promyelocytes ranged between 11 and 13 microm. Cells with smaller or larger nuclei were observed less frequently. Since the nuclear size depends on the cell cycle stage, a possibility exists that both granulocytic progenitors are mostly in the S and less frequently in G1 or G2 phase. Thus, the mean nuclear diameter, i.e. nuclear size, might be useful in helping to estimate the cell cycle stage of these cells in bone marrow smears at the single cell level. In addition, the results of the present study also indicated that the nuclear diameter in myeloblasts as well as promyelocytes of studied patients was not substantially influenced by the cytostatic therapy with imatinib mesylate.

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.

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.