Flow cytometric DNA content in myelodysplastic syndromes

DNA flow cytometric analysis of unfixed bone marrow cells stained with propidium iodide was carried out in 33 patients with untreated primary myelodysplastic syndromes. Patients with stable clinical course for up to 3 years had higher fractions of cells in S and G2 phases (22.7 +/- 12.4% and 12 +/- 3.6%) than those who developed acute leukemia and/or died early in the course of disease (14.4 +/- 8.5% and 6.6 +/- 4%). Median survival was more than 36 mo in patients with S + G2 cell fraction higher than 24%, and 14 mo in the remaining 16 patients with lower values (P less than 0.01). Analyses repeated after 3-24 mo showed no major changes in cell proliferation pattern in ten out of 11 patients. The remaining patient had sharp decrease in S and G2 cell fraction 3 mo before the transition into acute leukemia. The DNA index (DI) of bone marrow cells was calculated to assess ploidy. However, comparative evaluation of cytologic, cytogenetic, and flow cytometric data suggest that, under our experimental conditions, the DI may be influenced by factors such as the degree of chromatin compactness.     

Flow cytometric determination of ploidy and proliferative activity on isolated bone marrow particles and on total bone marrow aspirate

The nuclear DNA content distribution of peripheral blood (PB) and bone marrow (BM) cells was determined by propidium iodide flow cytometry in 33 patients who underwent BM aspiration for diagnostic purposes. Two types of BM samples were taken during every aspiration procedure: whole BM aspirate, composed of BM particles contaminated by PB cells; isolated BM particles. Proliferative activity was calculated as the percentage of cells with DNA content intermediate between the diploid (2n) and the tetraploid (4n) values (2n-4n%). Ploidy was expressed as the ratio between the modal channel of the G0-G1 peak of the probe and that of an internal reference standard (DNA index, DI). The 2n-4n% was very close to zero in all PB samples. It was significantly greater in BM particles (21.2 +/- 6.6%) than in whole BM aspirate (16.6 +/- 5.5%, p less than .0005), with a close correlation (r2 = 66; p less than .0001) between the two values. Aneuploid stem lines were found in BM but not in PB. The DI of BM stem lines were similar in whole BM aspirate and BM particles, but the percentage of aneuploid cells was usually higher in BM particles. The reduced proliferative activity and the lower percent of aneuploid cells found in whole BM aspirates, with respect to BM particles, can be attributed to the contamination of BM tissue by PB, which had a very low proliferative activity and did not show aneuploidy. BM particles are therefore an easily obtained and reliable sample for routine evaluation of proliferative activity and ploidy of BM cells by DNA flow cytometry.     

Mutations in ras genes in myelocytic leukemias and myelodysplastic syndromes

Acute myelocytic leukemias (AML) are characterized by a remarkably high incidence (approximately 30%) of point mutations affecting codons 12, 13, or 61 within ras genes. A predominant involvement of N-ras sequences has been established. Neither Philadelphia chromosome-positive chronic myelocytic leukemia nor other chronic myeloproliferative disorders show a similar frequency. However, a proportion of myelodysplastic syndromes, namely, the chronic myelomonocytic subtype (CMML) also show this molecular feature. The following is a brief discussion of the possible biologic and clinical implications of these observations.     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

Flow cytometric analysis of mouse hepatocyte ploidy

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.     

DNA ploidy and proliferative activity of human pulmonary epithelium

DNA ploidy and distribution has been determined in normal and abnormal bronchial, bronchiolar and alveolar epithelium from 22 patients, aged between 0 and 85 years, 9 of whom had received chemotherapy for malignant disease. The DNA ploidy was diploid in all the specimens examined. The S + G2/M fraction was significantly greater in diseased than normal bronchial trees. In the bronchial epithelium, mean values ± the standard deviation (SD) were 5.5 + 2.2% vs 1.1±0.6%, in bronchiolar epithelium 4.6 ± 1.6% vs 1.0 ± 0.9% and in alveolar epithelium 4.6 ± 1.6% vs 0.8 ± 0.5%. The highest S + G2/M value of 8.9% was obtained from inflamed bronchial epithelium. Polyploid cells up to the octaploid range occurred infrequently but their incidence was slightly increased to between 0.16% and 0.9% in diseased lungs and in patients who had received chemotherapeutic drugs. It was concluded that (1) non-cancerous pulmonary epithelium is diploid, that (2) pulmonary epithelium shows steady-state renewal at all ages and polyploid cells are rare under normal conditions and that (3) the S + G2/M fraction increases up to approximately 10% in reactive proliferative states.     

