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DNA content of human squamous cell carcinoma cell lines. Analysis by flow cytometry and chromosome enumeration.

Fifteen squamous cell carcinoma cell lines derived from nine patients were examined for DNA content by flow cytometry and chromosome counts. Using human peripheral blood leukocytes and nucleated trout and chicken red blood cells as standards, the DNA indexes of the squamous cell carcinoma cell lines were found to range from 1.1 to 3.3. The DNA content was a stable characteristic of individual cell lines in multiple passages over a seven-month period. Although flow cytometry could detect abnormal DNA content even in diploid tumor lines, the chromosome number correlated well with the DNA content by flow cytometry. In cases in which more than one cell line was established from the same patient, the individual cell lines were found to differ in their DNA content. The cell lines established from metastatic or recurrent tumors usually had a lower DNA content and chromosome number and exhibited a more aggressive in vitro growth pattern than the primary tumor or earlier recurrence. We hypothesize that "streamlined" and aggressive cell populations may evolve in vivo from more slowly growing hyperploid precursor tumor cell populations when in the course of random loss of DNA or chromosomes those that confer no growth advantage are lost, while those that do confer growth advantage are retained.

Animals↗

Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture.

Murine monoclonal antibodies (MAbs) against herpes simplex virus type 1 and 2 (HSV-1 and -2, respectively) nuclear antigens were used to identify cells infected with HSV-1 or -2 by indirect immunofluorescence in conjunction with flow cytometry after virus amplification of MRC-5 cell monolayers. The results indicate that MAbs Q1, Q2, and H-640 detect HSV-1- and HSV-2-infected cells, MAb SD-1 detects HSV-2- but not HSV-1-infected cells, and MAb 58-S detects HSV-1- but not HSV-2-infected cells. MAb Q1, which detects HSV-1- as well as HSV-2-infected cells, was used to detect HSV-infected cells after inoculation and overnight (16- to 20-h) incubation of MRC-5 monolayers with clinical samples suspected of containing HSV. In comparing the efficiency of flow cytometry with cytopathic effect (CPE) in tissue culture for detecting HSV in clinical samples, HSV was detected by flow cytometry in 77% of the cases where HSV was detected by CPE in tissue culture. In many cases, flow cytometry detected HSV from 1 to 3 days before HSV was detected by CPE.

Antibodies, Monoclonal↗

[Flow cytometry of thyroid tumors. Study of fresh tissue in 50 patients].

OBJECTIVES: A measurement of cell DNA content would be highly useful in determining the malignant nature of thyroid tumours in cases without distinctive features such as metastases, capsule invasion or emboli. Abnormal cell ploidy can be recognized with flow cytometry, but it is not known whether such results have diagnostic value. We therefore compared--in a double blind prospective study--the results of flow cytometry and pathologic diagnosis in fresh tumoural and non-tumoural thyroid cells. METHODS: Fifty unselected cold thyroid nodules were obtained from 50 consecutive patients (40 women, 10 men; age 18-80 years; mean 46) who underwent surgery within a 6 month period. Surrounding non-tumoural tissue was also obtained in 46 of them. Cell ploidy and the percentage of cells in each cell phase was determined with flow cytometry for both tumoural and nontumoural tissues. Two pathologists, unaware of the flow cytometric results, independently established the histologic diagnosis according to the WHO classification. RESULTS: The pathologic diagnosis was carcinoma in 7 cases (papillary carcinoma 6, vesicular carcinoma 1) and benign adenomas in 43 (29 macrovesicular, 11 microvesicular, 3 oncocytal). All the non-tumoural tissue samples were diploid. All 7 carcinomas were diploid and 10 of the 43 benign adenomas were aneuploid (4 near-diploid, 3 hyperploid, 1 near-tetraploid, 2 multiploid). The mean proliferation index was increased in 5 diploid tumours. CONCLUSION: These findings confirm that cell ploidy measured by flow cytometry is of no diagnostic value in the thyroid gland. It was also revealed that aneuploidy in adenomas may be related to tissue rearrangements of undetermined prognostic significance.

Adenoma↗

[Flow cytometry in exfoliative cytology of malignant tumor].

Exfoliative cells from 170 cases of malignant tumor and 70 cases of normal tissue were studied by flow cytometry. The results showed that 155/170 (91.2%) cases were positive and 15/170 (8.8%) were false-negative; 4/70 (6.0%) cases were false-positive. The authors consider that flow cytometry has achieved a diagnostic accuracy comparable to the conventional cytology. Flow cytometry is a useful supplementary method in the diagnosis of tumors.

