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PubMed1994

Congenital melanocytic nevi and DNA content. An analysis by flow and image cytometry.

BACKGROUND: Potential risk factors for the development of melanoma in congenital melanocytic nevi (CMN) are not well established. DNA aneuploidy may constitute such a risk factor but has not been sufficiently studied in CMN. METHODS: In the present study, DNA analysis of eight giant CMN, nine medium CMN (1.5-20 cm), and eight small CMN (< 1.5 cm) was assessed by flow cytometry and selected lesions (six nevi) by DNA image cytometry. DNA content was correlated with patient age, nevus size, and degree of cytologic atypia. RESULTS: DNA aneuploidy was detected by flow cytometry in two giant CMN from adult patients and in a small CMN from a child. DNA aneuploidy was not observed in any of the six CMN studied by image cytometry, although an increased S-phase was noted in a markedly atypical giant CMN. No DNA aneuploidy was detected in medium-sized CMN or in the CMN of nine patients 1 year of age or younger. CONCLUSION: In contrast to previous studies, it was observed that abnormal DNA content does tend to correlate with cytologic atypia, particularly in giant CMN with atypia or melanoma, in adults. Conversely, frank DNA aneuploidy in any CMN in children younger than 1 year of age, irrespective of histologic findings, was not detected. Finally, based on these limited studies, greater sensitivity of image over flow cytometry for detection of DNA aneuploidy cannot be verified.

Adolescent↗

PubMed1993

Flow and image cytometry for DNA analysis in bladder washings: improved concordance by using internal reference for flow.

Using flow or image cytometry, we compared the DNA distribution of cells from bladder washings from 52 patients with bladder cancer. For image cytometry, urothelial nuclei (recognized visually) were analyzed for DNA content using polymorphonuclear nuclei as internal diploid reference. For flow cytometry, two methods can be used: either all cells can be analyzed, as commonly performed, or urothelial cells can be analyzed alone, after specific detection. In this flow cytometry study, cells were doubly stained for panurothelial antigens T16 and for DNA. All the cells were first analyzed using peripheral lymphocytes as an external reference; 79% of the results were similar with results obtained from image analysis. For discordant results, flow-cytometric data were reprocessed to identify immunologically stained urothelial cells; one additional case became concordant with image cytometry when only urothelial cells were analyzed, with lymphocytes as diploid reference; a better concordance (94%) was found when the nonurothelial cells of the samples served as a diploid reference instead of peripheral lymphocytes. This suggests that we achieved an improvement of the flow-cytometric evaluations for ploidy assessment, and we conclude that, on these conditions, flow or image analysis can be considered as equivalent methods for DNA content studies of tumors.

Adult↗

PubMed1993

Multiparametric image cytometry of nevi and melanomas.

Multiparametric image cytometry was applied to 10 examples each of malignant melanoma and common, Spitz, and dysplastic nevus. DNA index, area, and 21 parameters describing chromatin texture were measured for 50 nuclei in each lesion. Linear discriminant analysis was used to derive discriminant functions based on the measured parameters. The analysis demonstrated that chromatin texture provides more diagnostic information than either DNA index or nuclear area. The discriminant functions allowed 68% of nuclei to be accurately classified among the four groups, and allowed 37 of the 40 lesions to be accurately classified as nevus or melanoma.

Analysis of Variance↗

PubMed1992

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.

Aneuploidy↗

PubMed1999

Influence of sample size on image cytometry of DNA ploidy measurements.

OBJECTIVE: To assess the number of nuclei required for significant image cytometry DNA ploidy measurements on one archival case of breast cancer. STUDY DESIGN: From one case of aneuploid DNA breast cancer, 18 subsets made up of 152-1,524 for the whole population of undamaged nuclei and made up of 74-735 epithelial nuclei had DNA measured. DNA ploidy type and five DNA ploidy indices, allowing DNA ploidy histogram interpretation were evaluated on each population. RESULTS: Three hundred nuclei were always sufficient for DNA typing, whereas reliable results for DNA ploidy indices required at least 750 nuclei. CONCLUSION: To DNA measure the above number of nuclei, fully automated image cytometry DNA ploidy measurements are required.

Aneuploidy↗

PubMed2001

Fourth updated ESACP consensus report on diagnostic DNA image cytometry.

