Cytophotometry of granulocytes in chronic granulocytic leukemia patients. Part I. Cell cycle distribution, S-phase transition and quantitative cytochemistry

In the chronic phase of CGL the proportion of granulocytes in S + G2 was lower (18.7 +/- 1.3% in marrow and 16.7 +/- 2.4% in blood) than in normal bone marrow (42.4 +/- 2.9%) as studied by Feulgen-DNA cytophotometry. During the blast crisis the percentage of S + G2 blasts was 39.3 +/- 8.4 in marrow and 38.7 +/- 7.8 in blood which was much higher than in acute myeloblastic leukemia patients (10.8 +/- 1.4 and 5.1 +/- 1.0). Thymidine labelling index values were lower than the percentage of cytophotometrically detected S-phase cells: up to 28% of cells with Feulgen-DNA content corresponding to S-phase did not incorporate 3H-thymidine. The rate of DNA synthesis remained constant during the S-phase but 3H-thymidine uptake increases towards the end of the S-phase. Morphometric parameters and quantitative cytochemical (PAS, Sudan, myeloperoxidase activity) characteristics of polymorphonuclear neutrophils were altered during the chronic phase of the disease but remained in the normal range during the blast crisis. Mature neutrophils in the blast crisis are assumed to originate from normal granulocyte progenitors.     

Elevation of an acid ribonuclease in serum of patients with chronic granulocytic leukaemia.

Ribonuclease (Ribonucleate nucleotide 2'-transferase E.C. 2.7.7.17) activity in serum of patients with chronic granulocytic leukaemia measured at pH 4.5-6.0 amounts to more than three times of that in serum of healthy subjects. At pH 6.0-8.0 the elevation of ribonuclease activity in serum of patients with chronic granulocytic leukaemia is less pronounced and amounts to about two times of that in normal ones. Using chromatography on CM Sephadex C-50 column, serum ribonuclease of both normal and chronic granulocytic leukaemia patients was separated into five distinct fractions. In serum of healthy subjects ribonuclease fractions denoted I-V contribute to 10; 21; 29; 22, and 18 percent of the total ribonuclease activity. In the serum of patients with chronic granulocytic leukaemia a decrease in ribonuclease fraction III to merely 17 percent and an increase in contribution of fraction IV to 32 percent of total ribonuclease activity could be observed. The comparison of each individual concentration of fraction in normal and leukaemia patients serum reveals, that ribonuclease fraction IV will increase about 3 times. A less pronounced increase could also be found for fractions I, II and V. However, ribonuclease fraction IV may be supposed to carry more than 50 percent of the whole extra load of ribonuclease present in the serum of chronic granulocytic leukaemia patients.     

Effect of recombinant human alpha interferon (INF) on chronic granulocytic leukaemia (CGL) clonogenic growth of bone marrow haematopoietic progenitors.

The effect of recombinant alpha interferon (INF) to the colony stimulating factor (CSF) production was examined with in vitro culture of the bone marrow of patients with chronic granulocytic leukaemia (CGL). It could be found that addition of interferon into a suspension of preincubated phytohaemagglutinin (PHA) lymphocytes from peripheral blood represents an inhibity factor for colony and cluster formation in autologic human marrow cultures.     

Primitive progenitor cells in the blood of patients with chronic granulocytic leukemia

We measured the number of blast colony-forming cells (Bl-CFC) in the blood of 11 patients with untreated chronic granulocytic leukemia (CGL). The culture system used detects three types of Bl-CFC (Types I, II and III) in normal marrow, of which Bl-CFC (I) are the most primitive and might represent the putative hemopoietic stem cell. The mean numbers of Bl-CFC (I) in CGL blood, normal bone marrow and normal blood were 134 +/- 29 (+/- SEM), 127 +/- 21 and 1.5 +/- 0 respectively per 1 X 10(6) mononuclear cells. These findings are consistent with the concept that CGL is due to a primary increase in stem cell numbers with secondary increases in committed progenitor and leukocyte numbers.     

Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes

Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420-430 nm and a trough at 380-400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes.     

