Double labelling to obtain S phase subpopulations: application to determine cell kinetics of diploid cells in an aneuploid tumour

Abstract. We studied the cell kinetics of the murine mammary carcinoma MCa-K using iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) given at different times as independently detectable labels of S phase cells. The presence of IdUrd and CldUrd, and the amount of DNA were measured by three-colour flow cytometry making it possible to define three subpopulations within S phase and to measure the progression through the cell cycle during the time following labelling. In DNA histograms of these subpopulations, the diploid and aneuploid cells (which had a DNA index of 1.7) are essentially completely separated. From appropriate combinations of cells labelled with IdUrd only, CldUrd only, or both, it was possible to construct separate DNA distributions for the labelled diploid and aneuploid cells at the times of administration of each label. The kinetics of the diploid and aneuploid cells could be calculated for individual tumours from these two time points without having to make corrections for the presence of the second population. The diploid and aneuploid populations had indistinguishable S and G₂+ M phase durations, T_S and T_G2+M, of about 9 and 2h; however, the potential doubling time values for the aneuploid and diploid populations were 30.2 and 101.2h respectively.     

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.     

Comparison of automated and manual techniques for analysis of DNA frequency distributions in bladder washings

Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant of the relatively low signal-to-noise ratios often found in DNA distributions obtained from clinical samples. Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S. A computer-based method has been developed for extraction of the above-mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight-line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer-based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer-based method. Correlation of percent hyperdiploid cell fraction was also excellent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

c-erbB-2/neu protein expression, DNA ploidy and S phase in breast cancer

Abstract. DNA content and c-erbB2/neu protein (p185) expression were evaluated by flow-cytometry and ELISA, respectively, in 166 specimens of primary breast cancer. A non-diploid DNA content was found in 88 tumours (53%), with the DNA index ranging from 0.7-2.7. S phase fraction (SPF) evaluation, performed in 130 cases, showed significantly higher values in aneuploid than in diploid tumours (median values, 17.3% and 5.8%, respectively). Thirty-six tumours (21.6%) showed p185 overexpression, while 45 (27.1%) and 85 (51.3%) showed intermediate and low expression, respectively. A good correlation ( P =0.0023) was found between DNA content and p185 positivity. Tumours with high p185 values were mainly aneuploid, while tumours with intermediate or low expression had variable degrees of DNA content. Furthermore, p185 concentration was significantly higher in aneuploid than in diploid tumours ( P =0.009). The highest rate of p185 (+) cases and the highest p185 concentrations occurred in triploid (1.3相似文献   

Dilution effect of nontumor cells on flow cytometric DNA S-phase fraction in breast cancer fine needle aspirates

OBJECTIVE: To analyze the proportion of nontumor cells in fine needle aspirates of breast carcinoma and its influence on flow cytometric S-phase fraction (SPF) estimation. STUDY DESIGN: We analyzed the proportion of nontumor cells in fine needle aspiration biopsy smears, performed flow cytometric analysis of DNA ploidy and SPF on freshly aspirated tumor material and analyzed histograms manually and automatically using Multi-Cycle AV software (Phoenix Flow Systems, San Diego, California, U.S.A.) for cell cycle analysis. We corrected SPF of diploid tumors for the dilution effect using an individually established percentage of nontumor cells (individual correction) and the mean proportion of nontumor cells in diploid tumors (factor correction). RESULTS: The proportion of nontumor cells ranged from 0.5% to 76.6% (mean, 12.6; SD, 15.7) in 55 diploid tumors and from 0.5% to 53% (mean, 8.6; SD, 8.9) in 84 aneuploid tumors (p=0.178). In 14 of 139 (10%) samples, the proportion of nontumor cells exceeded 20%. The mean SPF values of diploid tumors without correction were 4.9% (manually) and 6.5% (automatically) and of aneuploid tumors, 9.5% and 11.0%, respectively. In univariate Cox survival analysis, noncorrected SPF was a significant prognostic factor in overall survival (p < 0.001). Neither individual nor factor correction of SPF significantly changed its prognostic value. CONCLUSION: Fine needle aspirates contain low proportions of nontumor cells, having an insignificant dilution effect on SPF estimation. Most probably, SPF could be reliably estimated usingfreshly aspirated tumor material without any correction or adjustment.     