A task force of experts in the field of diagnostic DNA image cytometry, invited by the ESACP, and further scientists or physicians revealing experience in that diagnostic procedure (names are given in Addendum A), agreed upon the following 4th updated Consensus Report on Standardised Diagnostic DNA Image Cytometry during the 7th International Congress of that society in Caen, 2001. This report is based on the three preceding ones [6,14,17]. It deals with the following items:- Critical review and update of the definitions given in the 1997 Consensus Update;- Review and detailed description of basic terms, principles and algorithms for diagnostic interpretation;- Recommendations concerning diagnostic or prognostic applications in specific fields of tumour pathology. This update is not aimed to substitute the 1997 consensus, but to make necessary addenda and give more detailed descriptions of those items not unequivocally to interpret by potential users of the methodology.

Algorithms↗

PubMed1999

Density gradient centrifugation of colonic fluid after segmental lavage: a method of purification of exfoliative epithelial colonic cells for cytological interpretation and image cytometry in patients with long-standing ulcerative colitis.

OBJECTIVES: Patients with extensive, long-standing ulcerative colitis (UC) have an increased risk for developing colorectal cancer. In this study, we wanted to establish a method for retrieving cytological material after segmental colonic lavage for further cytopathological investigations and for performing DNA image cytometry. METHODS: Ten patients with long-standing and extensive ulcerative colitis and 10 patients without macroscopic abnormalities were investigated. After segmental colonic lavage during routine colonoscopy a three-layer (1.146, 1.075, and 1.046 g/ml, respectively) density gradient centrifugation of the retrieved colonic fluid was performed for isolation and purification of the epithelial cells. For identification of the epithelial cells flow cytometry with monoclonal antibody against cytokeratin and counterstaining with propidium iodine was performed. The smears obtained were stained for routine cytopathological interpretation and for DNA image cytometry. RESULTS: In eight of 10 UC patients and in nine of 10 control group patients adequate cytological material could be obtained. The band on top of the density gradient at 1.046 g/ml could be identified as the epithelial cells. Atypical cells were found in smears of three UC patients. In these patients and in one additional patient aneuploid stemlines could be detected. In smears of control group patients neither atypical cells nor aneuploidy were present. CONCLUSIONS: Isolation and purification of epithelial cells after segmental colonic lavage by using density gradient centrifugation was performed. This cytological material is adequate for cytopathological interpretation and for DNA image cytometry. Information about atypical cells and DNA aneuploidy as an additional marker of malignant transformation in UC patients was obtained. The combination of cytological examination and DNA image cytometry might improve the detection of UC patients with high risk for colorectal cancer.

Aneuploidy↗

PubMed1999

[Automated quantitative image cytometry of bronchial washings in suspected lung cancer: comparison with cytology, histology and clinical diagnosis].

INTRODUCTION: Automated image cytometry represents a new method for the quantitative analysis of nuclear structure and DNA-content of exfoliative airway epithelial cells. In the present investigation, we examined the correlation between automated cytometry, conventional cytology and histopathology with the final diagnosis as the "gold standard". METHODS: In 142 patients (100 males and 42 females) with suspected lung cancer and 50 controls (COPD, asthma), bronchial washings (5-10 ml) were obtained during bronchoscopy before taking biopsies for cytological and/or histological examinations. The washings were collected in 20 ml Saccomanno's fixative and centrifuged (500 g, 15 min). The cell pellet was resuspended in Saccomanno's solution. Two specimens were stained according to Papanicolaou and another two using the Feulgen reaction with thionine. Image cytometry was performed by means of a special, trainable classifier for exfoliative cells of the respiratory tract, using the Cyto-Sacant (Oncometrics, Vancouver). RESULTS: In the patients with suspected lung cancer we found numerous abnormal nuclei in 97 samples, 36 samples contained normal cells only, and 9 samples were insufficient. In our control group there was no sample with abnormal nuclei, and all washings were evaluable. Compared to the final diagnosis of lung cancer, we found a sensitivity of 90% (92/102) and a specificity of 84% (26/31). For histology sensitivity was 91% (73/80) and specificity 100%, while we found a sensitivity of 92% (92/100) and specificity of 100% for cytology. For automated cytometry the positive predicted value was 95%, the negative predicted value 71%. CONCLUSIONS: In the investigation of patients with suspected lung cancer, automated image cytometry of bronchial washings is a sensitive and reliable method for the detection of malignant changes in the tracheobronchial mucosa. The automated procedure seems well suited not only for analysing bronchial washings, but also for a screening procedure.

