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PubMed1998

[Correlation of flow and static cytometry; their application to the study of anaplastic lymphomas].

PURPOSE: DNA study by cytometric methods is one of the prognosis factors considered in malignant tumours. Flow cytometry (FCM) was the most frequently used techniques in cell suspensions. Image cytometry (ICM) was also applied in cellular smears and it is possible to measure the results with an Image Analyzer, which supposes a substancial advantage over DNA studies. To confirm the results and correlation of the two techniques a controversial subtype of lymphoid tumour was selected: Anaplastic large cell lymphoma (ALCL). MATERIAL AND METHODS: Fifty four cases of ALCL (23 classical type and 31 ACL-Hodgkin related) were studied. Cytometry was performed in paraffin-embedded tissues previously dewaxed, rehydrated and minced. FCM was done in suspensions incubated with ribonuclease A and stained with propidium iodide in an EPICS-C flow cytometer. ICM study was performed in Feulgen-stained smears and measured by an Image Analyzer CAS-200. RESULTS: All cases were aneuploid. ALCL were 30.5% hypodiploid (HpD) and 69.5% hyperdiploid (HrD) by FCM; 43.5% HpD and 56.5% HrD by ICM. ALCL-HR were 58% HpD and 42% HrD by FCM; 68% HpD and 32% HrD by ICM. There was a lack of correlation of 22% between both methods but it was not statistically significant. CONCLUSIONS: We can conclude the obtained results by FCM and ICM are almost similar.

Aneuploidy↗
PubMed1998

Critical aspects in the analysis of apoptosis and necrosis.

Despite that large number of methods to analyze cell death, in particular apoptosis, that have been developed, identification of the mode of cell death and its quantitation is not always simple or straightforward. The difficulties, potential pitfalls and traps in quantitation of dead cells, whether apoptotic or necrotic are reviewed. The following are common flaws in the measurement of cell death, which include incorrect assumptions, erroneous data interpretation and shortcomings of the methodology: 1) Misclassification of apoptotic bodies or chromatin fragments as individual apoptotic cells based on cellular DNA content analysis by flow cytometry; 2) Assumption that the quantity of fragmented DNA extracted from cells represents the frequency of apoptosis; 3) Conjecture that the apoptotic index represents the cell death rate; 4) Assumption that apoptotic cells must exhibit classical features of apoptosis e.g. internucleosomal DNA fragmentation; 5) Inadequacy of methods that presume to discriminate between late apoptotic and necrotic cells; 6) Possibility of a selective enrichment or loss of apoptotic cells during cell separation on density gradients, during trypsinization or other procedures of cell collection; and 7) Inability to distinguish between live, nonapoptotic cells phagocytizing apoptotic bodies and genuine apoptotic cells by flow cytometric methods. Many of the problems stem from the difficulty in identifying apoptotic or necrotic cells. Because apoptosis and necrosis have been originally defined based on morphological criteria it is essential to confirm the mode of cell death by microscopy. Laser scanning cytometry (LSC), which combines the advantages of flow and image cytometry, offers the possibility of morphological examination of apoptotic cells. By virtue of this attribute LSC appears to be the instrument of choice for analysis of apoptosis.

Apoptosis↗
PubMed1991

Characterization of conservatively resected renal tumors using automated image analysis DNA cytometry.

The DNA histograms of 57 conservatively resected renal tumors were studied using automated image analysis DNA cytometry (Leytas II). Forty-nine of the analyzed tumors were renal cell carcinomas, six were oncocytomas, one was an angiomyolipoma, and one was a renal cell adenoma. On the basis of their DNA histograms, diploid, tetraploid, and aneuploid tumors could be distinguished. Aneuploid tumors could be subtyped further according to the DNA content of the stem cell line as hyperdiploid, hypertriploid, or hypertetraploid. Eight of the tumors were characterized by a combination of diploid and hypertriploid stem cell lines. During a mean follow-up of 5 years, only the two patients with a pure hypertriploid tumor died of distant metastases. These results indicate that automated DNA image analysis cytometry is able to differentiate among several types of renal tumors with obviously different prognoses.

Adenoma↗
PubMed2000

The effect of hyperthermia on DNA in mouse HepA cells--an analysis with computer and observation with EM.

