Feasibility and reliability of flow-cytometry (fcm) DNA analysis of fresh and fixed urine samples

The qualitative results of FCM DNA analysis on fresh and fixed urine specimens (28 and 97, respectively) from 68 normal subjects and 10 patients with a past history of bladder cancer were compared. FCM DNA evaluability was not significantly different in fresh and fixed samples (63% vs 73%, respectively) whereas mean CV was significantly higher (7.3% vs 5.7%, respectively; p=0.04). A double FCM analysis on fresh and fixed urine was also performed in 16 cases. In this subgroup, the percentage of evaluable histograms from fixed urine specimens was slightly higher than that from fresh specimens. Aneuploid cases were found only in the fixed urine samples but the CVs from fresh and fixed cell suspensions did not differ. The absence of inflammatory cells with cytological analysis of the same samples was associated with low percentages of FCM evaluability and higher CVs. The use of fixed samples improves the quality of FCM DNA analysis permitting its use for screening programs.     

Flow cytometric analysis of DNA content in human bladder tumors and irrigation fluids

Flow cytometry (FCM) was used to study the DNA distribution of 99 tumor biopsy specimens and 41 bladder irrigation samples from patients with transitional cell carcinoma of the bladder. For tumor biopsy and cystectomy specimens, the frequency of aneuploidy increased with advancing tumor stage and grade. All T0 tumors were diploid. Twenty-seven percent of T1, 71.4% of T2, and 75% of T3 and T4 tumors were aneuploid. All Grade I tumors were diploid. Thirty percent of Grade II and 76.9% of Grade III tumors were aneuploid. The frequency of aneuploidy of tumors in the early stages (Ta, T1) is similar to the incidence of subsequent progression by these tumors described in the literature. For irrigation fluids, the relationship between grade and stage and the frequency of aneuploidy was similar to the relationship seen with tumor specimens. All four patients with only carcinoma in situ had aneuploid cells in their irrigations. The comparison of FCM data of bladder biopsy and bladder irrigation from the same cystoscopic evaluation suggests adequate representation of tumor cells in the irrigation fluids for almost all cases. The authors conclude that DNA ploidy analysis by FCM appears useful in a clinically important group of patients with aneuploid superficial tumors of moderate or high grade. Bladder irrigation analysis appears useful in the follow-up of patients with a history of carcinoma in situ and those with aneuploid tumors.     

Failure of anticytokeratin 18 antibody to improve flow cytometric detection of bladder cancer.

BACKGROUND. Bladder washing specimens containing inflammatory or squamous cells have been difficult to accurately analyze with single-parameter DNA flow cytometric (FCM) methods. METHODS. The anticytokeratin 18 antibody, CK5, was used in a multiparameter assay of 275 bladder washing and voided urine specimens to immunoselect only the bladder transitional cells for DNA analysis. RESULTS. Flow cytometric detection of transitional cell carcinoma was increased by immunoselection of CK5-positive cells in specimens from patients with disease. Unfortunately, a similar increase in hyperdiploid cells in pathologically benign specimens was observed, which resulted in a false-positive rate of 45%. In some instances, multiparameter FCM assays with CK5 could detect aneuploid cell populations not clearly evident by single-parameter analysis. CONCLUSIONS. However, the results from this study of the hyperdiploid cell fraction showed that the increased sensitivity resulting from the use of CK5 was not clinically useful because of the decrease in specificity.     

Bladder cancer diagnosis by flow cytometry. Correlation between cell samples from biopsy and bladder irrigation fluid

Results of flow cytometry (FCM) examinations of bladder irrigation specimens were compared with those of FCM examinations of cell suspensions from bladder biopsies of 44 urologic patients. The fluorescent dye, acridine orange (AO), was used to stain DNA and RNA differentially and abnormal urothelial cells were identified by their relative content of nucleic acids. Granulocytes and squamous cells could be distinguished from transitional cells in this procedure, and did not interfere with the analyses. Of 28 patients with papillary carcinoma, carcinoma in situ, and invasive carcinoma 27 were identified through FCM examination of irrigation cytology specimens; the one false-negative result was from a low-grade papillary carcinoma. Of 7 patients with papilloma, FCM examinations of irrigation specimens were positive in 4 and negative in 3. Results of FCM studies of biopsy specimens were in good but not complete agreement with those of irrigation specimens. In several cases, irrigation FCM disclosed tumor stemlines that were not identified in biopsy specimens. Discrepancies of this kind seemed most likely due to differences in sampling. Irrigation FCM seems to be a sensitive method for assessing multiple-site bladder tumors, and may be a useful technique for monitoring the course of conservatively managed bladder tumors.     