DNA ploidy and proliferative activity of human pulmonary epithelium

DNA ploidy and distribution has been determined in normal and abnormal bronchial, bronchiolar and alveolar epithelium from 22 patients, aged between 0 and 85 years, 9 of whom had received chemotherapy for malignant disease. The DNA ploidy was diploid in all the specimens examined. The S + G2/M fraction was significantly greater in diseased than normal bronchial trees. In the bronchial epithelium, mean values +/- the standard deviation (SD) were 5.5 +/- 2.2% vs 1.1 +/- 0.6%, in bronchiolar epithelium 4.6 +/- 1.6% vs 1.0 +/- 0.9% and in alveolar epithelium 4.6 +/- 1.6% vs 0.8 +/- 0.5%. The highest S + G2/M value of 8.9% was obtained from inflamed bronchial epithelium. Polyploid cells up to the octaploid range occurred infrequently but their incidence was slightly increased to between 0.16% and 0.9% in diseased lungs and in patients who had received chemotherapeutic drugs. It was concluded that (1) non-cancerous drugs. It was concluded that (1) non-cancerous pulmonary epithelium is diploid, that (2) pulmonary epithelium shows steady-state renewal at all ages and polyploid cells are rare under normal conditions and that (3) the S + G2/M fraction increases up to approximately 10% in reactive proliferative states.     

Flow cytometric evaluation of anti-herpes drugs

A rapid means of screening drugs for toxicity and anti-herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV-1 (multiplicity of infection 1-10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4-n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.     

Stem cell transplantation for leukemias following myelodysplastic syndromes or secondary to cytotoxic therapy

Two main forms of therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) have been recognized. The most frequent type, occurring after treatment with alkylating agents, is characterized by abnormalities of chromosomes 5 and/or 7 and t-MDS/AML following treatment with topoisomerase II inhibitors and is associated with molecular aberrations of MLL (11q23) and AML-1 (21q22). Individuals with certain polymorphisms associated with impaired detoxification of cytotoxic agents have an increased risk of developing MDS or AML after treatment of unrelated cancers. Multidrug chemotherapy is less effective for patients with MDS, or AML following MDS, or t-MDS/AML when compared with primary AML, and results in lower complete remission (CR) rates and lower long-term survival. Patients with good risk cytogenetic features, such as t(15; 17), t(8; 21) and inversion 16 are an exception as their treatment outcome is comparable with primary AML patients. Patients who attain a polyclonal and/or a cytogenetic CR may be candidates for autologous stem cell transplantation. For the remaining patients, the only curative option is allogeneic stem cell transplantation with stem cells from a histocompatible sibling or an alternative donor. Reduced intensity conditioning regimens may be considered for patients older than 50 years or patients with comorbidities. The advice is to treat patients early after diagnosis and preferably before progression as these patients have the highest chance of a favorable outcome.     

Flow cytometric determination of DNA ploidy level in nuclei isolated from paraffin-embedded tissue

Nuclei, isolated from paraffin-embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA-derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffin-embedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices (DI) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P less than 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S-phase nuclei between paraffin-extracted and fresh samples (r = 0.5875, P less than 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM-derived DNA parameters in relation to diagnosis and prognosis of neoplasms.     

Flow cytometric analysis for ploidy level differentiation of 45 hairy vetch accessions

Determining the ploidy of plant germplasm is a necessary step in breeding or genetic studies in species. The purpose of this research was to determine the presence of ploidy level differentiation of hairy vetch (Vicia villosd) germplasm. Flow cytometry and root tip chromosome squashing methods were employed to assess 45 accessions labeled V. villosa available through the USDA germplasm collection. Flow cytometry determined that 43 of the accessions were 2C, one accession was 4C, and one accession was 6C. Analysis of accessions by root tip chromosome counts indicated that all accessions were diploid. The 2C accession contains 14 chromosomes and their chromosomes were approximately one-half and one-third in size as compared to the chromosomes of the 4C and 6C accessions, respectively. The 4C accession was observed to have 16 chromosomes and the 6C accession was observed to have 14 chromosomes. The large-scale differences in DNA amounts were due to chromosomal size variability as opposed to ploidy differences. This revealed the incidence of species misidentification of these two V. villosa accessions to be Vicia pannonica. All the V. villosa accessions were observed to be diploid and have similar DNA amounts. Flow cytometry proved to be useful in the efficient assessment of these accessions.     

