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Biomedical subjects

M R Melamed

Publications and source records attributed to M R Melamed.

At least 19 recordsLinked to original sources
PubMed1979

Subpopulations of human peripheral blood cells: analysis of granulocytic progenitor cells by flow cytometry and immunologic surface markers.

Normal human peripheral blood cells were separated into different populations based upon isopycnic sedimentation, E rosetting, and EAC rosetting. Each population was characterized according to morphology, surface markers, granulocytic colony formation in semi-solid media, and stainable RNA content by acridine orange (AO) flow cytometry. These techniques enrich for a population of cells that is characterized by a lymphoid morphology, a high granulocytic-macrophage progenitor cell cloning efficiency, a lack of surface markers, and a high stainable RNA content not found in the other two populations of peripheral blood lymphocytes (T cells and B cells). The stainable RNA content serves as a new metabolic marker for the population of cells in which the preponderance of granulocytic progenitor cells reside.

B-Lymphocytes↗
PubMed1979

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry.

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.

Cell Count↗
PubMed1979

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxy-uridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes.

Cell Cycle↗
PubMed1979

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.

Cytological Techniques↗
PubMed1979

Quantitation of lymphocyte response to PHA by flow cytofluorometry. III. Heterogeneity of induction period.

Early events in phytohaemagglutinin (PHA) stimulation of mouse splenocytes have been quantitated by using flow cytometry and supravital staining with acridine orange (AO). Increasing percentages of single cells with increased metachromatic (red) AO staining were demonstrated in cultures stimulated by PHA for up to 24 hr. These differences in staining could be eliminated by fixation with 1:1 ethanol/acetone before staining. Stimulated cells showed an increase in nonspecific esterase activity as measured by flow cytometry after supravital staining with fluorescein diacetate (FDA). The data reported show a heterogeneity in the per cell response of mouse splenocytes to PHA. The relationship between these data and the mechanism of mitogen stimulation is discussed.

Acridine Orange↗
PubMed1979

Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry.

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.

Cell Cycle↗
PubMed1979

The coexistence of acute myeloblastic leukemia and diffuse histiocytic lymphoma in the same patient as demonstrated by multiparameter analysis.

Measurement of cellular DNA content by flow cytometry demonstrated presence of two distinct aneuploid neoplasms in a patient who developed acute myeloblastic leukemia (AML) 4 mo after diagnosis of a diffuse histiocytic lymphoma (DHL). A lymph node aspirate contained peroxidase-negative, "null," hyperdiploid (2.6C) DHL cells, while the bone marrow (BM) contained 84% primitive peroxidase-positive tetraploid AML cells (4.0C). Minor populations of hyperdiploid HDL and normal diploid cells could be detected by flow-cytometry in the BM, and all three populations were also seen in the peripheral blood.

Adult↗
PubMed1979

Regional lymph node reactivity in explanted bladder cancer of mice as measured by flow cytometry.

The reactivity of lymphocytes in lymph nodes draining the site of a transplantable experimental bladder tumor (MBT2 in C3H/HeJ mice) has been measured in a multiparameter flow cytometry system. Acridine orange was used as a nucleic acid probe. This dye intercalates in helical DNA, emitting green (530 nm) fluorescence upon exposure to blue light; it stacks to single-stranded RNA, emitting red (640 nm) fluorescence. The relative magnitude of the increase of lymphocyte DNA and RNA has been evaluated simultaneously in tumor-draining nodes, in nondraining nodes of the same animal, and in untreated control animals. Stimulation of the regional node lymphocytes could be observed after 20 days but not after 10 days. It was uniformly high at 35 days. The transcriptive response (increased proportion of lymphocytes with high RNA) was more pronounced than the proliferative (increased proportion of lymphocytes with more than diploid DNA). The histological changes in the stimulated nodes resembled closely those described by others in human tumor-draining nodes. The described method has the advantage of being simple, rapid, and able to measure a representative part of the whole-cell population.

Animals↗
PubMed1979

Lymph node reactivity to experimental bladder tumor in preimmunized animals as measured by two parameter flow cytometry.