Comparative DNA Analysis by Image Cytometry and Flow Cytometry in Non-small Cell Lung Cancer

To determine whether image cytometry (ICM) is advantageous for clinical DNA analyses of tumor cells, nuclear DNA contents measured by ICM were compared with those by flow cytometry (FCM), using 46 samples of non-small cell lung cancers. ICM was performed on smear specimens of fresh materials (f-ICM) and cell suspensions obtained from paraffin-embedded tumors (p-ICM). The same cell suspensions were also analyzed by FCM (p-FCM). Aneuploid rates/coefficient of variation (CV) of f-ICM, p-ICM, and p-FCM were 76.1/4.90, 71.7/5.01 and 60.9/5.31%, respectively. There was a high correlation in the DNA indices between p-ICM and p-FCM (r=0.80). In the comparative DNA analysis, there were seven discordant samples. Six of them were estimated as aneuploid by p-ICM, but they were miscounted as diploid or undefinablc (impossible) by p-FCM. This was caused by measuring condensed nuclei or debris. All "impossible" samples in p-FCM were squamous cell carcinoma with necrosis. In cell cycle analysis, the S and S+G2/M phase fractions in diploid samples were higher in p-ICM than those in p-FCM ( P < 0.005), because the GO/G1 phase (2N) fraction presented by FCM was composed of cancer and non-malignant cells in diploid cancers. In ICM, they can be separately measured by means of morphological selection. These findings indicated that ICM is superior to FCM, especially for the practical DNA measurement of a few cancer cells and in the evaluation of the proliferation rates.     

Flow cytometry for detection and evaluation of urinary bladder carcinoma.

Flow Cytometry (FCM) DNA assays of bladder irrigation specimens are now recognized as a clinically useful and reliable means of detecting and monitoring carcinoma of the bladder. This technique, which identifies carcinoma by the presence of an aneuploid population of cells, can be carried out on specimens obtained in an outpatient or hospital setting and is easily performed in any medium-sized laboratory. It is most sensitive to superficial and high grade tumors. Overall, nearly 80% of superficial carcinomas of bladder will have positive flow cytometry, comparing very favorably with conventional cytology. Until now, the widest clinical application of FCM has been in monitoring the conservative treatment of stage 0-1 flat and papillary carcinomas, but newly developed dual parameter measurements are capable of quantifying proliferative activity, oncogene expression, growth factor receptors, and other cellular features that may better characterize the biologic potential of these tumors and can be expected to aid in the selection and timing of treatment.     

An in vitro clonal assay for bladder cancer: Studies of the biologic potential of the urothelium and determination of in vitro sensitivity to cytotoxic agents

We have applied an in vitro colony-forming assay system to the study of primary urothelial explants obtained transurethrally by bladder barbotage and tumor biopsy from 91 patients. Urothelial cells obtained by bladder barbotage from patients with bladder cancer and from a “control” group of individuals exhibit differential capacity to clone in agar. Of samples from bladder cancer patients, 88% formed colonies, whereas only 27% of these from a “control” group did so. Of samples from a “suspicious” patient group, 70% formed colonies. In vitro drug sensitivity studies of cells obtained by biopsy of solid tumors demonstrate a spectrum of drug sensitivity to cytotoxic agents.     

细胞周期检测点的流式细胞术分析

背景与目的:真核细胞的细胞周期运行遵循着严格的时相次序,这种精密的延缓和强制的次序,由细胞监控机制——检测点来完成,检测点功能的丧失会导致肿瘤的发生。传统的细胞周期检测点分析多数基于群体细胞DNA直方图的流式细胞术。本研究以晚G1期检测点为模式,建立一种新的细胞周期检测点分析方法——Cyclins/DNA双参数流式细胞术,并评价应用该方法进行细胞周期检测点分析的可行性和优越性。方法:将紫外线照射诱导后的人类急性淋巴细胞型白血病细胞株(MOLT-4)于不同时间点收获固定,分两组进行流式细胞仪检测:一组用DNA直方图法,Modifit软件分析计算G0/G1期细胞总数;另一组应用CyclinE/DNA双参数流式细胞术对G1晚期细胞CyclinE的荧光强度、阈值及G0/G1各亚期细胞数量进行定量分析,并比较两组实验结果。结果:用DNA直方图法观察到紫外线照射后0～4hG0/G1期细胞总数基本不变(5300～5500),6h后开始上升(6241,12.6%)。而用CyclinE/DNA双参数法观察到:(1)G1晚期细胞的CyclinE荧光强度于照射后短时间内便开始变化,1h上升为341.2(对照295.1,15.6%),6h上升为577.6(95.7%),此时CyclinE阈值上升到5.4(0h为2.0);(2)G0/G1各亚期细胞数目变化趋势不明显:G1晚期细胞数6h时略为减少(此时凋亡率为5.61%),G1早期细胞数缓慢上升。结论:CyclinE/DNA双参数流式细胞术将CyclinE的荧光强度及表达阈值定量化,对于细胞晚G1期检测点的检测比传统的DNA直方图法更为敏感和精确。     