Adult↗

PubMed1999

Clinical value of DNA image cytometry in effusions with atypia.

Malignant cells in serosal effusions provide essential information about the extent of malignant disease. The main aim of this study was to examine the additional diagnostic value of DNA image cytometry for cases with uncertainty in the cytological diagnosis. In addition, the feasibility of automated nuclei detection was investigated. Out of 457 cases, 33 samples in 32 patients were diagnosed with "atypia" (probably benign) and 21 as "suspicious for malignancy." DNA image cytometry was performed on these 54 cases and on an additional group of 14 cytologically malignant cases. The results show that automatic classification is useful for separation of control cells, i.e., lymphocytes and neutrophilic granulocytes from other mononuclear cells. In 21 cases an insufficient number of control cells were measured. Seventy-two percent of the cytologic malignant cases were aneuploid. In contrast, in none of the cases with "atypia" and in only 2 of the cases "suspicious for malignancy" was aneuploidy present (2 of the remaining 32, 6%). From the cases with follow-up, a malignancy in the pleural fluid was present in 2 out of 17 cases with "atypia" and in 5 out of 10 with a "suspicious for malignancy" cytologic diagnosis, respectively. In conclusion, the additional diagnostic value of DNA image cytometry in cases with a cytological diagnosis of "atypia" or "suspicious for malignancy" is limited. Diagn. Cytopathol. 1999;21:112-116.

Adult↗

PubMed1996

Confocal 3-dimensional DNA image cytometry in thick tissue sections.

We present a three-dimensional confocal DNA image cytometry (3-D CICM) method for analysis of DNA content in 30-40-microns-thick sections of routinely processed paraffin-embedded specimens. A comparison of DNA ploidy profiles obtained by 3-D CICM and conventional DNA image cytometry (ICM) on tissue sections sections showed significantly higher numbers of cells with high DNA content in DNA histograms by 3-D CICM. As estimated by 3-D CICM, the size of nuclei frequently exceeded the thickness of tissue sections used in conventional ICM, which suggested that many nuclei measured by this technique may be incomplete. This artifact was excluded in 3-D CICM by automatic rejection of cut nuclear profiles. This and the favorable ratio of tissue thickness to nuclear size in 3-D CICM permitted the DNA quantitation even in large cells with highly increased DNA ploidy values such as megakaryocytes and Reed-Sternberg cells of Hodgkin's disease. Additionally, 3D-CICM allowed evaluation or morphometric parameters and 3-dimensional reconstruction of studied cells.

Bone Marrow↗

PubMed1995

Bleaching of melanin before image cytometry of the DNA content of pigmented skin tumors.

OBJECTIVE: To study the effect of bleaching of melanin with KMnO4 on the results of DNA image cytometry in pigmented skin tumors. STUDY DESIGN: Image cytometry of nuclear DNA content was performed on sections from 14 melanocytic skin tumors stained with Feulgen stain both with and without prior bleaching with KMnO4. RESULTS: The nuclear staining intensity of Feulgen stain was lower in the bleached sections, but this did not significantly affect the evaluation of ploidy. Heavy pigmentation caused some false peaks in the histograms (4 of 28 measurements made on unbleached slides). CONCLUSION: Bleaching of sections with KMnO4 can be useful when heavy melanin pigmentation would make DNA measurements impossible or difficult in image analysis cytometry. Bleaching is not advisable when only lightly pigmented tumors are analyzed if nuclei obscured by any pigment granules are to be avoided. In large series containing both bleached and nonbleached specimens, statistical analysis of these groups should be separated.

DNA, Neoplasm↗

PubMed1989

DNA determination in endometrial carcinoma by flow and image cytometry.