Methodologic aspects of DNA assessment by means of image cytometry in tumors of the salivary glands. A comparison between the results obtained using sections and cytospin preparations from the same paraffin-embedded specimens

The image cytometric nuclear DNA assessments on paraffin-embedded tissue sections and on Cytospin preparations of disaggregated specimens from the same cases were compared in 98 salivary gland tumors, including 21 acinic cell carcinomas, 29 mucoepidermoid carcinomas, 21 adenocarcinomas and 27 adenoid cystic carcinomas. The histogram type (diploid, tetraploid or aneuploid) and the number of cells with DNA values greater than 2.5c (expressed in relative units) were considered as variables in the correlation. A high correlation between the results in different specimens was found in acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas; the histogram type and the number of cells with DNA values greater than 2.5c were essentially the same between specimen types in these three tumor entities. The cases of adenoid cystic carcinomas showed a considerably lower degree of correlation: in 8 of the 27 cases, the Cytospin preparations yielded diploid histograms, while the tissue sections yielded aneuploid histograms. The number of cells with DNA values greater than 2.5c was notably lower in the Cytospin preparations from adenoid cystic carcinoma; the reasons for this exceptional behavior of the cells of adenoid cystic carcinoma are discussed. These findings demonstrate that paraffin-embedded specimens of different tumor entities, even from the same organ, can be affected differently by disaggregation procedures. While retrospective studies on disaggregated paraffin-embedded specimens can yield reliable results, comparative assessments using both DNA analysis techniques, as in this study, should be performed before a large number of cases is evaluated.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

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

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

Applicability of liquid-based cytology to the assessment of DNA content in cervical lesions using static cytometry

OBJECTIVE: To evaluate the nuclear DNA content of cervical lesions in liquid-based cytologic specimens prepared for static cytometry. STUDY DESIGN: The DNA content of cervical lesions was evaluated in cervical samples prepared with the Autocyte PREP liquid-based cytology system (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.). A series of 47 samples stained with the Papanicolaou method (chronic cervicitis, n = 15; cervical intraepithelial neoplasia [CIN] 1, n = 25; CIN 2, n = 5; CIN 3, n = 2) were collected from consecutive women enrolled in an ongoing screening study at Leonor Mendes de Barros Hospital, S?o Paulo, Brazil, in 2002. Each residual sample was processed according to the Feulgen-thionin method (TriPath Imaging). Ploidy evaluation was performed using the CAS 200 image analysis system and Quantitative DNA Measurement software 3.0 (version 8.1) (Becton Dickinson, San Jose, Califoria, U.S.A.). Cellular ploidy was analyzed from atypical nuclei, and the DNA index was obtained using histograms for interpretation. RESULTS: All chronic cervicitis cases were diploid. Of the CIN 1 cases, 44% were diploid, 12% tetraploid, 32% aneuploid and 12% polyploid (diploid plus tetraploid). CIN 2 lesions were diploid in 60% and aneuploid in 40% of cases, whereas all CIN 3 lesions (100%) were aneuploid. CONCLUSION: The liquid-based cytologic samples proved to be suitable and highly useful for DNA analysis by image cytometry, which was capable of discriminating CIN 3 lesions from CIN 1 and 2 but not CIN 1 from 2 lesions. Aneuploidy was closely associated with CIN 3 lesions.     