Colony formation in urinary bladder carcinoma. Relationship to DNA flow cytometry, stage and histopathology

Tumour cells from 74 biopsies from human urinary bladder carcinomas were cultivated in a semisolid system (Courtenay and Mills method). In 51 cases DNA flow cytometry (FCM) was performed. Significant growth was obtained in 53 of 74 cases (71.6%), and good quality DNA histograms from FCM were obtained in all 51 attempts. No correlations between clinical or histopathological data and plating efficiency (PE) were found, nor between ploidy and PE. However, a very high S-phase (greater than 15%) correlated with a low PE. This work shows that bladder carcinoma cells can be studied in a semisolid agar system. It also suggests that there exists no correlation between PE and DNA FCM data or clinical or histopathological data.     

Clinical application of flow cytometric analysis to predict results of radiation therapy

This includes review of recent papers about clinical application of flow cytometry referred to sensitivity and effectiveness of radiation therapy and also our study to develop the new method to detect the DNA damage by irradiation. In view point of nucleoid damage with radiation therapy, FCM showed usefulness in using forward scatter histogram. The nucleoid induced to measure DNA damage here was originally developed by Cook et al, and thereafter, by Milner who first applied FCM to detect the damage. The nucleoids from HeLa cell and U 138 cell line were prepared following their methods. Control nucleoids showed a dose-dependent reduction in the ethidium bromide fluorescence. On the other hand, nucleoids obtained after irradiation showed a restricted reduction of it. The nadir of the fluorescence reduction seems to be a dose-dependent in radiation dose. This results indicate that FCM would be a useful tool for a rapid analysis of nuclear damage in near future. Other indicators, such as DNA index, DNA histogram, were discussed from the recent papers.     

Cytogenetic abnormalities in benign lymphoid hyperplasia: a dual-parameter study using chromosome analysis and flow cytometry

This is a prospective study of lymphoid tissue showing benign reactive hyperplasia (18 lymph nodes and 2 tonsils), using cytogenetic analysis of cells stimulated with T- or B-cell mitogens. The reason for this study was the detection of an abnormal chromosomal population in cells from an enlarged lymph node excised from a 7-year-old female who on further investigation was found to be clinically well and after one year's close follow-up had not developed any further signs or symptoms of malignancy. In addition, DNA content was measured by flow cytometry (FCM) on fresh cell suspensions in 17 cases and fixed cell suspensions in 3 cases. Structural and numerical clonal chromosome abnormalities were found in 9 of the 20 samples, but no common specific defect was identified. FCM showed an abnormal DNA content in 10 of the 20 samples studied; 3 of these showed clonal chromosome abnormalities. Surface membrane immunoglobulin studies were carried out on 15 of the 20 samples using cell suspensions and frozen tissue sections. In 5 of the 15 cases, monoclonal surface immunoglobulin was detected. There was no direct correlation between the surface membrane immunoglobulin studies and the chromosome and FCM analyses. We conclude that aneuploidy is a common feature in reactive lymphoid tissue, but both cytogenetics and FCM are needed to identify it.     

Presumptive downstaging from preoperative irradiation for bladder cancer as determined by flow cytometry: Preliminary report

