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Comparison of flow cytometry to routine testicular biopsy in male infertility.

We compared DNA flow cytometry to morphologic evaluation of routine testicular biopsies as methods of monitoring spermatogenesis. The study group consisted of 14 azoospermic men and 5 others who underwent testicular surgery unassociated with fertility problems. The findings for both studies were divided into three groups: normal, moderately abnormal, and markedly abnormal. Correlations between the findings from routine biopsy and flow cytometry were good. Of 9 patients having normal testicular morphology, 7 had normal ploidy classes by DNA flow cytometry while 2 had moderately abnormal histograms. Of 5 cases with moderately abnormal morphology, 1 had normal, 1 had moderately abnormal, and 3 had markedly abnormal ploidy distributions. In 5 cases described as Sertoli cell only, all DNA histograms were markedly abnormal, consisting almost exclusively of diploid cells. DNA flow cytometry of testicular biopsies and aspirates has been demonstrated to be a rapid, reproducible, and objective approach in evaluating the infertile male and is a promising method to investigate spermatogenesis in an outpatient clinic in lieu of formal testis biopsy.

Biopsy, Needle↗

Flow cytometry as a predictor of response and progression in patients with superficial bladder cancer treated with bacillus Calmette-Guerin.

Simultaneous bladder wash flow cytometry, voided urinary cytology and cystoscopic examinations were performed at 3-month intervals during a median of 18 months (range 5.5 to 50 months) in 65 patients receiving intravesical bacillus Calmette-Guerin treatment for superficial bladder cancer. Of the 65 patients treated 36 (56 per cent) had a complete response, 12 (18 per cent) had no response and 17 (26 per cent) had progression. Results of examinations at 6 months suggested that a negative bladder wash flow cytometry (29 of 36 patients, r equals 0.73, p less than 10(-7) is a strong predictor of response to bacillus Calmette-Guerin, comparable with cytological (r equals 0.60, p less than 10(-7) or cystoscopic (r equals 0.38, p less than 0.005) examinations alone or combined with cytology (r equals 0.74, p less than 10(-7)). At 6 months a positive bladder wash flow cytometry (r equals 0.44, p less than 0.0005) is as strong a predictor of disease progression as a positive cystoscopic examination (r equals 0.43, p less than 0.0005). The combination of bladder wash flow cytometry and voided urinary cytology is not superior to positive bladder wash flow cytometry alone. Median estimated interval to progression for these patients treated with bacillus Calmette-Guerin was 38 months. In the subgroup with positive bladder wash flow cytometry at 6 months the median interval to progression was 30 months. With a negative bladder wash flow cytometry at 6 months the probability of survival free of progression at 30 months was 85 per cent (p less than 0.01). Thus, negative bladder wash flow cytometry at 6 months is a strong predictor of response to bacillus Calmette-Guerin and also survival free of progression.

Administration, Intravesical↗

[Correlation of results of flow cytometry and morphologic findings in the diagnosis of malignant B-cell lymphoma].

BACKGROUND: Immunophenotyping of malignant lymphomas becomes necessary for the correct classification, for the design of therapy and for prognosis projection (WHO). Although the spectrum of classic immunohistochemical (IHC) examinations in paraffin embedded or frozen sections has recently considerably extended, IHC should preferably be combined with flow cytometry. The main advantage of flow cytometry is a synchronous application of two or more antibodies marked with various fluorochromes in one sample. The method is limited by utilizing native material only. METHODS AND RESULTS: The flow cytometry combined with histological and IHC investigations were used in diagnosis of primary non-Hodgkin's lymphomas of B-cell origin (B-NHL) and for bone marrow staging or restaging. We studied 90 patients with confirmed or suspected B-NHL and we found a good correlation in 89% of samples of primary lymphomas and in 85% of bone marrow samples when IHC and flow cytometry results were compared. The overall efficacy of the flow cytometry determination in lymphoma infiltration of the samples was 89%. CONCLUSIONS: Immunophenotyping utilizing flow cytometry contributes to diagnosis and classification of B-cell lymphomas in the significant proportion of investigated patients. In some cases it is even unnecessary to employ IHC examination of tissue sections. The method is especially suitable for determination of monoclonal populations of B-cells by detection of cell surface markers because it is more specific and sensitive than IHC. The immunophenotyping by flow cytometry as an auxiliary method and in correlation with morphological findings it can make the diagnosis of B-cell lymphomas faster and more specific.