Endometrial carcinomas from 29 patients were analyzed by flow and image cytometric techniques with respect to DNA-content. Good agreement of ploidy value was obtained in 22 of the cases. A correlation was demonstrated between percentage cells above 2.5 C in image cytometry and S-phase as determined by flow cytometry. The interpretation was that cells with major chromosomal abnormalities may also have a high proliferative rate. These two features cannot be separated by image cytometry, whereas this is claimed to be the case according to the terminology used in interpretation of flow cytometric data. Our findings underline the need for a clarification of the terminology used in cell measurements on clinical material as well as proper choice of cytometric method to be used in conjunction with diagnostic cytology. The possible clinical value of the descriptors chosen from the two methods awaits follow-up of the patients.

DNA, Neoplasm↗

PubMed1994

Quality control in image cytometry: DNA ploidy.

DNA ploidy has become a commonly performed quantitative image cytometry test in microscopic pathology. This has led to the need to develop quality control procedures to aid in assuring uniform and reliable test results. There are a number of unique issues related to the emerging technology of image analysis and its routine use as a quantitative microscopic assay that require consideration before establishing a quality control program. Previous considerations of this topic have primarily related to measurement issues, e.g., accuracy comparisons to other methodologies, calibration of instrumentation, sources of measurement error, and the interpretation of measurements results. Although these issues are critically important, with more routine usage the focus is now turning to quality control of the overall testing process in everyday use. Control charts and methods of controlling the total measurement process such as have been used in clinical chemistry, need to be established. As one of the first image assays in pathology, quality control procedures established now for DNA ploidy measurements could help shape the development of this field, especially as pathology transitions from being a subjective visual microscopic inspection process to a quantitative measurement process. This paper discusses these issues as they relate to overall quality assurance for the DNA ploidy test and describes a quality control program developed for an active breast cancer testing laboratory specializing in image cytometry tests, including DNA ploidy. The quality control program includes calibration control charts, control charts for internal diploid controls, check samples, and computerized individual histogram interpretation.

Biopsy, Needle↗

PubMed1993

Methodological aspects of DNA image cytometry in normal bone marrow smears.

The possibility of using DNA image cytometry in hematological material was evaluated in 27 Feulgen stained normal bone marrow smears. The 95% normal limits of DNA index (DI) determined separately for each cell population in bone marrow were 0.89-1.15, 0.93-1.09 and 0.92-1.12 for blasts, granulocytes and lymphocytes, respectively. The mean DI for blasts, granulocytes and lymphocytes was 1.02, 1.01 and 1.02, respectively. In the blasts population the mean percentage of cells in S+G2/M phase was 38.1% (S.D. 12.9%, range 14.3-65.6%), whereas the corresponding values for granulocytes and lymphocytes were below 1%. Calculation of DI and G0/G1 C.V. obtained in bone marrow smears subjected to Feulgen hydrolysis (5 N HCl, 22 degrees C) for different periods of time revealed the presence of a plateau of results between 60 and 120 min for all of the cell types. The intraobserver and interobserver reproducibility was confirmed by duplicate measurements of each subpopulation (P > 0.05). A comparison of DI and G0/G1 C.V. using smears from the same donors stained with the Feulgen method either straight away or after destaining from May-Grunwald-Giemsa confirmed that destained smears can be used, provided applying appropriately prepared reference cells. We conclude that image cytometry can be used reliably to analyze DNA content in hematological material.

Adolescent↗

PubMed1998

Image cytometry of breast carcinomas that are DNA diploid by flow cytometry: time to revise the concept of DNA diploidy?

OBJECTIVE: To investigate whether breast carcinomas found to be DNA diploid by flow cytometry (FCM) are still diploid if reassessed by image cytometry (ICM). STUDY DESIGN: In a series of 286 breast cancers analyzed by FCM there were 100 (35%) cancers that were classified as DNA diploid. Fourteen of the 100 diploid cases were selected for further analysis with ICM because the patient had died of breast cancer within 11-84 months after the diagnosis (a group with unfavorable outcomes), and 19 cases were selected at random from the cases who had no recurrence of cancer during follow-up of six or more years (a favorable group). RESULTS: Eleven (33%) of the 33 cases turned out to be DNA nondiploid, with a DNA index > or = 1.2 when analyzed by ICM. Nine of the 11 DNA aneuploid samples by ICM were found among the 14 patients with unfavorable prognoses and only 2 among the 19 patients with favorable outcomes (P = .002). The five-year survival rate of the women with DNA diploid cancer by both methods was 86%, whereas that of patients with DNA aneuploid cancer by ICM was 36% (P = .002). CONCLUSION: The results show that some breast carcinomas classified as DNA diploid based on FCM are not DNA diploid by ICM and that such carcinomas are associated with poorer outcomes than the ones that are DNA diploid also by ICM. The prognostic significance of DNA ploidy in breast cancer may need to be reexamined in studies where both FCM and ICM are used.