Lymph node lymphocyte reaction to an explanted, transplantable mouse bladder tumor (MBT-2) was investigated by flow cytometry in animals previously immunized with irradiated tumor cells. Nodal lymphocytes in representative samples from four different lymph node sites were differentially stained for DNA and RNA with the fluorescent dye acridine orange; cell proliferation and the increase in RNA content were measured. Immunization abrogated tumor growth; one immunization reduced tumor take to 25 per cent of the animals, and two immunizations to 14 per cent. Lymphocyte reactivity to the tumor was reflected both by an increase of DNA synthesizing cells and by diploid cells with high RNA. The latter response was more pronounced and thus the more sensitive parameter for measuring immunologic lymph node reactivity. The juxtatumoral node displayed the most pronounced reactivity, but all node sites showed some degree of reaction.

Animals↗
PubMed1978

RNA content in human lymphocyte subpopulations.

Human peripheral blood lymphocytes are stained with the metachromatic dye acridine orange and the fluorescence of individual cells is measured by flow cytometry. The relative content of stainable RNA per cell is estimated by comparison with RNase-treated cells. Non-T and T lymphocytes have different mean quantities of RNA per cell, and these classes exhibit different distributions of RNA content. Non-T cells have a unimodal distribution with a sharp peak and exponential distribution towards higher RNA values. T cells have a bimodal distribution with two separate peaks. When T cells having receptors for IgG (Tgamma cells) and IgM (Tmu cells) are separated, each of these cell populations displays a unimodal distribution. Of these three lymphocyte subpopulations, Tgamma cells have the lowest content of RNA per cell. Non-T cells have slightly higher RNA content than Tgamma, and Tmu cells have twice as much RNA as Tgamma cells. The RNA content, which surely relates to the different functions of these lymphocyte subpopulations, may also be a useful marker for rapidly distinguishing the lymphocyte subpopulations.

Acridines↗
PubMed1978

Radiation-induced soft-tissue and bone sarcoma.

From the records of Memorial Hospital of the past 50 years, 47 cases with an established diagnosis of radiation-induced sarcoma were identified and divided into two groups: the first included 20 cases of soft-tissue sarcoma arising from irradiated tissues, and the second comprised 27 cases of bone sarcoma arising from normal bones in the irradiated field. Medians for the latent periods from irradiation to diagnosis of bone and soft-tissue sarcoma were 11 and 12, years, respectively. In bone sarcomas, the latent period was longer after larger radiation doses and children appeared to be more susceptible to cancer induction than adults. Criteria for establishing the diagnosis of radiation-induced sarcoma and the magnitude of the risk of bone sarcoma are discussed.

Adolescent↗
PubMed1978

Accuracy of chest film screening by technologists in the New York early lung cancer detection program.

A study of the feasibility of using specially trained radiologic technologists to screen chest radiographs was undertaken as part of an early lung cancer detection program. In their initial examination, 8,000 men had posteroanterior and lateral chest films which were prepared and evaluated by two specially trained technologists prior to interpretation by a radiologist. The technologists' accuracy in screening was subsequently assessed by comparison with the radiologist's interpretation and with clinical follow-up information. There were differences in the level of suspicion of the two technologists, but both were effective in selecting a subset of the screened population that contained the men with radiologically identifiable lung cancer.

Humans↗
PubMed1977

Nucleic acid content and cell cycle distribution of five human bladder cell lines analysed by flow cytofluorometry.

Five human bladder cell lines, four derived from tumor tissue and the fifth originating from presumed benign transitional epithelium, were examined by flow cytofluorometry to estimate the DNA and RNA content per cell during exponential and stationary phases of growth. A new staining technique was employed using acridine orange to differentially stain DNA and RNA in unfixed cells made permeable to the dye and other reagents by treatment with detergent at low ph. Stemline chromosome numbers for each cell line correlated well with relative DNA content of the G1 population as measured by this technique. In addition, the simultaneous measurements of DNA and RNA per cell yielded cell cycle distributions for each cell line. The ratio of stainable RNA/DNA was lower for all cell lines derived from bladder tumors as compared to the presumed normal cell line, indicating high nuclear/cytoplasmic ratios for the former.

Cell Division↗