Aneuploidy↗

[HLA B27 phenotyping using flow cytometry].

Results of typing for HLA B27 antigens of 106 patients in whole blood with a FITC monoclonal anti HLA B27 antibody and analysis by flow cytometry are compared to the results obtained by the classical microlymphocytoxicity test. By flow cytometry analysis, there are not false negative result, but, because of cross reactions of the used monoclonal antibody with other specificities of the CREG B7 group, false positive result may be encountered. The flow cytometry analysis for typing HLA B27 antigens using a monoclonal antibody can be a good screening technique: so it's rapid and moreover the negative results can be accepted. The positive results must be confirmed.

Antibodies, Monoclonal↗

Sensitivity of hemozoin detection by automated flow cytometry in non- and semi-immune malaria patients.

BACKGROUND: Cell-Dyn automated blood cell analyzers use laser flow cytometry technology, allowing detection of malaria pigment (hemozoin) in monocytes. We evaluated the value of such an instrument to diagnose malaria in febrile travelers returning to Berlin, Germany, the relation between the instrument's performance and the patients' immune status, and the capacity to increase its sensitivity. METHODS: Malaria diagnosis was routinely established by thick-film microscopy. The patients' immune status was determined by an indirect fluorescent antibody test. Hemozoin detection was performed with a Cell-Dyn 3000. To assess the capacity for sensitivity increase, the relative frequencies of pigment-containing monocytes were determined for a subgroup of patients with the Cell-Dyn 3000 in comparison with a flow cytometer specifically adapted to rare-event analysis. RESULTS: Of 403 patients screened microscopically, 107 had malaria. Overall sensitivity with the Cell-Dyn 3000 reached 48.6% (73.7% in semi-immune and 28.6% in nonimmune individuals; P < 0.0001). Specificity was 96.2%. The detection limit was at a relative concentration of 2 x 10(-4) pigment-containing monocytes (PCMs). By employing rare-event flow cytometry, the detection limit decreased to 3.25 x 10(-5), thus yielding a considerably increased sensitivity for the subgroup studied. CONCLUSIONS: The correlation between immune status and relative concentration of PCMs explains the failure of the routine instrument for nonimmune patients and its significantly higher sensitivity for semi-immune individuals. The technique can be significantly improved by rare-event flow cytometry.

Adolescent↗

Cytoplasmic IgM in leukemic B cells by flow cytometry.

The presence of intracellular cytoplasmic immunoglobulin M (IgM) in leukemic cells from patients with acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) was investigated by flow cytometry. The objective of the study was to develop a reproducible flow cytometric method. A Burkitt's lymphoma-derived B-cell line, Daudi, and a pre-B ALL, Nalm-6, served as prototypes. Normal B cells and cells from patients with chronic myelogenous leukemia (CML) were used as negative controls. Cytoplasmic mu was expressed in 77.3 +/- 7.5% (n = 10) of Nalm-6 cells. CALLA+ ALL and CML cells lacked cytoplasmic mu. The surface-membrane immunoglobulin on the viable B cells was blocked with purified goat anti-human IgM. Subsequently, the B cells were fixed in cold absolute methanol and stained with a fluorescein-conjugated goat anti-human IgM to demonstrate cytoplasmic IgM. After the surface-membrane IgM was blocked, normal B cells had no cytoplasmic IgM (0.3-0.5% positive cells) detectable by flow cytometry. However, the peripheral blood lymphocytes from five patients with CLL and the Daudi cells contained cytoplasmic IgM, ranging from 7.8 to 76.7% and 45.7 to 89.3%, respectively. We conclude that cytoplasmic mu in Nalm-6, CLL, and Daudi cells can be easily and rapidly demonstrated by flow cytometry.

Animals↗

Correlation between endoscopy, histopathology, and DNA flow cytometry in patients with gastric dyspepsia.