Adult↗

Determination of testicular function after torsion by DNA flow cytometry of serial fine-needle aspirates.

OBJECTIVE: To determine the efficacy of DNA flow cytometric analysis of testicular percutaneous fine-needle aspirates in the assessment and follow-up of testicular function after torsion, and to determine the relationship between the duration of torsion and testicular injury. MATERIALS AND METHODS: Three groups of 15 adult rats underwent a 720 degrees torsion, with fixation of the mesorchial ligament, for 1, 3 or 5 h. Bilateral aspirations, performed 7, 21 and 35 days after torsion were examined by flow cytometry. Testes were harvested and evaluated histologically using Johnsen's scoring. RESULTS: Irreversible testicular injury occurred in all three groups of rats, with loss of function after 1 h and loss of viability after 3 and 5 h. The results from flow cytometry suggested significant contralateral testicular injury (P < 0.025) but this was not supported by the histological evaluation. There was a strong correlation between the testicular function assessed by flow cytometry and by Johnsen's scoring of histological specimens (r2 = 0.95). CONCLUSION: The assessment of testicular aspirates by flow cytometry allows testicular function to be followed after torsion in rats, and potentially in humans. Using DNA flow cytometry, the temporal course of the twisted testis in the adult rat was determined; contralateral testicular injury following the reversal of torsion could not be excluded.

Animals↗

Tissue-specific markers in flow cytometry of urological cancers. III. Comparing chromosomal and flow cytometric DNA analysis of bladder tumors.

Thirty-seven transitional-cell carcinomas (TCC) of the urinary bladder were analyzed by DNA flow cytometry (FCM). After labelling of the cell suspensions with antibodies to cytokeratin, the cytokeratin-positive cells and the non-epithelial cytokeratin-negative cells could be analyzed separately. After estimation of S- and G2M phase, 3/17 cases (18%) with a normal DNA index showed elevated proliferative levels, among cytokeratin-labelled suspensions only. Of these 17 cases, 14 showed chromosomal abnormalities. The remaining 20 cases were abnormal, irrespective of the technique used. Although immuno-labeling of tumor cells for cytokeratin in FCM increases the sensitivity of this method in detecting aneuploid tumors or tumors with high proliferation fractions, the discriminating power of chromosomal analysis of TCC is greater than FCM.

Carcinoma, Transitional Cell↗

Flow cytometry demonstrates bacteriocin-induced injury to Listeria monocytogenes.

Flow cytometry was used to study the effect of the bacteriocin leucocin B-TA11a on Listeria (L.) monocytogenes. Mixed proportions of dead and live control populations were analyzed by flow cytometry to determine detection limits of the Dead/Live Baclight Bacterial Viability KitTM. High correlations for flow cytometric detection of defined proportions of live or dead cells in mixtures between 10 and 100% of dead (r2 = 0.97) or live (r2 = 0.99) cells were obtained. However, mixtures containing less than 10% of either live or dead control cells gave correlations below 0.72. The growth of L. monocytogenes in the absence and presence of leucocin B-TA11a was analyzed by flow cytometry with Baclight, plate counts, and optical density measurements. Although leucocin B-TA11a initially inhibited listerial growth, the uptake of both Baclight dyes suggested that cells remained viable but became leaky, possibly indicating bacteriocin-induced pore formation in the target membranes.

Bacteriocins↗

Analysis of residual disease in chronic lymphocytic leukemia by flow cytometry.

