Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

Flow cytometric evaluation of antiviral agents against human herpesvirus 6

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.     

Flow cytometric analysis of head and neck carcinoma DNA index and S-fraction from paraffin-embedded sections: comparison with malignancy grading

Archival, paraffin-embedded, pathology specimens representing pretreatment tissue biopsies from 73 patients with epidermoid carcinoma of the head and neck were analyzed for DNA Index and %S-phase cells by flow cytometry and were scored for quantitative histomorphology. The DNA fluorescence/light scatter size patterns derived from paraffin-embedded specimens were shown to be essentially the same as those from mechanically disaggregated, ethanol-fixed cells obtained from the same tissue specimen. Patterns ranged from lymphocyte-like to highly abnormal DNA Index cytokinetic patterns. The DNA Index values ranged from 0.70 to 3.50 (median 1.42), with an aneuploidy frequency of 63/73 (86%). DNA distribution %S ranged from 4% to 45% (mean 19), with the microscopic malignancy grading showing broad heterogeneity (mean 2.1, range 1.0-3.0, where 1.0-1.7 = well differentiated, 1.8-2.3 = moderately differentiated, 2.4-3.0 = poorly differentiated). Cross-comparison of these data showed that (1) the tumor %S was dependent on DNA Index (higher %S at higher ploidy), (2) low to high malignancy tumors were randomly distributed between diploid/near diploid tumors and high-degree DNA abnormality tumors, and (3) proliferative activity values broadly overlapped between low to high malignancy scored tumors. However, those carcinomas characterized by high DNA Index (greater than or equal to 1.50) and high %S-phase fractions (greater than or equal to 20) had a five fold higher incidence of high-degree malignancy, invasive tumors than diploid/near diploid (%S less than or equal to 19) tumors.     

Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors

DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA-indices measured after staining with propidium iodide and with 4',-6-diamidino-2-phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1.67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA-indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and well-differentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA-indices of greater than 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.     

Flow cytometric sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS‐sorting of the obtained cell suspension that utilizes morphological features, integrin‐β1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.     

Flow cytometric immunophenotyping and comparison with immunocytochemistry in small round cell tumors.

OBJECTIVE: To quantitate different antigens by flow cytometric immunophenotyping (FCI) in small round cell tumors (SRCTs) and to compare the FCI technique with immunocytochemistry (IC). STUDY DESIGN: IC and FCI were performed on 24 consecutive cases of SRCT on fine needle aspiration biopsy material using a panel of antibodies--e.g., cytokeratin (CK), leukocyte common antigen (LCA), desmin, epithelial membrane antigen, neuron-specific enolase, chromogranin, retinoblastoma gene product, neuroblastoma clone (NB84a (NB), vimentin and Mic-2 gene product. IC was done by indirect immunoperoxidase and FCI by indirect immunofluorescence onflow cytometry. RESULTS: In Ewing's sarcoma, with the help of FCI, positive results were obtained in an additional 4 samples in CK, 2 samples in actin and 3 samples in desmin. Similarly, one each sample was additional positive regarding Mic-2 and vimentin by IC. In cases of neuroblastoma with the help of FCI, additional positive results were obtained in one each sample of CK, LCA and NB and two in actin. Combined use of FCI and IC helped to show chromogranin positivity in an additional two cases. Divergent differentiation was noted in four cases of Ewing's sarcoma, one neuroblastoma and two peripheral neuroectodermal tumors. CONCLUSION: FCI technique is sensitive, more objective and quantitative in comparison with manual absorbance-based microscopic detection of enzyme immunohistochemistry products. FCI may determine divergent differentiation in SRCTs.     

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.     

Comparative chromosome analysis of nine squamous cell carcinoma lines from tumors of the head and neck

The chromosomes of nine cell lines from human squamous cell carcinomas of the head and neck were examined. Five of the lines were derived from patients who had received chemotherapy and radiotherapy, one line was derived from a lymph-node metastasis from one of these patients, and three lines were from patients who had not been treated. The cell lines from the untreated patients had lower modal chromosome numbers than those of all but one of the lines from the treated patients. A structural abnormality involving chromosome 1 with breakpoints located in p13, p22, q21, or q32 was found in all cases. The NRAS and SK oncogenes are located in or near some of these segments. In four of the cell lines the nucleolus organizer regions (NORs) were 10-30 times larger than the NORs of the normal human chromosomes. Double minutes (DM) and homogeneously staining regions (HSR) were found in four of the lines.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

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.     