Diagnostic and prognostic value of the terminal deoxynucleotidyl transferase (TdT) activity in treating the blastic phase (bp) of chronic granulocytic leukaemia (CGL)

The blastic cell phenotypes of 26 cases of CGL in blastic phase were estimated and the patients were treated with different schemes. The following methods of typisation of the blast cells were used: cytochemical stainings (POX, Sudan B, PAS, nonspecific esterase), estimation of TdT activity, and in 11 patients the testing with monoclonal antibodies of VI series. Using these methods 10 patients (38%) with lymphoid form of the blastic phase, 11 (43%) with the myeloid type and 5 patients (19%) with undifferentiated type were diagnosed. In the group of lymphoblastic type a longer survival time and complete remissions were observed. High TdT activity in blastic cells did correspond with favourable response to Vincristin and Prednison. The introduction of TdT assessment into the diagnosis of CGL allows the cells to be classified more precisely, thus helping in defining the prognosis and in the choice of treatment programme.     

The ultrastructural localization of human neutrophil alkaline phosphatase in normal individuals during pregnancy and in patients with chronic granulocytic leukaemia

Summary The intracellular localization of alkaline phosphatase was determined in human neutrophils by electron microscope cytochemistry. In normal individuals, the largest and most intense deposits of reaction product were seen in a unique cytoplasmic granule population termed phosphasomes. Lighter deposits were seen on nuclear membranes, some intracytoplasmic membranes lining vacuoles and granules and occasionally in focal patches on the internal surface of the plasma membrane.In cell isolated from women in the third trimester of pregnancy, activity was found in the same intracellular sites but there were, on average, more alkaline phosphatase-containing granules per cell than in the cells from non-pregnant individuals. Neutrophils from pregnant women were also characterized by the presence of large deposits of reaction product on the external surface of the plasma membrane (extramembranous). This activity had properties characteristic of the placental isoenzyme of alkaline phosphatase, found in serum during pregnancy.Neutrophils from patients with chronic granulocytic leukaemia, showing normal mature morphology, contained significant amounts of granule reaction product but there were fewer phosphasomes per cell than in normal individuals. In morphologically immature cells, reaction product was present in nuclear membrane, endoplasmic reticulum and large granules. These results were in agreement with previous biochemical data confirming a quantitative lack of alkaline phosphatase in chronic granulocytic leukaemia.     

Factors influencing myeloid stem cell (CFU-C) survival after cryopreservation of human marrow and chronic granulocytic leukemia cells

Human marrow stem cells obtained from 20 patients (9 with nonhematological malignancy, 11 with acute leukemia in remission) and peripheral blood stem cells from 27 patients with chronic granulocytic leukemia were cryopreserved in 10% dimethyl sulfoxide (Me₂SO). It was found that the optimal cooling rate for the human myeloid stem cells (CFU-C) ranged from 1 to 3 °C per minute. The myeloid stem cells (CFU-C) maintained their viability for up to one year of storage in liquid nitrogen, after an initial 20% reduction due to the freezing procedure. Myeldoid stem cells survived better when thawed and diluted at room temperature (RT) than at 4 °C. However, the viability of thawed stem cells decreased when stored at RT for more than 1 hr. The viability of stem cells cryopreserved in bags and ampoules was similar. No differences were noted in the surivial of normal human marrow stem cells and cells from patients with chronic granulocytic leukemia when cryopreserved under similar conditions.     

Cytophotometry of granulocytes in chronic granulocytic leukaemia patients. Part II. Effects of chemotherapy on cell kinetics and cytochemical parameters of granulocytes

A successful course of chemotherapy (myelosan or combined regimens) results in a two to eight fold decrease of the percentage of S + G2 cells in the bone marrow and peripheral blood and in the restoration of normal morphometric and cytochemical patterns of polymorphonuclear neutrophils in the chronic phase of CGL. In those patients of the chronic phase not responding to chemotherapy and in blast crisis the proportion of S + G2 cells did not change after a course of chemotherapy. Cytostatic drugs killed cells in all phases of the cell cycle. The initial response to chemotherapy was a slight increase of the proportion of S + G2 cells in peripheral blood, apparently as a result of the block in cell cycle transition which was followed by a rapid drop of S + G2 proportion. During chemotherapy such phenomena as an appearance of tetraploid mature neutrophils and of aneuploid clones were occasionally observed.