A computer analysis system, automated imaging cytometry (AIC), flow cytometry (FCM) and EM, were used to study the relationship of apoptosis and the change of DNA in mouse HepA cells subjected to hyperthermia. Our results showed that pyknotic rate in the 44 degrees C group was significantly increased (P < 0.01) when compared with that in the 37 degrees C group, both the diploid rate and aneuploid rate in the 44 degrees C group were significantly decreased (P < 0.01) when compared with that in the 37 degrees C group. The pyknotic rate determined by ALC was in conformity with the apoptotic index (AI) determined by FCM. Under EM, ultrastructures of HapA cells appeared typical apoptosis changes in the 44 degrees C group. We are led to conclude that (1) when the temperature is 42 degrees C or 44 degrees C for 2 h, it did not promote the necrosis of HepA cells; (2) when the temperature is 42 degrees C for 2 h, it did not obviously induce the apoptosis of HepA cells, and the 44 degrees C for 2 h obviously induced the apoptosis of HepA cells; (3) AIC method can be used as an aid for the measurement of apoptotic index. Further study is needed to established it as a novel, simple and convenient method for determining the apoptosis.

Animals↗
PubMed1995

Radial scar versus tubular carcinoma of the breast. A comparative study with quantitative techniques (morphometry, image- and flow cytometry).

The present study is focused on the differential diagnosis between radial scar (RS) and tubular carcinoma (TC) using morphometrical and cytophotometrical analysis (static and flow cytometry) of a number of histologically well-established RS cases (17 lesions) compared with 6 early infiltrating small TC with sclerotic stroma and pseudo-RS fields. One case displayed both RS and TC foci in contiguity. Mean nuclear area was larger in the group of tubular carcinomas (51.0 mu 2) than in the case of radial sclerosis (38.30 mu 2). We also found a larger number of aneuploid cases in tubular carcinomas measured by image cytometry, but both types of lesions were diploid when measured by flow cytometry; only one case of radial scar resulted aneuploid.

Adenocarcinoma↗
PubMed1992

Interactive cytometry, chance or evil of bias?

Interactive selection of a limited number of cells in imaging cytometry for determining the DNA histogram of breast cancer cells as the best known prognosticator at the moment, implies statistical and systematic sampling problems. Analysis of histograms of 361 breast cancer aspirate specimens measured in two laboratories demonstrate the expected high statistical variations in view of the only 100 cells measured per case but also slight systematic differences. Controlled systematic sampling without pathological bias results in a somewhat higher malignancy grading than selective biased sampling. For this finding we have no explanation. The main result is, however, that we did not find the expected contrary which makes the argument for at least this application invalid that expert pathologists are needed for reliable interactive sampling.

Adenocarcinoma↗
PubMed2004

Cytologic and DNA-cytometric very early diagnosis of oral cancer.

BACKGROUND: The aim of this study was to evaluate the diagnostic accuracy of exfoliative cytology (EC) and DNA-image cytometry applied to suspicious oral lesions compared with synchronous histology. METHODS: Brush- and scalpel biopsies were obtained from 98 patients with suspicious oral lesions. In cases, in which EC revealed malignant or suspicious cells, nuclear DNA-contents were measured using a TV image analysis system. RESULTS: Among 98 oral lesions both cytological and histological diagnosis showed no sign of malignancy or dysplasia in 75. In 23 cases cytology yielded tumor cell-positive (15), suspicious (four) or doubtful (four) results. DNA-cytometry showed aneuploidy in 19 of these. The comparison between cytological diagnosis combined with DNA-cytometry and biopsy-histology resulted in a sensitivity of 100% and a specificity of 97.4%. CONCLUSION: In conclusion, cytology with DNA-cytometry is a highly sensitive, specific and non-invasive method for the early diagnosis of oral epithelial neoplasia, showing excellent compliance among patients.

Adult↗
PubMed1993

Standardization and quality control of quantitative microscopy in pathology.

Standardization and quality control of quantitative microscopy techniques are distinct but related concerns. The first deals with the great variety of quantitative methods, measured features, and even response variables used in investigation of biological or clinical processes. The latter deals with reproducibility of results from those investigations across time and test performance sites. Though distinct, efforts for standardization and quality control are inherently interactive. Consensus on standard methods, instrumentation, and data analysis is hard to achieve in fields developing as rapidly as quantitative microscopy. Consensus is possible, however, on the issues that affect test performance and interpretation. For example, issues of specimen type, fixation, processing, and staining affect image cytometry just as they do flow cytometry. Raw data acquisition issues include area sampling rules and fidelity of optical and sensor systems (light wavelength, glare/stray light, lens aberrations, numerical aperture, depth of focus, scan precision, pixel spacing and depth, sensor linearity, and stability). Intermediate data issues are primarily related to image foreground/background segmentation techniques--automated versus manual, object-specific versus field-based. Data reduction and interpretation procedures also provide many roads for divergence from uniformity. Each of these issues must be considered in terms of its effect on comparability and utility of quantitative microscopy results. Quality control for quantitative microscopy is as important as standardization for its use in research programs and with clinical specimens. The sine qua non of quality control is comparison of experimental results against a known "correct" value to estimate accuracy, and against other experimental results to estimate precision.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonic Neoplasms↗
PubMed1990

A prospective comparison of DNA quantitation by image and flow cytometry.

Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type. The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan). For 81 cases, there was excellent correlation between the two methods. For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry. Three cases had aneuploid peaks by flow cytometry that were not identified by image. Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation. Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.

Adenocarcinoma↗
PubMed1993

[Image analysis DNA cytometry in medullary thyroid gland cancer. Comparison with histomorphologic parameters and prognostic effect].

In 24 patients with a medullary thyroid cancer a histopathological reexamination was performed. In 16 patients the DNA content was measured. Patients with a diploid or hypotriploid tumor had a 5-year survival rate of 100% in contrast to 48% of patients with a triploid or hypertriploid aneuploid tumor. Aneuploid tumors were more often found in patients with a high pre- or postoperative calcitonin level and lymph node metastasis. No correlation was found between the DNA content and the patients age, sex, preoperative CEA value, tumor size, pT4 stage, invasion of vessels calcitonin immunoreactivity, silver or amyloid staining, and distant metastasis.

Adolescent↗
PubMed1997

Evaluation of autofocus functions in molecular cytogenetic analysis.

This work describes a systematic evaluation of several autofocus functions used for analytical fluorescent image cytometry studies of counterstained nuclei. Focusing is the first step in the automatic fluorescence in situ hybridization analysis of cells. Thirteen functions have been evaluated using qualitative and quantitative procedures. For the last of these procedures a figure-of-merit (FOM) is defined and proposed. This new FOM takes into account five important features of the focusing function. Our results show that functions based on correlation measures have the best performance for this type of image.

Algorithms↗
PubMed1994

Automated cytometry of fibre size and spatial distribution in the superficial masseter muscle of the rat at three ages.

An image cytometry program was applied to sections of the superficial masseter muscle of female and male 21-, 42- and 105-day-old rats. Lesser diameter and spatial distribution of more than 6000 muscle fibres were automatically measured in digital images from muscle sections stained for myofibrillar ATPase activity. In this muscle, only type 2A, 2B and 2C fibres were observed. At the three ages and in both sexes, 2A fibres were found to have the largest diameter and were the most frequent (> 54%). In the 21-day-old animals, females presented larger diameters than did males; in contrast, in the 105-day-old animals, the three fibre types were larger in males than in females. At all ages and in both sexes, type 2A occupied 32 to 80% more cross-sectional area than type 2B. Most images (98%) showed a random spatial distribution of their fibre types. All fibre types grew in diameter with age. The coefficient of variation of the diameter was age-independent and remained under 23%. The finding of an age-independent variable could have a practical application: an increase of the coefficient of variation (> 25%) can be considered as pathological, even without a perfect age-matched control.

Aging↗
PubMed1993

Quantitative microscopical and confocal laser scanning microscopy for intermediate endpoint biomarkers in breast cancer: potential and reproducibility.

Diagnostic quantitative pathological (QP) determinations are increasingly used in our hospital. The number of requests for QP for reference materials is rising rapidly. This is understandable; quantitative assessments have a strong prognostic value and can be very reproducible, depending on the care taken with a number of factors including cell and tissue processing, application of the appropriate stains, and the measurement protocol used. As to the latter, systematic random sampling gives the best intra- and interobserver agreement (with correlation coefficients between observers for certain features > or = 0.94). Flow cytometric determinations are often regarded as more reproducible than interactive morphometry due to the high speed of the assessments, the large number of objects measured per specimen, and the lack of observer interaction. Indeed, flow cytometrically assessed DNA ploidy is very reproducible, even though the % S-phase fraction is much more variable. Unlike image cytometry (ICM), visual inspection of cells is not easily accomplished with flow cytometry (FCM). With ICM, the fully automated measurement of DNA in thousands of cells is possible in 3-5 minutes, with a very low coefficient of variation (< or = 2% for the diploid and tetraploid peak of liver cell nuclei). ICM also allows measurement of texture features. However, quantitative immunohisto/cytochemical determinations may not always be as reproducible as sometimes believed. Recently, we found large variations in the measurements, made by a commercially available image processing instrument, of the estrogen and progesterone receptors, Ki-67, cathepsin D, and neu protein overexpression in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1994

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↗
PubMed1993

Detection of false DNA aneuploidy and false DNA multiploidy in flow cytometric DNA analysis.