Flow cytometric S-phase fraction measurement in breast carcinoma: Influence of software and histogram resolution

BACKGROUND: S-phase fraction (SPF) measurement by flow cytometry is a clinically useful prognostic factor in patients with breast carcinoma. Standardized SPF determination is essential. As part of a multicenter study, we evaluated the influence of the choice of software and histogram resolution (256, 512, or 1,024 channels) on SPF quantification. METHODS: One hundred thirty-three DNA histograms were analyzed in three laboratories with Modfit 5.2, Modfit LT, and Multicycle AV software. Strict rules for histogram interpretation and software management were applied. The following five options were compared: MF 5.2 1024, MF 5.2 256, MF LT 256, MC AV 256, and MC AV 512. RESULTS: In the DNA diploid and aneuploid groups, SPF distributions were not statistically different among the five options. Excellent quantitative correlations were obtained between pairs of options. When using tertiles as cutpoints for SPF classification, concordance rates ranged from 79.7% to 93.2% for DNA diploid samples and from 87.8% to 95.9% for DNA aneuploid samples, the best results being obtained with software working with a similar histogram resolution. CONCLUSIONS: Standardized use of commercially available software, including the choice of histogram resolution, provides comparable SPF results.     

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation ( CV ) from 2 to 16%; (ii) constant measurement CV or CV 's increasing from G₁ to G₂ phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.
The fractions of cells in the G₁, S, and (G₂+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G₁ fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

Discrimination between precancerous and cancerous lesions of the uterine cervix by DNA measurements on tissue sections

Morphologically typical uterine cervical biopsies were separated into normal cervices, condylomas and cervical intraepithelial neoplasias (CIN) grades I, II and III. At least 100 nuclei per lesion were measured on 4 micron Feulgen-stained sections using a Zeiss microspectrophotometer, with a variant of the plug method used to compute the nuclear DNA content. DNA distribution histograms were then decomposed into subsets of diploid, tetraploid, octoploid and aneuploid cells. The decomposition, which assumed a log-normal model of polydiploidy distribution, led to the identification of six indices: (1) the percentage of diploid cells, (2) the percentage of tetraploid cells, (3) the percentage of octoploid cells, (4) the percentage of aneuploid cells with DNA contents less than tetraploidy, (5) the percentage of aneuploid cells with DNA contents between tetraploidy and octaploidy and (6) the percentage of aneuploid cells with DNA contents greater than octoploidy. These indices, along with the mean nuclear radius, the 5c exceeding rate and the 2c deviation index, generated a nine-dimensional space. Two methods of discriminant analysis on this space showed discriminating powers of 78.22% and 87.13%, respectively, as compared to the original diagnoses. The most discriminating variable in both analyses appeared to be the percentage of octoploid cells.     

DNA microspectrophotometry of bone sarcomas in tissue sections as compared to imprint and flow DNA analysis

The aim of the present study was to establish an upper limit of diploidy for microspectrophotometric (MSP) DNA measurements in sections of mesenchymal tissue analyzing DNA data of a large number of normal cell populations. The reliability of this upper limit of diploidy for discriminating between diploid and hyperploid bone sarcomas was tested by analyzing the same tumors by MSP in imprint preparations and flow cytometry (FCM). The median DNA value of control cells in tissue sections was given arbitrary value of DNA index (DI) 1.0, denoting the diploid DNA content. The proportion of cells with DNA values exceeding DI 1.25 (greater than DI 1.25) was determined for each normal cell population. The maximum percentage of cells with DNA values exceeding DI 1.25, encountered by analysis of 91 normal cell populations in tissue sections, was 31%. This percentage was set as an upper limit of diploidy. Hence, tumors with a higher percentage of cells greater than DI 1.25 were classified as hyperploid. When we applied this criterion, 31 of 36 sarcomas analyzed by MSP in tissue sections were hyperploid, which was in complete agreement with FCM and MSP in imprints of the same tumors. Apart from discriminating between diploid and hyperploid tumors, an attempt was made to determine peak DNA values of sarcomas analyzed in tissue sections. Peak DNA values, as defined by a minimum of 30% of the cells within a class width of DI 0.25, could be determined for 23 of 36 tumors. These peak DNA values correlated well with corresponding peaks obtained by FCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.