We have investigated the value of both conventional and quantitative flow cytometry to detect minimal residual disease in 21 CLL patients in remission including bone marrow histology: eight in complete remission (CR), 11 in nodular partial remission (nPR) and two in PR. The techniques used were double immunostaining with CD5 and CD19 and quantitative estimation of the number of both antigens with standard microbeads. Reference values were established on normal peripheral blood and bone marrow controls. Patients were considered in 'immunological' remission when the percentage of CD5+ CD19+/total CD19+ cells was <25% in PB and <15% in BM. In six of the eight patients in CR, CLL cells were still detectable by flow cytometry. Only two patients, that underwent allogeneic bone marrow transplant, achieved immunological remission. CLL samples showed significantly higher CD5 and lower CD19 antigen density than normal controls (P < 0.001). Persistence of residual disease was a predictor of time to progression. None of the two patients in immunological remission relapsed within a period of 13 and 33 months, whilst two of the six patients in CR with positive flow cytometry relapsed 3 and 6 months after achieving CR. This study demonstrates that flow cytometry contributes to increase the sensitivity of the clinicohematological criteria to detect residual malignant cells in CLL patients and may be useful to monitor disease status following treatment.

Adult↗

[Flow cytometry results in patients with ulcerative colitis and Barrett esophagus. An additional diagnostic tool in the early diagnosis of a malignant transformation?].

In this review the basic principles of flow cytometry for the measurement of DNA content are described. In addition to cell cycle phase distributions DNA ploidy can be analysed by flow cytometry. Flow cytometric results in patients with ulcerative colitis and Barrett's esophagus seem to indicate that besides histologic examinations flow cytometry may play a role in surveillance programs of patients with an increased risk of a malignant transformation. These first interesting results, however, have still to be confirmed in long term studies.

Barrett Esophagus↗

[Three- and two-color flow cytometry for immunologic surveillance of toll-like receptor 4 after renal transplantation].

OBJECTIVE: To compare the characteristics of three- and two-color flow cytometry for immunologic surveillance of toll-like receptor 4 (TLR-4) after renal transplantation. METHOD: The expressions of CD14, TLR4 and CD80 in peripheral blood monocytes were measured in 10 patients after renal transplantation by three- and two-color fluorescent staining flow cytometry, respectively. The percentages of TLR4 and CD80 in CD14-positive monocytes were calculated, and the characteristics of two methods of flow cytometry were compared. RESULTS: The two detection methods did not significantly differ in their detection of TLR4 (P=0.198) and CD80 (P=0.872) expressions in CD14-positive monocytes, and the measurements of the two methods for the same sample showed positive correlation (r=1, P=0.000 for TLR4 and r=0.999, P=0.000 for CD80). Three-color flow cytometry was also capable of detecting monocytes positive for both TLR4 and CD80. Compared with two-color flow cytometry, three-color flow cytometry provided more information and required less blood sample and monoclonal antibody. CONCLUSIONS: Three- and two-color flow cytometry have almost the same accuracy in immunologic surveillance of TLR4 after renal transplantation. The former method also provides information of the simultaneous expression of TLR4 and its down stream molecule CD80, which could be critical for imploring the mechanisms by which TLR4 takes part in alloimmunity through modulating CD80 expression, and requires less blood sample and monoclonal antibody.

Adult↗

Flow cytometry's contribution to the measurement of cell functions.

Flow cytometry has important advantages over conventional techniques. It is rapid, highly sensitive and allows multi-parametric analysis and cell sorting. Potential exists for the measurement of many cell functions by flow cytometry. The technique can be used to determine cell viability, intracellular calcium and pH, membrane potential, enzyme activities, membrane fluidity and endocytosis. Numerous examples are given on the applications of flow cytometry for cell functions measurements in the fundamental and biomedical fields.

Animals↗

Analysis of micronucleated cells by flow cytometry. 3. Advanced technology for detecting clastogenic activity.