Although false DNA aneuploid peaks have previously been described in normal tissue, criteria for distinguishing them from 'true' near-diploid peaks have not been established. Normal thyroid (n = 4) and kidney (n = 1) tissue were allowed to autolyze over a fixed period of time and DNA content was analyzed by flow cytometry (FCM). Autolysis was associated with the development of distinct separate G0/G1 peaks which had low DNA indices (1.09-1.18) and showed decreased forward light scatter (FSC) when compared to fresh tissue. Using DNA content and FSC measurements similar false DNA aneuploid peaks were identified in 29/94 surgical specimens. These cases included both benign and malignant lesions from thyroid (n = 63) with the remaining 31 neoplastic cases being from breast (16), lymphoma (8), sarcoma (4), lung (2) and uterine (1) tissue. In addition, false DNA multiploidy was identified. None of these cases showed histological evidence of necrosis. In a parallel comparison study using image cytometry (ICM) on the thyroid nodules, the presence of false DNA aneuploidy was supported. Investigators should routinely employ quality control criteria to identify possible cases of false DNA aneuploidy when measuring DNA content using FCM.

Adolescent↗
PubMed1988

DNA cytometry of pure dysgerminomas of the ovary.

The value of DNA cytometry for predicting the malignant potential of pure dysgerminomas of the ovary was investigated. Feulgen-thionin DNA image cytometry and propidium iodide DNA flow cytometry were performed in isolated nuclei from paraffin-embedded tissue of 25 dysgerminomas. Nineteen were in clinical stage Ia1, and six were in stage III (two in IIIa and four in IIIb). Eight patients showed recurrences during a 5-year follow-up period and one patient died of the disease. The DNA index (DI; the modal DNA value compared with that of normal cells) of the tumor cells was computed from the image and flow DNA histograms. Comparable DI values, ranging from 1.29 and 3.23, were found with both types of cytometry. No correlation was found with the clinical stage or the recurrence state. Furthermore, it was striking that, although values were found strongly deviating from the normal diploid content (DI = 1.0) suggesting an unfavorable prognosis, the survival rate was relatively high. It can be concluded that prediction of the clinical course of pure dysgerminomas by DNA cytometry does not seem feasible.

Cell Nucleus↗
PubMed1993

Quantitative morphologic assessment of nuclei extracted from paraffin for DNA flow cytometry.

We present a method for quality control of flow cytometric DNA content studies using nuclei extracted from paraffin-embedded tissue. This method is based on a quantitative morphologic assessment of extracted nuclei. Cell nuclei prepared from 22 paraffin-embedded tumors known to contain discrete diploid and aneuploid stemlines were deposited onto poly-L-lysine-coated glass slides. Nuclei were stained with Diff-quic and examined by light microscopy. Two hundred nuclei were counted and classified based on morphologic appearance into tumor and nontumor groups. Classification criteria included differences in nuclear size, nuclear chromatin structure, the degree of nuclear chromatin condensation, and the presence of nucleoli. Excellent agreement was found between two independent observers (R = 0.989) on the classification of nuclei. The relative number of tumor nuclei on the morphologic preparation was compared with the relative number of aneuploid cells in the DNA histogram. Good agreement was observed (R = 0.975) in all but three cases in which the relative number of tumor nuclei was underrepresented by the percentage of aneuploid nuclei in the DNA histogram. In each case, further analysis by image cytometry demonstrated a diploid and aneuploid component of the tumor cell population. This quantitative method of morphologic examination of the preparation ultimately analyzed by flow cytometry offers several distinct advantages, including: (a) identification of peaks in the DNA histogram, (b) assessment for selective loss of cell nuclei in the extraction process, and (c) identification of biologic heterogeneity in tumor populations.

Cell Nucleus↗
PubMed1990

[Diagnosis of prospective malignancy of cervix dysplasia using DNA cytometry].

134 borderline lesions of the cervix uteri (CIN I/II) were investigated by using the newly developed diagnostic method DNA-image cytometry (MIAMED-DNA, Wild-Leitz, Wetzlar). The demonstration of aneuploid cells served as a marker for prospective malignancy. 18 out of 35 cases, which proved to be CIN III in the follow-up, had a malignant DNA diagnosis. The sensitivity of the diagnostic DNA cytometry for prospective malignancy was therefore 51.4%. All 43 cases with a negative DNA diagnosis proved to be negative in the follow-up, so that the specificity of DNA cytometry was 100%. Suspicious DNA diagnosis in 26% of the cases proved to be CIN III in the follow-up. DNA cytometry seems to be a reliable method to predict the biological behaviour of borderline lesions of the cervix uteri in everyday practice.

Carcinoma in Situ↗