BACKGROUND AND AIM: Current evidence suggests a possible relationship between DNA ploidy status and Ki-ras gene mutations in human cancers. However, the conventional method does not enable accurate determination of DNA ploidy status of a tumor cell. The present study attempts to clarify whether Ki-ras gene mutations are associated with DNA ploidy status in sporadic colorectal carcinomas using a crypt isolation technique coupled with DNA cytometric sorting. METHODS: Polymerase chain reaction and single-strand conformation polymorphism and direct sequencing were used to analyze Ki-ras gene mutations in 82 sporadic colorectal carcinomas: 21 diploid, 12 aneuploid, and 49 multiploid. In addition, microsatellite instability (MSI) was assessed using seven microsatellite markers to study the relationship to Ki-ras mutations. RESULTS: Ki-ras mutations were found in 12 of 21 diploid carcinomas and in 8 of 12 aneuploid carcinomas. In contrast, Ki-ras gene mutations were detected infrequently in the 34 multiploid carcinomas examined, 8 of which were seen in diploid populations and 10 in aneuploid populations. On the other hand, Ki-ras gene mutations were inversely correlated with MSI, which was found in diploid carcinomas only. CONCLUSIONS: The low frequency of Ki-ras gene mutations that we observed in multiclonal colorectal carcinomas suggests that development of multiclonal colorectal carcinoma may involve a mechanism different from that involved in the development of diploid or aneuploid colorectal carcinomas.     

Intratumoral variations in DNA distribution patterns in mammary adenocarcinomas

Correlated flow-cytometric (FCM) and microspectrophotometric (MSP) techniques were applied to investigating whether intratumoral variations in the DNA distribution patterns of 21 primary mammary adenocarcinomas can occur. Although neoplastic cell populations with both diploid and tetraploid (i.e., euploid) distribution patterns could be found in varying proportions in some of the tumors, there was no evidence in any tumor nodule for the presence of euploid populations in one part and aneuploid populations in another. This statement was based on the results of the MSP technique, where the assessments were made on cytodiagnostically identified neoplastic cells. Also, when applying the FCM technique the statement was found to be essentially valid; only one of the tumor nodules showed a DNA distribution pattern that, by means of the criteria used in this procedure, was defined as being both euploid and aneuploid. Here, however, the technique consists of assessments made on a great number of microscopically non-identified cells. It was concluded that when conflicting reports are given from different laboratories on the prognostic value of the cytochemically assessed DNA distribution patterns in breast carcinomas, they are not likely to be attributed to intratumoral DNA heterogeneity but, rather, to differences in the methods used and in the criteria applied for the so-called ploidy assessments.     

Automated identification of diploid reference cells in cervical smears using image analysis

BACKGROUND: Acquisition of DNA ploidy histograms by image analysis may yield important information regarding the behavior of premalignant cervical lesions. Accurate selection of nuclei for DNA measurement is an important prerequisite for obtaining reliable data. Traditionally, manual selection of nuclei of diagnostic and reference cells is performed by an experienced cytotechnologist. In the present study, a method for the fully automated identification of nuclei of diploid epithelial reference cells in Feulgen- restained Papanicolaou (PAP) smears is described. METHODS: The automated procedure consists of a decision tree implemented on the measurement device, containing nodes with feature threshold values and multivariate discriminant functions. Nodes were constructed to recognize debris and inflammatory cells, as well as diploid and nondiploid epithelial cells of the uterine cervix. Evaluation of the classifier was performed by comparing resulting diploid integrated optical densities with those from manually selected reference cells. RESULTS AND CONCLUSION: On average, automatically acquired values deviated 2.4% from manually acquired values, indicating that the method described in this paper may be useful in cytometric practice.     

A method of DNA histogram analysis by computer and its evaluation by simultaneous combination with 3H-thymidine autoradiography

A method for determining the fractions of cells in the G1, S, and G2 + M phases of the cell life cycle, by quantifying DNA histograms derived from static fluorescence cytophotometry, was evaluated by simultaneous combination with 3H-thymidine autoradiography. DNA histograms were obtained by cytofluorometry on the Feulgen-stained autoradiographs of HeLa cells, and mouse and rat hepatocytes, after DNA labelling with 3H-thymidine. The synthetic histogram determined by "sum of discrete normal curves" technique was fitted to the experimental data according to a weighted least-squares method by a desk-top computer (HP 85F). The mean relative percent deviations of estimated cell cycle phase fractions from the actual phase fractions determined directly on an autoradiograph was 6.6 +/- 3.3%.