BACKGROUND/AIMS: Gastric cancer has a poor prognosis, this is partly due to the advanced stage in which the tumor is diagnosed. The objective of this study is to elucidate the clinical significance of DNA flow cytometry and study its impact on monitoring the progression of gastric precancerous lesions in patients with gastric dyspepsia, and to correlate between endoscopic and histopathological findings with results of DNA flow cytometry. MATERIAL AND METHODS: A total of 92 cases underwent upper gastrointestinal endoscopy, 69 males with mean age 44.0 years and 23 females with mean age 38.7 years. Based on the endoscopic appearance, patients under study were classified into: 15 cases with endoscopic normal mucosa (EN), 26 cases with endoscopic gastritis (EG), 43 cases with duodenal ulcer (DU), and 8 cases with gastric ulcer (GU). Two antral biopsies were taken for histopathology and DNA flow cytometry. RESULTS: Chronic gastritis (CG) was present in 12 (80%) of EN cases. In DU patients, CG was present in 42 (97.7%) of cases, and it was associated with intestinal metaplasia (IM) in 11 (25.6%), and with dysplasia in 9 (20.9%) of these cases. While in GU patients, CG was present in all cases. Two (13.3%) of endoscopic normal cases revealed DNA aneuploidy in specimens with CG. The incidence of aneuploidy increases as the endoscopic findings changes from EG (15.4%), DU (16.3%) to GU (37.5%), and as the histopathological changes progresses from chronic atrophic gastritis (CAG) (18.2%), IM (21.7%) to dysplasia (33.3%). CONCLUSION: DNA aneuploidy is a useful marker for recognizing the presence of abnormal cells in epithelial lesions of the stomach, and for monitoring the progression of gastric lesions. Patients with gastric dyspepsia should not only be subjected to endoscopy but also to biopsy and DNA flow cytometry to allow the early detection of malignant transformations in gastric precancerous lesions.

Adult↗

Cloning of DNA libraries from mouse Y chromosomes purified by flow cytometry.

To purify mouse Y chromosomes by flow cytometry, a male cell line containing the Robertsonian translocation Rb(9.19)163H has been established by SV40 transformation. Flow karyotypes obtained from these cells exhibit a well-isolated peak of fluorescence corresponding to the single Y chromosome, clearly distinct from that of chromosome 19. From this peak, 650,000 chromosomes were sorted, and two restriction fragment libraries were constructed from the DNA of the sorted chromosomes. The characterization of several Y-specific fragments has shown that the Y DNA was enriched at least 36-fold. Furthermore, given that there are likely homologies between the X and Y chromosomes, we can assume that this calculated value of the purification factor is an underestimation and that the Y DNA was more highly purified by flow sorting.

Animals↗

Transitional cell carcinoma of the renal pelvis and ureter in Taiwan. DNA analysis by flow cytometry.

BACKGROUND: The incidence of upper urinary tract tumors is relatively high in southern Taiwan. DNA analysis by means of flow cytometry is not well investigated with regard to tumors of the upper urinary tract and the differences in DNA ploidy between transitional cell carcinomas in endemic and nonendemic areas. METHODS: A retrospective nuclear DNA ploidy analysis by flow cytometry comprised 41 formaldehyde solution-fixed, paraffin-embedded tissue specimens of transitional cell carcinoma of the renal pelvis and ureter. The preparation of nuclear suspensions from paraffin-embedded tissue blocks and staining were modified by means of the techniques of Hedley and Vindeløv. RESULTS: There was no statistical correlation between DNA ploidy, histologic grade, and pathologic stage, however, 82% of the DNA nondiploid tumors showed tumor progression. In contrast, only 46% of the DNA diploid tumors revealed tumor progression. Among Grade 2 tumors, 85% of the DNA nondiploid tumors showed postoperative tumor progression, whereas only 31% of the DNA diploid tumors showed tumor progression. Seventy-nine percent of the nondiploid patterns were present in patients native to the Pa Chang Valley, where the so-called "blackfoot disease" and urothelium tumor are endemic, whereas only 22% appeared in patients living in other areas. CONCLUSIONS: DNA flow cytometry can identify a group of patients with poor outcome unpredictable by pathologic examination, and is an important tool in research into the pathogenesis of cancer.

Carcinoma, Transitional Cell↗

Age-related changes in the proliferative kinetics of phytohemagglutinin-stimulated lymphocytes. Analysis by uptake of tritiated precursors of DNA, RNA and proteins, and by flow cytometry.