Flow cytometric analysis of adriamycin-perturbed mouse mammary tumors.

The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.     

Flow cytometric analysis of human breast tumors and assessment of in vitro chemosensitivity by clonogenic assays

We report a double-agar clonogenic system adapted to human breast cancer. We optimized the conditions for cell growth and clonogenicity with respect to hormones (insulin, estradiol, progesterone) and components of the extracellular matrix (collagen, laminin and fibronectin). Using our experimental improvements, 67% of the breast tumor samples received were grown successfully. Tests on 21 tumors with three agents: Doxorubicin, Methotrexate and 5-Fluorouracil permit objective discrimination of the in vitro pharmacosensitivity of human breast tumors. Flow cytometric analysis reveal that 64% of the tumors were diploid and 36% were aneuploid. The aneuploid tumors grew better in the double agar layer system used for the clonogenic assay. The diploid tumors were especially rich in estrogen (ER⁺) and progesterone (PR⁺) receptors whereas the aneuploid tumors were mostly estrogen and progesterone receptors negative (ER^–/PR^–). Finally, we noted no difference in drug responsiveness depending on the tumor ploidy and steroid receptor content.Abbreviations DCC dextran coated charcoal - DI DNA index - DXB Doxorubicin - ECM extracellular matrix component - ER estrogen receptors - FCM flow cytometry - 5-FU 5-Fluorouracil - HTSCA human tumor stem cell assay - MTX Methotrexate - PBC primary breast carcinoma - PI proliferative index - PR progesterone receptors - SPF S phase fraction     

Flow cytometric discrimination of human semen cells

An improved method for differential staining and high resolution flow cytometric measurement of human semen cells is presented. Using a mild pretreatment with citric acid/detergent and staining with DAPI, the new procedure provides excellent preservation and good discrimination of all cells which are present in normal and pathological semen samples.     

Flow cytometric cell division tracking using nuclei

BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle.     

Flow cytometric immunophenotypic characteristics of plasma cell leukemia

The aim of this prospective study was to define the flow cytometric characteristics of simultaneously investigated bone marrow and peripheral blood plasma cells antigens expression in 36 plasma cell leukemia (PCL) patients. The immunophenotypic profile of plasma cells was determined with a panel of monoclonal antibodies. The antigen expression intensity was calculated as relative fluorescence intensity (RFI). Bone marrow plasma cells showed expression of particular antigens in the following proportion of cases: CD49d 100%, CD29 94%, CD54 93%, CD44 83%, CD56 60%, CD18 26%, CD11b 29%, CD11a 19%, CD117 27%, CD71 30%, CD126 100% and CD19 0%, while the expression of those antigens on peripheral blood plasma cells was present in the following percentage of patients: CD49d 100%, CD29 96%, CD54 93%, CD44 95%, CD56 56%, CD18 50%, CD11b 53%, CD11a 29%, CD117 26%, CD71 28%, CD126 100% and CD19 0%. The expression of CD54 was significantly higher than that of adhesion molecules belonging to the integrin b2 family: CD11a, CD18 and CD11b, on both bone marrow and peripheral blood cells (p < 0.01). Expression of CD18, CD11a and CD11b was differential between two cell compartments: lower on bone marrow and higher on peripheral blood cells. We found that plasma cells in the bone marrow of patients with plasma cell leukaemia showed significantly greater granularity and size than those in the peripheral blood (p = 0.0001 and p = 0.04, respectively). However, no differences in cell size or granularity were revealed between bone marrow plasma cells from patients with PCL and multiple myeloma. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 (LFA-1) or CD56 may explain hematogenic dissemination characterizing PCL. The following pattern of adhesion molecule expression according to the proportion of plasma cells expressing a given antigen in peripheral blood and bone marrow and arranged in diminishing order may be established: CD49d > CD44 > CD54 > CD29 > CD56 > CD18 > CD11b > CD11a. Immuno-phenotyping of plasma cells in PCL, as in multiple myeloma, might be useful in detecting minimal residual disease in cases with aberrant antigen expression and for selecting therapeutic agents towards specific membrane targets.     

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