Breast Neoplasms↗

PubMed1990

Image cytometry of aneuploidy, growth fraction (MoAb Ki-67) and hormone receptors (ER, PR) immunocytochemical assays in breast carcinomas.

DNA nuclear content was assessed in human breast carcinomas (n = 132) using image cytometry. Optical density histograms of Feulgen stained cell imprints from fresh tissue samples, subsequently frozen for immunocytochemical assays, were determined by the SAMBA system and used for the DNA index, the ploidy balance (PB) and the proliferation index (PI) computation. The three parameters were correlated to (i) histological data (tumour grade, vascular and/or lymph node invasion) and to (ii) growth fraction (Ki67), hormone receptor antigenic sites (ER, PR) and intramedullar (bone marrow) biopsies and anti-KL1-positive epithelial cells. It was shown that 57% of breast carcinomas were aneuploid. Aneuploidy PI significantly correlated to the criteria of poor prognosis such as high tumour grade, vascular and lymphatic invasion and to increased Ki67-positive cells, and the absence of or low ER and PR. Since image cytometry is easy to handle and perfectly suitable for current diagnostic practice in pathology departments, particularly for tumour cell ploidy assessment and standardized analysis of immunostaining procedures with morphological control of the preparation, we conclude that image cytometry, as performed with the SAMBA, must be regarded as a relevant tool for prognosis evaluation and therapy guidance in individual patients.

Aneuploidy↗

PubMed1996

Prognostic value of DNA image cytometry in colorectal carcinoma.

OBJECTIVE: To investigate the diagnostic sensitivity and prognosis predicting of DNA image cytometry in colorectal carcinoma. STUDY DESIGN: We studied the ploidy status and other DNA cytometric parameters in 68 patients with colorectal carcinoma. In addition, clinical-histologic and follow-up information was collected for at least five years. RESULTS: DNA histograms were available in all cases, showing a diploid DNA distribution pattern in 6 (8.8%), tetraploid in 21 (30.9%), hyperdiploid in 20 (29.4%) and hypertetraploid in 21 (30.9%). The differences in the correlation study between cytometric parameters and pathologic features were not statistically significant. Ploidy status and DNA malignancy grade were individually related to five-year survival (P < .005 and P < .05). CONCLUSION: The data show that DNA image cytometry can provide valuable prognostic information on colorectal carcinomas and may prove useful in guiding adjuvant therapy in these patients.

Adenocarcinoma↗

PubMed1996

A strategy for multiple immunophenotyping by image cytometry: model studies using latex microbeads labeled with seven streptavidin-bound fluorochromes.

Multiple immunophenotyping is aimed at identifying several cell populations in a single labeling procedure by their ability to bind combinations of specific labeled antibodies. The present work demonstrates the simultaneous discrimination by using image cytometry of aminomethylcoumarin acetate (AMCA), Lucifer yellow (LY), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Texas red tandem (Red613), peridinin-chlorophyll protein (PerCP), and allophycocyanin (APC), which were all bound to latex beads as streptavidin-conjugated fluorochromes. This has been the result of a step-by-step optimization of the several factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. First, 14 streptavidin-conjugated fluorochromes were evaluated by using spectrofluorometry. A primary selection was then made of ten spectrally separable dyes that could be evaluated by using image cytometry. These dyes were bound to latex particles, and specific filter combinations were assembled to minimize crosstalk between fluorophores while preserving sufficient fluorescence intensity and counting statistics. Potential probe associations were then assessed by measuring the emissions of all fluorochromes that were detected by each filter combination. The resulting crosstalk matrix served as the basic tool both for final selection of the optimal filter combination and for dye set (composed, in this case, of the seven fluorochromes described above) and for mathematical correction of residual spectral overlap. Next, an image cytometry system was adapted to collect seven images of matched brightness with the selected combination of excitation/emission filters and dichroic mirrors. Finally, seven-parameter synthetic images were generated by digital image processing.

Automation↗