We have examined the age-related changes in the kinetics of lymphocyte proliferative responses to phytohemagglutinin by flow cytometry and by the uptake of tritiated thymidine, uridine and leucine. The lymphocyte suspensions used in these experiments were obtained by umbilical cord blood samples of full-term normal newborns and from the peripheral blood of young (under 30-year-old) or aged (more than 70-year-old) healthy donors. The results indicate that the cord blood lymphocytes were activated by phytohemagglutinin to incorporate tritiated precursors of proteins, RNA and DNA at a more rapid rate than the lymphocytes from young or old donors in the first 3 days of in vitro culture. Flow cytometry confirmed higher percentages of activated cycling cells in umbilical cord blood after 24 h of culture. The lymphocytes from old donors incorporated significantly lower amounts of tritiated precursors of DNA and RNA than lymphocytes from young donors at the third day of culture. The uptake of tritiated leucine by lymphocytes from old donors was significantly reduced up to the sixth day of culture. On the contrary, at the eight day of culture, lymphocytes from old donors incorporated significantly higher amounts of labeled DNA, RNA and protein precursors. In agreement with these findings, flow cytometry demonstrated a trend towards higher percentages of cycling cells in the lymphocyte cultures from old donors after 6 and 8 days. These results indicate an age-related decline in the rate of lymphocyte blastogenesis after in vitro PHA stimulation and suggest that after a delayed PHA-induced activation, the lymphocytes from aged donors have a near-normal ability to proliferate.

Adult↗

Quantitative measurement of DNA content in gastric carcinoma; flow cytometry and video image analysis.

The DNA content of 48 gastric carcinomas from archival material was analysed by static and flow cytometry. By image analysis 81.3% of the tumours were aneuploid and ploidy was related to stage (P = 0.024) and lymph node metastasis. A trend for better survival (> 12 months) was observed in patients with diploid tumours (P = 0.058). The mean 5c exceeding rate (5cER) was significantly related to tumour stage (P < 0.05) and patient's survival (P = 0.018). In contrast, by flow cytometry only 43.7% of these tumours were aneuploid and these were more often associated with lymph node metastasis (59.3%) but no relationship was observed with any other parameters or patient's survival. In this series, image analysis appears to be more sensitive than flow cytometry in detecting small aneuploid populations. It may give additional prognostic information. It is, however, a time-consuming technique.

Aneuploidy↗

Rapid G+ count and subpopulation assessment of the intestinal bacteria in Apodemus sylvaticus and Mus musculus by flow cytometry.

We report a novel application of calcein-acetomethyl ester in flow cytometry for rapid estimation of the number of G+-bacteria in rodent feces (Apodemus sylvaticus and Mus sp.f. muridae). We also use the combined application of flow cytometry and Syto-13 or Sypro Orange staining to count rapidly the total bacterial population and to describe bacterial subpopulations in the intestine.

Animals↗

Microbial determinations by flow cytometry.

Recent improvements in the optics and electronics of flow cytometry systems, as well as in staining techniques, permit the assay of such minute cellular constituents as the DNA and protein contents of micro-organisms. To assess the usefulness of this technique, DNA and protein content distributions were determined in Escherichia coli, Lactobacillus brevis, Lactobacillus casei, Chlorella kessleri 8k, Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe and Euglena gracilis. Investigations of the DNA content distributions of polyploid strains of Saccharomyces cerevisiae indicated that the method can be used to determine ploidy. The rapidity of flow cytometry measurements allows accurate determinations in large populations.

Bacteria↗

Prognostic value of DNA flow cytometry in the locally recurrent, conservatively treated breast cancer patient.

PURPOSE: This study attempted to determine the prognostic value of DNA flow cytometry in the treatment of patients with locally recurrent, conservatively treated breast cancer. METHODS AND MATERIALS: Of 433 patients with clinical stage I and II breast cancer treated with conservative surgery and radiotherapy at Yale-New Haven Hospital before January 1985, 50 patients experienced an ipsilateral breast relapse as a first site of treatment failure. Using standard flow-cytometric techniques, DNA ploidy, DNA index, and S-phase fraction (SPF) were measured for 38 of the 50 (76%) paraffin-embedded specimens available for analysis. RESULTS: At a median postrecurrence follow-up of 5.8 years, the 5-year and disease-free survival rates following ipsilateral breast treatment failure were 48% and 54%, respectively. Sixty-three percent of the recurrent tumors were DNA diploid and 37% were aneuploid. Both DNA ploidy and SPF were statistically significant prognostic indicators for 5-year survival and disease-free survival after local recurrence. The 5-year survival rate of the DNA diploid population was 64%, compared with 15% in the aneuploid population (P < .02). Patients with low SPF (< 12%) experienced an 83% 5-year survival rate, compared with a 24% 5-year survival rate in patients with high SPF (> or = 12%) (P < .03). Ploidy and SPF were combined to define the categories of favorable (diploid, low SPF) and unfavorable (diploid, high SPF or any aneuploid subgroups). Patients in the favorable category experienced an 89% 5-year postrecurrence survival rate and a 100% disease-free survival rate, whereas patients in the unfavorable category had a 24% 5-year survival rate and a 32% disease-free survival rate (P < .01). The flow cytometry as a factor correlated with other clinical parameters previously shown to be of prognostic significance in this patient population. In a multivariate analysis, flow cytometry was a statistically significant and independent prognostic factor for disease-free survival following local recurrence. CONCLUSIONS: DNA ploidy and SPF as measured by currently available flow-cytometric techniques show promise as a tool in determining prognosis for the patient with locally recurrent breast cancer. Implications of these findings with respect to issues of adjuvant systemic therapy at the time of local recurrence are discussed.

