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THE CELL BODY SPACE OCCUPIED BY THE NUCLEUS DURING THE CELL DIFFERENTIATION IN HUMAN LYPHOCYTIC, 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.


Introduction
The morphological relationship of the nucleus and cytoplasm was a subject of a great interest since the end of the 19 th and onset of the last century (Hertwig R 1903, Sharp 1921). This relationship was expressed by the "Kern-Plasma-Relation", i.e. nuclear-cytoplasmic index (Hertwig R 1903, Hertwig G 1929, Ries and Gersch 1953. The changing equilibrium of both these main cell compartments was considered to precede the cell division. Then, the changing proportion of the nucleus in the cell body was apparent in the course of the cell differentiation and maturation or aging (Sharp 1921, Hertwig G 1929, Bessis 1973. Concerning the calculation of the nuclear -cytoplasmic ratio or index there are several calculation formulas depending on the specimen preparations and field of interest. In basic research or classical cytology, the nuclear-cytoplasmic index was calculated by dividing the nuclear volume by the cytoplasmic volume subtracted from the nuclear one (Robertis de and Robertis de jun 1987). In the clinical and hematological cytology, the nucleus to cell ratio was calculated dividing the nuclear size estimate by the cell size. Such simple calculation appeared to be very useful in both light and electron microscopy if the number of measured cells was adequate to reduce the variability (Ochiai and Eguchi 1987, Doughty 2012). It should be added that in the clinical cytology or histology the simple nuclear to cytoplasmic ratio appeared to be a very important tool to estimate the cell proliferation potential and cell malignancy (Cardozo 1954). In addition, the nuclear size related to the cell body might be also useful to detect further cell abnormalities or pathology in various cell lineages including hematological disorders. At his occasion it should be also mentioned that the nucleus to cell body ratio multiplied by 100 just reflected the rough cell space estimate occupied by the nucleus.
Since it was generally believed that the cell differentiation and maturation is accompanied by the reduction of the nuclear size in the cell space, the present study is dealing with the nucleus to cell body ratio (Nu/CBR) in differentiation steps of human lymphocytic, granulocytic and erythroid cell lineages. These cell lineages appeared to be very convenient models because differentiation steps of non-leukemic or leukemic lymphocytic lineages were easily recognizable and accessible in diagnostic peripheral blood smears of blood donors (BDs) and patients with chronic lymphocytic leukemia (CLL). The differentiation steps were also easily identified and accessible in diagnostic bone marrow smears of selected patients with chronic myeloid leukemia

Material and methods
Nu/CBR (Ochiai and Eguchi 1987) during the cell differentiation was studied in lymphocytic, granulocytic and erythroid cell lineages of 4 blood donors, 4 patients suffering from CLL, 4 patients with CML and 4 patients with RA of MDS. Lymphocytic cell lineages were studied in peripheral blood smears, granulocytic (neutrophilic) and erythroid cell lineages were studied in bone marrow smears. All peripheral blood and bone marrow samples were originally taken for diagnostic purposes with the approval of the Institute authorities. Since blood smears of blood donors did not contain lymphocytic progenitors or a satisfactory number of immature lymphocytes for measurements, these cells were replaced by stimulated and de-differentiated Tlymphocytes with phytohemagglutinin in vitro (Kalousek and Křížková 2000, Smetana et al.

2020).
To get such cells, isolated lymphocytes of the peripheral blood of BDs were cultured for 48 hrs. in RPMI 1640 medium with inactivated 10% FBS and phytohemagglutinin (10µg/mL-Sigma) at 37°C in 5% atmosphere with CO2. For microscopy, cytospins of these cells were prepared using the cytocentrifuge Shandon Cytospin 2 (Shandon Southern Products UK).
The largest diameter of nuclear and cell body outlines was measured in micrograph of

Granulocytic (neutrophilic) and erythroid cell lineages (Graph 2, 3)
The granulocytic lineage in CML patients. Granulocytic progenitors, i.e., myeloblasts were characterized by a large nucleus that occupied more than 70 percent of the cell space (Tab. 1).
The further differentiation stepspromyelocytes were characterized by a slightly smaller nuclear size in the less enlarged cell space (Tab. 1). The last step of the proliferating pool of differentiating granulocyticneutrophilic cells such as myelocytes possessed a significantly smaller nucleus that occupied the cell space. (Tab. 1).

The erythroid cell lineage in CML patients.
Erythroid progenitors, i.e., proerythroblasts possessed a large nucleus that occupied more than 70 per cent of the cell space (Tab. 1). Nuclei in further differentiation steps comprised significantly smaller portion of the cell space. However, such change was not significant due to the large variation coefficient (about 11 per cent). The largest nuclear size reduction in the cell space was noted in advanced and terminally differentiated nuclear stages of smallest erythroblasts (Table 1).  (Table 1). However, the reduction of the cell space occupied by the nucleus was less apparent. The cell size reduction was significant only in intermediate and terminally differentiated cells (Table 1).

Discussion
Since the onset of the last century, it is generally accepted that nuclear size is generally proportional to that of the cytoplasm and reflects the equilibrium of these both main cell In the non-leukemic erythroid lineage of patients with CML there was a fluent reduction of the Nu/CBR reflecting the decreasing cell space occupied by the nucleus. Sub-terminal and terminal steps of this lineage were characterized by the small cell space occupied by the nucleus that possessed only micronucleoli as the only one nucleolar type (Smetana et al. 1975). It was not surprising that in RA of MDS the reduction of the cell space containing the nucleus was less apparent. and statistically not significant. The ineffective erythropoiesis in this blood disorder possessed not only normoblastic cell lineage but also macro-and megaloblastoid erythroblasts (Bessis 1973, Cazzola and Malcovati 2005). In late megaloblastoid erythroblasts the nucleus still occupied a large space of the cell body, such as 69.5±9.4 per cent (additional data), that was not substantially different from erythroid progenitors (Table 1). Table 1. The approximate cell space estimate occupied by the cell nucleus (expressed by the nucleus to cell body maximal diameter ratio multiplied by 100) during the differentiation of human lymphocytes, neutrophils and erythroblasts*