Flow cytometric evidence for multiple ploidy levels in the endosperm of some gymnosperm species

 Considered to be haploid tissue, the endosperm of coniferous trees has been extensively used by forest geneticists. Using laser flow cytometry, we show that endosperm ploidy level depends on the systematic position. The Abies, Cedrus and Pinus species tested exhibited uniform haploid endosperm compared to the diploid DNA content of the corresponding embryo. Endosperm of Cupressaceae contained multiple ploidy levels: Cupressus arizonica, Juniperus oxycedrus and Thuja orientalis endosperms exhibited a mixture of haploid–diploid nuclei, while C. atlantica and C. sempervirens endosperms contained six ploidy levels: 1C, 2C, 3C, 4C, 5C and 6C. Physiological and genetic implications of this original feature are discussed. Received: 17 August 1996 / Accepted: 18 October 1996     

Internuclear chromosome distribution of dysplastic megakaryocytes in myelodysplastic syndromes is dependent on the level of ploidy

Megakaryopoiesis is largely disturbed in myelodysplastic syndromes (MDS), and megakaryocytes (MKs) frequently show multinucleation. Here, we investigated dysplastic mono-, bi-, and multinuclear MKs (n = 169) of seven patients with MDS and one patient with myelodysplastic/myeloproliferative neoplasm by sequential multilocus FISH. Analysis of binuclear MKs with a combined DNA content of 4 N (n = 46) indicated a significantly even (symmetric) chromosome distribution between the two separate nuclei (p = 0.0223), which suggests bipolar spindle orientation and symmetric chromosome segregation during the first endomitotic cell cycle. In contrast, multinuclear MKs of higher ploidy (>4 N, n = 108) demonstrated a significantly uneven (asymmetric) chromosome distribution between the separate nuclei (p = 0.0248). Thus, the internuclear chromosomal distribution of dysplastic MKs depends on the level of ploidy. In addition, centrosomal aberrations were not found in dysplastic MKs. Our results indicate that megakaryocytic multinucleation in MDS originates from dysregulated endomitosis, including restoration of karyokinesis.     

Flow cytometric analysis of DNA content for ploidy determination in human solid tumors.

Flow cytometric studies of cellular DNA content were conducted in 26 patients with a variety of neoplasms. Cell dispersal was achieved with pepsin treatment, and a combination of ethidium bromide and mithramycin was used as DNA specific staining procedure. All measurements were conducted with a new sheath flow chamber in a PHYWE ICP 11 pulse cytophotometer. All but one patient with multiple myeloma had unimodal tumor cell DNA distributions. With human granulocytes as reference standard, 24 of 26 tumors were aneuploid; and of these, 23 showed varying degrees of hyperdiploidy. Except for one patient, ploidy abnormalities were stable on repeat examination.     

DNA profiling by arrayCGH in acute myeloid leukemia and myelodysplastic syndromes

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent two distinct but related myeloid haematological neoplasms. At diagnosis, a substantial proportion of cases show cytogenetic and molecular genetic markers whose range of specificity is highly variable. Most specific reciprocal translocations, as t(8;21)(q21;q21) or t(15;17)(q22;q21), have been extensively studied and are currently introduced in clinical diagnosis. Two other major groups remain to be better characterized at the genetic and genomic level: cases with normal karyotype and cases with complex aberrations. Comparative genomic hybridization (CGH) performed on chromosomes was the first approach taken and nearly 300 cases studied by this technique have already been reported. Array based CGH has also been applied to a smaller number of cases. Both types of genomic studies have confirmed that recurrent genomic losses and gains can almost exclusively be found in cases with complex karyotype. In most cases with normal karyotype (as well as in others with single chromosome aberrations as trisomy 8), arrayCGH has been able to unveil small DNA copy number changes whose recurrence is very low. Recently, single- nucleotide-polymorphism based arrays have been used in AML showing that loss of heterozygosity (LOH) is a common feature in normal karyotype leukemia.     

Flow cytometric measurements do not reveal different ploidy levels in Minthostachys (Lamiaceae)

Ten of the 17 species of the taxonomically difficult Andean mint genus Minthostachys (Lamiaceae) were submitted to flow cytometric measurements of nuclear DNA content to test the hypothesis of the occurrence of different ploidy levels within the genus. Nuclear DNA content was found to vary from 1.643 to 1.775 pg, i.e by only ca. 8% between individual accessions, thus providing no evidence for polyploidy in Minthostachys. While these results do not preclude the possibility that the genus contains polyploid species nor the occurrence of heteroploidy with nearly identical nuclear DNA contents, they suggest that polyploidy did not play a major role in its diversification.     

Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34.