Adult↗

Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals.

The effectiveness of toxicology biomonitoring programs could be improved by the addition of sensitive biomarkers. In this study the cell viability and sensitivity of phagocytic function of phagocytes from bivalves (Mya arenaria) to selected heavy metals were measured by flow cytometry, a novel approach. Hemocytes (phagocytes) collected from bivalves by puncture of the posterior adductor muscle were incubated in vitro for 18 h in hemolymph containing 10(-9)-10(-3)M of cadmium chloride, zinc chloride, mercuric chloride, methylmercury chloride or silver nitrate, before determining their capacity to phagocytose fluorescent latex beads by flow cytometry. Heterogeneity of the hemocyte cell population was determined by forward scatter (FSC) and side scatter (SSC) cytometric profile which showed two distinct cell populations. At low doses (10(-9), 10(-8) M), all the metal compounds studied stimulated phagocytic activity except silver nitrate. At higher levels of exposure (10(-6), 10(7) M), all metals caused a significant concentration-related decrease in hemocyte phagocytosis activity. From the concentration of each metal inducing 50% suppression (IC50) of the phagocytic activity, the immunotoxic potential of metals with respect to phagocytic function can be ranked in the following increasing order: ZnCl2 < CdCl2 < AgNO3 < HgCl2 < CH3HgCl. Parallel analysis of hemocyte viability showed that suppression of phagocytosis by heavy metals was not solely related to a decreased cell viability. These results reveal the high but different degree of sensitivity of the phagocytosis activity of bivalves with respect to heavy metals, as measured by flow cytometry, and demonstrate that flow cytometry is a potentially useful tool in ecotoxicological monitoring.

Animals↗

A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture.

The increasing frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections in hospital and community settings highlights the need for effective anti-MRSA agents that will not contribute to the growing problem of antibiotic resistance. Lactic acid bacteria (LAB) are known to exclude various pathogens through multiple mechanisms. In vitro models studying interactions of pathogens and LAB in mixed cultures use selective agar plates to quantify changes in target populations. We applied commercially available S. aureus-specific polyclonal antibodies conjugated with fluorescein isothiocyanate (FITC) for this purpose, producing a bright green signal that clearly differentiates S. aureus from LAB species when mixed cultures are analyzed by flow cytometry and fluorescent microscopy. Flow cytometry of mixed cultures revealed a much larger population of MRSA cells than was detectable using selective agar plates. To our knowledge, this is the first time immunofluorescent flow cytometry has been applied to the study of competitive exclusion in mixed bacterial populations over time.

Animals↗

Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy.

Artificial unilateral cryptorchidism was performed in golden hamsters which were then held for different periods of time. The non-operated side was used as a control. At various times from 4 to 15 days, hamsters were killed, testes were removed and weighed, single cell suspensions were prepared for flow cytometry analysis and seminiferous tubules were fixed for confocal microscopy. Using DNA staining by propidium iodide or acridine orange followed by flow cytometry analysis, a marked decrease in the haploid condensed cell fraction was detected at the beginning stages of experimental cryptorchidism. In correlation with flow cytometry results, spermiogenic arrest at stages IX and X of seminiferous epithelium was detected in these animals by confocal microscopy and there were no mature forms of haploid cells in the cryptorchid testis. In the testis with more severe damage, there were almost no haploid cells in the seminiferous tubules of cryptorchid animals. In addition, a significant decrease in tetraploid cell fraction and an increase in S-phase fraction was obtained in severe cases. This may be explained by cell arrest before entrance into meiosis. Destruction of tubule structure and cell arrangement were also observed by confocal microscopy in such cases. In conclusion, flow cytometry, combined with confocal analysis, added useful information about spermatogenesis disturbances in cryptorchid testis and it may be used as diagnostic tools in other cases of spermatogenic disorders.

Animals↗