Presumptive tumor downstaging was evaluated in 28 patients with grade 11 or III, solid, muscle-infiltrating bladder cancer (clinical category T3) treated by integrated irradiation (2000 rad to the whole pelvis in 5 days) and cystectomy (1–14 days later) by comparing the results of flow cytometry (FCM) on barbotage specimens obtained before and after irradiation (at the time of cystectomy) and the results of pretreatment clinical stage (T category) and post cystectomy pathological stage (P category). The patients were divided into three groups: (1) P > T, (2) P = T, and (3) P < T. All of the patients in this study had positive FCM specimens with an aneuploid stemline in the pre-irradiation specimen. A complete radiation response (CRR) was defined by FCM as disappearence of the aneuploid stem cell line. Of the 5 patients in the P < T group, 4 showed a CRR; of 20 patients in the P = T group, 8 showed a CRR; of the 3 patients in the P > T group, none showed a CRR. The proportion of patients in the various T/P groups is consistent with that previously observed in patients receiving integrated irradiation (2000 rad in 5 days) and cystectomy (1–14 days later). The overall downstaging response of 43 %, as determined by FCM, correlates well with the pathological downstaging rates of 40%–68% reported by others following high dose (4000–5000 rad) integrated irradiation cystectomy regimens; however, it is more than the 27 % rate reported with the low dose short course (2000 rad in 5 days) regimen. The correlation of the FCM findings with clinico-pathological downstaging is consistent with the possibility that FCM may be useful in identifying a favorable radiation response.     

Development of an agar-methyl cellulose clonogenic assay for cells in transitional cell carcinoma of the human bladder.

We report the development of a clonogenic assay for progenitor cells in transitional cell carcinoma of the bladder. Colony growth has been demonstrated from cells obtained both from surgical biopsies and from bladder barbotages. Electron microscopic and karyotypic evidence supports the contention that these progenitors represent a part of the population maintaining the tumor in vivo. Colony growth occurred in 9 of 11 surgical biopsy samples and in 6 of 6 bladder barbotage samples. Plating efficiency ranged up to 0.7%, and colony size was in some instances greater than 1000 cells. The assay appears potentially useful for analysis of the biology of human transitional cell carcinoma.     

Comparison of autoradiography and DNA histograms in assessing S-phase cells from patients with myeloid leukemias

The studies reported here were designed to determine whether DNA histogram analysis provided the same estimate for the percentage of cells in S-phase as does 3H-TdR labeling index (LI). For DNA histogram analysis high resolution flow cytometry was employed together with 3 different computer based methods for histogram analysis. When tissue culture cells were employed there was a good correlation (r = 0.89) between the estimates for the percentage of cells in S-phase by the LI and DNA histogram techniques. In contrast, when 1138 bone marrow or peripheral blood specimens obtained from leukemic patients were studied the correlation was poor. For example when 243 pretherapy bone marrow aspirate cells were studied by both methods the correlation was only 0.46. A direct comparison of the clinical relevance of the two methods for measuring S-phase cells when high dose cytosine arabinoside therapy was used to treat patients demonstrated that only LI measurements provided clinically useful information. In conclusion, DNA histogram analysis provides an inaccurate estimate of the percentage of cells in S-phase.     

A comparative study of DNA ploidy in 115 fresh-frozen breast carcinomas by image analysis versus flow cytometry.

BACKGROUND. Qualitative and quantitative analysis of cellular DNA content may be clinically useful in the prognostic evaluation of certain types of malignant tumors, including breast carcinoma. Flow cytometric (FCM) analysis has been the most frequently used procedure for DNA analysis, but it requires a reasonably large tissue sample. Computer-based image analysis (IA) now allows imprint, cytospin, and needle aspiration smear preparations and other small tissue samples to be used. METHODS. To resolve concern about the diagnostic efficacy of small tissue samples in the use of IA, the authors performed a comparative study of FCM analysis and IA using 115 fresh-frozen breast carcinomas. Feulgen-stained imprint preparations for IA and single-cell suspensions from the same fresh-frozen tissue for FCM analysis were used, and the respective histograms were compared. RESULTS. The results were concordant in 90.4% (104 of 115) of the cases, but 11 specimens yielded discordant data. IA provided histograms with a somewhat lower resolution and a relatively high coefficient of variation for the G0/G1 peak, thus rendering occasional tumors, which were near-diploid aneuploid by FCM analysis (four cases), not amenable to diagnosis by aneuploid characterization. In three additional cases, FCM analysis showed aneuploid hyperdiploid (two cases) and multiploid (one case) histograms, but IA only demonstrated a diploid peak. Conversely, in four other cases, aneuploid peaks were recognized only by IA. CONCLUSIONS. Computerized IA has significant advantages over FCM analysis, including lower cost, the ability to analyze very small specimens, the capability of detecting rare high ploidy cells, the capacity to classify cellular populations according to specific morphologic type, and the fact that no destructive enzyme or chemical digestion is required for specimen preparation, thereby preserving the integrity of fragile cells.     