Under optimum conditions, flow cytometry (FCM) can provide a powerful technology for analyzing rare micronucleated cells in the peripheral blood. Our efforts in this endeavor have been directed toward a careful and meticulous optimization of experimental conditions, in order to achieve high resolution and high accuracy before introducing biological variation. We have achieved high resolution (Tometsko et al., 1993a) wherein the micronucleus signal is moved 100-fold upfield and away from the DNA deficient red blood cell (RBC) peak. In addition, we have demonstrated the high accuracy of our flow cytometry method in scoring rare micronucleated cells (Tometsko et al., 1993b). In the course of our studies, we rigorously pursued conditions which minimized experimental noise, demanding that FCM-scoring accuracy should approach theoretical limits. Thus, we laid the foundation for detecting clastogen activity with great sensitivity. The experiments described herein extend the previous studies by using high-speed flow cytometry to detect a clastogen-induced increase in MN cells in the total erythrocyte population. Methyl methanesulfonate (MMS) served as a model clastogen in these studies. This manuscript describes our development of a suitable blood-sampling regimen, the advantages of obtaining initial blood samples before dosing, the sex-linked difference in background micronucleus levels in BALB/c mice, and the analysis of a clastogen-induced biological response in male and female mice. As described, our flow cytometry method is able to provide a quantitative analysis of the net change in micronucleated cells (delta MN) for each mouse.

Animals↗

Cytokine flow cytometry: understanding cytokine biology at the single-cell level.

In the past 4 years, cytokine flow cytometry has emerged as the premier technique for enumeration of cytokine producing T cells. The multiparameter capability of flow cytometry permits the simultaneous detection of two or more cytokines within a single cell, allowing true Th1 vs. Th2 determination. The high throughput inherent to flow cytometry has enormous advantages when applied to clinical research questions previously not amenable for study using labor intensive techniques such as ELISPOT, limiting dilution and T cell cloning. Furthermore, cytokine flow cytometry allows the study of individual T cells directly ex vivo, minimizing artifacts due to long term culture. As such, cytokine flow yields unique insights into cytokine biology heretofore not possible. We have used cytokine flow cytometry to examine coexpression of cytokines within the memory/effector CD4+, CD27- subset. Doing so, we have found distinct cytokine producing subsets that correlate with the previously described Th1, Th2 and Th0 subsets. The majority of cytokine producing cells were of these first two subsets with fewer cells coexpressing IFN-gamma and IL-4. These results validate the Th1/Th2 hypothesis and demonstrate specific subsets of cytokine producing T cells in fresh ex vivo human T cells.

Cytokines↗

Flow cytometry with or without cytochemistry for the diagnosis of acute leukemias?

Ninety-three (93) cases of acute leukemia were assessed using flow cytometry and cytochemistry and assigned to one of four categories: myeloid, lymphoid, biphenotypic, and non-diagnostic. In leukemias designated as ALL or AML by both methodologies, there was lineage agreement in all but 3 of 71 cases (95.8%). However, when nondiagnostic or biphenotypic diagnoses made by either methodology were included, complete agreement occurred in only 77.4% of cases. Of 37 cases designated myeloid origin by flow cytometry, 33 (89.2%) were read as myeloid by cytochemistry. The four discordant diagnosis were read as lymphoid (2) or as non-diagnostic (2). Eighty percent of lymphoid leukemias were diagnosed as such by both flow cytometry and cytochemistry; one early B cell ALL was diagnosed as myeloid and 8 as non-diagnostic. Fifty percent (50%) of flow cytometry defined T-cell ALL were considered non-diagnostic by cytochemistry as compared to 17% of the total ALL group. Of the remaining four designated non-T cell ALL by flow cytometry and non-diagnostic by cytochemistry, three were read by flow cytometry to be standard pre-B ALL and one an early B-cell ALL. Only 2/9 leukemias considered biphenotypic by flow were identified as such by cytochemistry. Given (1) the potential importance of non-lineage expression in the prognosis of myeloid and lymphoid leukemias, (2) cytochemistry's impaired ability to diagnose biphenotypic, T-cell, and promyelocytic leukemias, and (3) the increased costs incurred in diagnosis when both modalities are used, perhaps it is time to re-examine the utility of performing both flow cytometry and cytochemistry as initial testing for leukemia categorization.

Adolescent↗

Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry.

