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.     

Nile red simultaneous staining of intracellular lipids and membrane network in human muscle cultures

We have applied a new fluorescent probe, Nile red, on normal and pathological human muscle derived cultures and compared the results with corresponding human muscle sections. In normal human muscle cultures, Nile red strain has proved useful for visualization of both intracellular lipids and membrane network. Similar patterns have been observed in muscle cultures derived from lipid storage and mitochondrial myopathies. Moreover, abnormalities in pathological muscle cultures could be revealed by establishing more advanced culture systems.     

Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis.

A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.     

Flow cytometric determination of carbohydrates in human erythrocytes

Carbohydrate components known from biochemical analysis to be present in peripheral normal human erythrocytes so far could not be detected cytochemically. By periodic acid oxidation followed by Schiff pararosaniline (SO2) staining, however, a specific fluorescent signal can be obtained, strong enough to allow measurement by flow cytometry. Dimethylsuberimidate fixation results in low autofluorescence and low staining of unoxidized cells. By treating erythrocyte ghosts similarly, it is found that about 20% of the signal is present in the membrane, most probably due to glycophorins. The main signal resides in the matrix of the fixed erythrocyte and may be due to traces of glycogen and to the glycosylation of proteins, especially hemoglobin.     

Flow cytometric determination of carbohydrates in human erythrocytes

Summary Carbohydrate components known from biochemical analysis to be present in peripheral normal human erythrocytes so far could not be detected cytochemically. By periodic acid oxidation followed by Schiff pararosaniline (SO₂ sstaining, however, a specific fluorescent signal can be obtained, strong enough to allow measurement by flow cytometry. Dimethylsuberimidate fixation results in low autofluorescence and low staining of unoxidized cells. By treating erythrocyte ghosts similarly, it is found that about 20% of the signal is present in the membrane, most probably due to glycophorins. The main signal resides in the matrix of the fixed erythrocyte and may be due to traces of glycogen and to the glycosylation of proteins, especially hemoglobin.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.     

An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat anti-BrdUrd monoclonal antibody from Sera-lab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti-BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas-Red conjugated goat antimouse.     

Standardized method for the determination of human erythrocyte membrane adenosine triphosphatases

A standardized assay is described for the simultaneous determination of Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and Ca²⁺-ATPase in human erythrocyte (RBC) membrane preparations. Membranes were prepared by lysis of RBCs in hypotonic buffer, and ATPase activity assays were based on the measurement of ³²P-labeled inorganic phosphate release from [γ-³²P]ATP. The results obtained by this method were compared with those of colorimetric determination of inorganic phosphate and of ATP hydrolysis with high-performance liquid chromatography. The activity of the three enzymes was measured in RBC membranes obtained from 30 normal subjects. Repeated sampling of individuals over a 4-month period showed that interindividual differences were substantial, but that in each individual enzymatic activity was maintained in a narrow range by presumed homeostatic mechanisms. Statistical analysis of the data showed no interdependence of the three enzymes; a correlation of activity with age, sex, or phase of the menstrual cycle was not apparent. The values obtained for the Ca²⁺-ATPase did not follow a normal distribution, and it is suggested that this enzyme has two phenotypic variants. The described method is sufficiently precise and economical to be recommended for adoption as standard procedure in clinical research.     

Flow cytometric double labeling technique for screening of multidrug resistance.

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.     

A technique for simultaneous antikinetochore immunofluorescence staining and Q-banding in chromosomes from human lymphocytes

Antikinetochore immunofluorescence staining has been used in several studies to determine whether a second kinetochore is present, active, or both, in multicentric chromosomes. All of these studies have used tissue culture cells, and contended with the problem of obtaining well spread, banded metaphase chromosomes without affecting the kinetochore staining. We have adapted hypotonic, centrifugation and chromosome staining techniques to obtain simultaneous Q-banding and bright kinetochore staining of chromosomes from human lymphocytes.     

Propidium iodide staining of cytoautoradiographic preparations for the simultaneous determination of DNA content and grain count

Summary A new method is described for staining cell nuclei with propidium iodide in preparations that have been processed for autoradiography. The permeability of the stripping film to the dye molecule has been studied, as have the staining conditions, in order to optimize the stoichiometry of the DNA-dye interaction. A procedure has also been set up to allow the simultaneous measurement of DNA content and grain count on the same cell.     

Flow cytometric identification of microorganisms by dual staining with FITC and PI.

The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.     

Flow cytometric analysis of peripheral blood erythrocyte chimerism in alpha-thalassemic mice.

A rapid and reliable method for longitudinal studies on the degree of red cell chimerism following bone marrow transplantation of alpha-thalassemic recipient mice is presented. Blood obtained by tail clipping from transplanted mice was analyzed by measuring forward light scatter (FLS) distribution of red cells using a flow cytometer. Amplification and threshold of FLS were specifically adjusted. For flow cytometric analysis, the red cells needed to be suspended in hypotonic saline (103 mmol/l NaCl). Osmotic fragility testing showed that lysis of erythrocytes did not significantly influence the measurements. Flow cytometric measurement allowed for a rapid determination of the degree of red cell chimerism.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).