New sensitivity test using flow cytometry

Summary Flow cytometry (FCM) has been used to evaluate not only the malignancy of tumor cells but also the effects of chemotherapy. Here a new application of FCM for selecting the best antineoplastic agent in the chemotherapy for brain tumors is reported.Through our preliminary study using established brain tumor cell lines, the system for this sensitivity test was developed. Antineoplastic agents were placed in contact with monolayer-cultured cells; then cell viability and changes in the DNA histogram were analyzed by FCM. Cell viabilities were measured with the fluorescein diacetate (FDA) staining method, and the DNA histogram was analyzed by the propidium iodide (PI) staining method. The best antineoplastic agent was determined based on changes in cell viability and cell cycle. In other words, when markedly decreased viability as compared with that of the control, is measured by FCM, then the agents can be considered to have had a cytocydal effect on the tumor cells, and thus the sensitivity of the agents is able to be evaluated. If the viability of the tumor cell is observed to be similar to that of the control, the cytostatic effects of the agents are able to be evaluated only if a marked change is observed in the DNA histogram.After the preliminary study, this system was applied clinically to malignant brain tumor cases, resulting in success in selecting the best antineoplastic agent for each individual case. Our sensitivity test using this FCM established in vitro system has much potential value for clinical use.     

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.     

DNA flow cytometry applied to fine needle sampling of human breast cancer

Breast cancer cells can be obtained directly from the patient with minimal trauma by fine needle sampling (FNS). A method was developed that enabled us to prepare tumor cell nuclei for ploidy determination by flow cytometry (FCM). Fine needle sampling was performed on 235 patients with clinically suspected malignancy. Two hundred sixteen specimens (92%) produced enough material for assessment; 206 were diagnosed as cytologically malignant. In 41 patients surgical specimens from the same tumors were available. Thirty-eight of these specimens (93%) were classified according to ploidy. No significant correlation was found between aneuploidy and clinical stage (size and lymph node involvement). The comparison of DNA histograms from 21 primary breast tumors and homolateral axillary lymph nodes showed mostly similar patterns. On the contrary, eight of nine synchronous bilateral cancers were shown to have different ploidy. Flow-cytometry-derived DNA histograms of fine needle samples could be a valuable tool in the management of breast cancer.     

Characterization of bladder papilloma by two-parameter DNA-RNA flow cytometry

Two-parameter flow cytometry (FCM) studies of 0.9% NaCl solution bladder irrigation specimens were performed on 48 patients with histologically orderly or atypical papilloma of the urinary bladder in order to assess the value of RNA as a possible second parameter, along with DNA, in the detection of bladder tumors. DNA, RNA, and nuclear diameter measurements were obtained for each of 5000 cells/sample, and analyses were based on the distributions of those values. With the use of DNA content alone, 22 cases (46%) were classified positive by FCM. With RNA content as an additional parameter, 40 cases (83%) were positive. Two cases were suspicious, and 6 cases were normal by both parameters. Of 28 patients with papillomas showing histological atypia, 16 patients had positive DNA histograms, including 3 patients with aneuploid stemlines, but 24 of the 28 patients had positive RNA histograms. Of 20 patients with orderly papillomas, 6 patients had positive DNA histograms, including 3 patients with aneuploid DNA stem cell lines, but 16 of the 20 patients had positive RNA histograms. Thus, the probability of positive DNA histograms is higher in atypical papillomas (57%) than in orderly papillomas (30%), whereas elevated (positive) RNA is more characteristic of all papillomas without distinction between those that are histologically atypical (86% positive) or orderly (80% positive). For patients at risk of developing papillary bladder tumors, two-parameter DNA-RNA FCM appears to offer greater diagnostic sensitivity than does FCM based on DNA content alone.     

Predictive value of DNA measurements in bladder washings. Comparison of flow cytometry, image cytophotometry, and cytology in patients with a past history of urothelial tumors

Comparative DNA ploidy measurements were carried out by flow cytometry and by image analysis on cells in 71 bladder washing specimens from 50 patients with past histories of bladder tumors. Among the specimens classified as diploid or questionable by flow cytometry, 14 showed the presence of aneuploid DNA values documented by image analysis. In 18 of the 50 patients, recurrent tumors were observed during a relatively brief period of follow-up. In 15 of them the DNA pattern was aneuploid and in three it was questionable. In nine of the 15 patients, both methods of DNA analysis disclosed aneuploidy, but in six patients aneuploidy was detected by image analysis only. A combination of DNA aneuploidy, whether observed by flow cytometry, image analysis, or both, and of positive or suspicious urine cytology is highly predictive of recurrence of high grade bladder tumors. Image analysis of DNA content in bladder washings adds information of clinical value above and beyond that obtained by flow cytometry.