OBJECTIVE: To analyze human articular chondrocyte cell-associated matrix aggrecan, hyaluronan (HA) and type II collagen metabolism using flow cytometry, and to compare the results obtained for aggrecan with classic(35)Sulfate incorporation methods and an enzyme linked immunosorbent assay (ELISA). DESIGN: Human articular chondrocytes obtained from five donors were cultured in gelled agarose and tested for their response to different concentrations of interleukin-1beta (IL-1beta). Synthesis and distribution of aggrecan in the cell-associated matrix (CAM), in the interterritorial matrix and in the nutrient medium of the chondrocytes in culture were analyzed using(35)Sulfate incorporation. The results were expressed as pg SO(4)incorporated in aggrecan per 1 x 10(6)cells/h. Flow cytometry with FITC-conjugated monoclonal antibodies against aggrecan and type II collagen, and with the biotinylated hyaluronic acid binding protein (b-HABP), was used to investigate the synthesis and accumulation of aggrecan, type II collagen and HA in the CAM of the cultured cells. The packing of these macromolecules in the CAM of the chondrocytes was assessed by measuring the mean fluorescence intensity (MFI) of the cell sample due to the binding of the specific monoclonal antibodies or b-HABP used. ELISA was used in parallel to quantify CAM aggrecans after these macromolecules were brought into solution with guanidinium chloride. Detection of aggrecan by flow cytometry was compared with(35)S-incorporation in chondrocytes from two subjects and with ELISA in a further two donors. RESULTS: IL-1beta suppressed aggrecan synthesis by chondrocytes in agarose. An IL-1beta dose-dependent suppression of(35)S-aggrecan in the CAM reflected the changes in the interterritorial matrix. IL-1beta-induced aggrecan breakdown was followed by a rise in(35)S-aggrecan metabolites in the incubation media of the cells in culture. Flow cytometry and ELISA confirmed this decreased accumulation of aggrecan in the CAM of the chondrocytes. The results obtained with flow cytometry were closely related to those obtained with ELISA.(35)S-incorporation, on the other hand, indirectly measures the glycosaminoglycan content of the aggrecan and does not necessarily reflect the absolute amount of aggrecan molecules. Therefore, the effects of IL-1beta on cell-associated aggrecan, where assessed with(35)S-incorporation, did not correlate with the results of the flow cytometric assays. Flow cytometry enabled the detection of an impaired synthesis and accumulation of HA and of type II collagen in the CAM of the cultured chondrocytes. IL-1beta-induced changes in CAM aggrecan and hyaluronan closely agreed. CONCLUSIONS: Flow cytometry offers an efficient tool to study the metabolism of the chondrocyte CAM. The MFI has been used as a parameter to quantify the ECM molecules in the CAM.

Aggrecans↗

Analysis of atresia in bovine follicles using different methods: flow cytometry, enzyme-linked immunosorbent assay, and classic histology.

This study was performed in order to validate flow cytometry as an acceptable method for analyzing follicular atresia in bovine granulosa cells by comparing it to two other techniques, histology and ELISA. Ovaries from 35 nontreated cows, all at different times of their estrous cycle, and 12 superovulated cows were collected. Superovulation treatments began between Days 9 and 12 (Day 0 = estrous), and animals were administered 8 doses of FSH-P (32 or 20 mg) at 12-h intervals over 4 days with or without the addition of 1 mg of prostaglandin s.c. on the third day. Animals were slaughtered after the last FSH-P injection. Granulosa cells from 133 follicles from non-treated cows and 85 follicles from superovulated cows were analyzed. Follicular diameters ranged from 2 to 20 and 2 to 16 mm, respectively. Because of the ample amounts of cells collected, it was possible to perform more than one technique for each follicle. Flow cytometry detected in most follicles a subpopulation of cells that possessed less DNA than normal, viable cells (referred to as -G1 cells). Histological classes used (established in previous work) were nonatretic (< or = 5% picnotic nuclei), slightly atretic (> 5 to < 15% picnotic nuclei), and atretic (> or = 15% picnotic nuclei). A strong linear correlation existed between the percentage of picnotic nuclei and the percentage of -G1 cells (R = 0.86; p < 0.001) with granulosa cells from follicles from nontreated cows. In some cases, flow cytometry detected a certain percentage of cells with reduced DNA content while histology revealed very few picnotic nuclei, indicating a higher sensitivity of flow cytometry. Superovulation decreased considerably the percentage of atretic cells seen with both techniques. The linear correlation was not as strong because follicles from superovulated animals represent a very homogenous population (R = 0.54; p < 0.001). The ELISA technique coincided with flow cytometry as seen in the strong correlation between the two techniques (R = 0.91; p < 0.001). Flow cytometry appeared to be very effective and rapid in evaluating the atretic states of follicles from nontreated and superovulated cows. Strong correlations existed between this method and histology and ELISA.