BACKGROUND: Flow cytometry is potentially suited to the chemosensitivity analysis of peripheral blood or bone marrow subpopulations in patients with leukaemia and myelodysplastic syndromes. METHODS: The use of the fluorescent dye 7-amino-actinomycin (7AAD) on unfixed cells to measure loss of viability at a range of cytosine arabinoside (ara-C) doses was evaluated. A six-tube flow cytometric assay for measuring the sensitivity to ara-C of CD45/side-scatter-gated or of CD34-positive leukemic blasts with 7AAD was established, using fixed stained normal mononuclear cells as an internal standard for quantitation of viable cells following culture. RESULTS: 7AAD dose response curves for 10 patients with acute myeloblastic leukemia (AML) showed a wide range of sensitivities at 2.5-5 microM araC (3.7-97%, mean 54% of control cell viability at 2.5 microM and 4.1-94.6 %, mean 27% at 5 microM). Parallel assays for ATP bioluminescence agreed reasonably well with the 7AAD method, r(s) = 0.78. The chemosensitivity of CD45/SSC-gated blast cells at 2.5 microM araC showed no consistent relationship with the ungated cell populations, such that CD45/SSC-gated blast sensitivity of seven samples ranged from 86% more to 38% less than that of the total population. Similarly, the chemosensitivities of the CD34-gated subpopulations ranged from 51% more to 78% less than those of the total populations. CONCLUSIONS:These results emphasize the necessity of measuring the chemosensitivity of the population of interest rather than of the sample as a whole in heterogeneous clinical material.     

Biology of myelodysplastic syndromes

Myelodysplastic syndromes (MDS) represent a diverse spectrum of disorders ranging from refractory anemia to a preleukemic state. Peripheral cytopenias, cellular marrow, dysplasias and dysfunctions of myeloid and lymphoid cells constitute hematological hallmarks, and are caused by ineffective hemopoiesis. Investigations of cell cultures and cellular functions indicate that the disease originates in a stem cell pluripotent to all myeloid cells and possibly lymphoid cells as well. The disease commonly runs a chronic indolent course, often terminating in acute leukemia or nonleukemic death, notably infections and/or hemorrhage due to cytopenias and cellular dysfunctions. Clonal expansion or clonal evolution appears to be related to the disease progression with a greater degree of malignancy. However, the initial sequence of events causing damage to stem cells is still undefined.     

Flow cytometric evaluation of leukocyte function in rat whole blood

The aim of this study was to establish a standard flow cytometric method to measure the phagocytic function of and intracellular hydrogen peroxide (H2O2) production by rat leukocytes. Thirty-six adult, male Sprague-Dawley rats were included in this study. Whole-blood specimens from the inferior vena cava were collected in a heparinized tube and ethylenediaminetetraacetic acid (EDTA) anticoagulated tube. The phagocytic function of and intracellular H2O2 generation by leukocytes were measured with FACS Vantage trade mark flow cytometer (Becton Dickinson, San Jose, CA), using fluorescent microspheres and dihydrorhodamine-123 as probes, respectively. Several conditions were optimized in this study, including anticoagulants (heparin and EDTA), fluorescent probes (0.75- and 1.72-microm-diameter microspheres), incubation time, and concentration of the chemicals used in the experiment. Neutrophils, monocytes, and lymphocytes could be clearly defined and separated in whole blood by flow cytometry and tested for phagocytosis and intracellular H2O2 generation without the need for further purification and handling of the cells. Intracellular H2O2 production by and phagocytic function of neutrophils and monocytes were inhibited in EDTA-anticoagulated blood compared with heparin- anticoagulated blood (P < 0.01). Neutrophils showed similar phagocytic function to 0.75- and 1.72-microm microspheres, but monocytes showed weak phagocytic activity to 1.72-microm beads compared with 0.75-microm beads (P < 0.01). In conclusion, a flow cytometric method to measure the phagocytic function of and intracellular H2O2 production by rat leukocytes has been developed. Quantitative flow cytometric analysis of rat leukocyte function is convenient and feasible and provides a reliable and rapid assay to assess phagocytosis and intracellular H2O2 production by rat neutrophils and monocytes.     

The nature of blast cells in myelodysplastic syndromes evolving to acute leukaemia

The blast cells from nine patients with an overt acute leukaemia following a previous myelodysplastic syndrome (MDS) are analyzed with a panel of monoclonal antibodies as well as by morphological and cytochemical criteria. By integrating the results obtained with these three approaches the leukaemia in 6 patients was assessed as myeloid-granulocytic and/or monocytic-, in two as mixed- megakaryoblastic/myeloid- and in one as lymphoid. A good correlation between morphology, cytochemistry and immunological markers was observed in 7 out of the 9 cases. In three cases a noteworthy percentage of J5+ cells was detected. The exceptional finding of lymphoid as well as megakaryocytic and myeloid transformations suggests that the target cell for these leukaemias could be a pluripotent stem cell.