Animals↗

Impact of the new Beckman Coulter Cytomics FC 500 5-color flow cytometer on a regional flow cytometry clinical laboratory service.

Calgary Laboratory Services (CLS) in Alberta, Canada, is the regional reference laboratory providing flow cytometry services for southern Alberta and southeastern British Columbia. As a busy reference flow laboratory we provide flow cytometry immunophenotyping for investigation and diagnosis of acute and chronic leukemias, lymphomas, immunodeficiencies, neuroblastoma, platelet disorders, and interstitial lung disease (ILD). Because of increasing workload and the continual effort to improve the service to our health care providers, CLS invested in the new Beckman Coulter Cytomics FC 500 5-color flow cytometer. In addition to time and labor savings due to reduced maintenance and operating system design, this new flow cytometer automates many of the previous manual steps involved in quality control and flow cytometric analysis. It also incorporates 2 lasers and is capable of measuring 5-color antibody combinations in a single tube, enabling us to reduce the number of tubes and overall costs, giving us better gating options for minimal residual disease analysis. We present the first published evaluation, an assessment of the overall productivity and cost impact of the new state-of-the-art Cytomics FC 500 flow cytometer. Implementation of the Cytomics FC 500 has resulted in a 20% reduction in reagent costs and shorter turnaround time for analysis and diagnosis. This instrument has allowed us to reduce our acute leukemia panel from 17 to 13 tubes, our lymphoma panel from 13 to 7 tubes, and our ILD panel from 4 to 2 tubes. The availability of 2 lasers provides more flexibility in choosing antibodies and conjugates to customize immunophenotyping panels. It also allows us to use the DRAQ5 dye and simultaneously analyze the immunophenotype and DNA content of cells with very little compensation. Many of the arduous, time-consuming flow operator tasks often associated with previous generation flow cytometry instruments, such as color compensation, list mode analysis, sample repeats, and interpretations, have been substantially reduced with the Cytomics FC 500 5-color flow cytometer. In conclusion the Cytomics FC 500 5-color flow cytometer is a major advance in flow cytometry instrumentation and has reduced our overall reagent costs by 20%, provided better information and speedier turnaround time to our health care professionals. It is an ideal flow cytometer for any busy clinical or research flow cytometry service.

Automation↗

Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis.

BACKGROUND: Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS: A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS: Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS: Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.

Animals↗

Flow cytometry for followup examinations of conservatively treated low stage bladder tumors.

A series of 26 conservatively treated patients with low stage bladder tumors was examined later by outpatient cystoscopy, conventional cytology and flow cytometry. Based on those findings the patients were separated into 5 groups. In the 4 patients in group 1 all findings persistently were negative. The 4 patients in group 2 had initially normal cystoscopy with progressively more abnormal flow cytometry and intermittently positive cytology; they subsequently suffered tumors. The 10 patients in group 3 had positive cystoscopy and flow cytometry, and intermittently positive cytology. The 6 patients in group 4 had negative cystoscopy with intermittently positive cytology and persistently positive flow cytometry. In all 6 cases flow cytometry was more sensitive than conventional cytology in detecting early and, sometimes, cystoscopically occult neoplasms. In the 2 patients in group 5 cystoscopically visible papillomas and low grade papillary carcinomas were found with negative flow cytometry and negative or suspicious cytology. The papillary tumors in these 2 patients were believed to be composed principally of benign or atypical epithelium, with too few malignant cells to be detected by present flow cytometry techniques. Our preliminary results suggest that flow cytometry may be a valuable tool for followup of patients who have had conservatively treated low stage bladder tumors and are at risk for development of carcinoma. In addition, the techniques may be used to monitor changes in the proportion of benign and malignant cells and, thus, can be of help in following the course of newly forming papillomas, papillary carcinomas or carcinoma in situ in papillomas or flat epithelium.

Aged↗