Image Cytometric DNA Analysis Research Articles

Comparison of nuclear texture analysis and image cytometric DNA analysis for the assessment of dysplasia in Barrett's oesophagus

Background:Dysplasia is a marker of cancer risk in Barrett's oesophagus (BO), but this risk is variable and diagnosis is subject to inter-observer variability. Cancer risk in BO is increased when chromosomal instability is present. Nucleotyping (NT) is a new method that uses high-resolution digital images of nuclei to assess chromatin organisation both quantitatively and qualitatively. We aimed to evaluate NT as a marker of dysplasia in BO and compare with image cytometric DNA analysis (ICM).Methods:In all, 120 patients with BO were studied. The non-dysplastic group (n=60) had specialised intestinal metaplasia only on two consecutive endoscopies after 51 months median follow-up (IQR=25–120 months). The dysplastic group (n=60) had high-grade dysplasia or carcinoma in situ. The two groups were then randomly assigned to a training set and a blinded test set in a 1 : 1 ratio. Image cytometric DNA analysis and NT was then carried out on Feulgen-stained nuclear monolayers.Results:The best-fit model for NT gave a correct classification rate (CCR) for the training set of 83%. The test set was then analysed using the same textural features and yielded a CCR of 78%. Image cytometric DNA analysis alone yielded a CCR of 73%. The combination of ICM and NT yielded a CCR of 84%.Conclusion:Nucleotyping differentiates dysplastic and non-dysplastic BO, with a greater sensitivity than ICM. A combination score based on both techniques performed better than either test in isolation. These data demonstrate that NT/ICM on nuclear monolayers is a very promising single platform test of genomic instability, which may aid pathologists in the diagnosis of dysplasia and has potential as a biomarker in BO.

Image cytometry accurately detects DNA ploidy abnormalities and predicts late relapse to high-grade dysplasia and adenocarcinoma in Barrett's oesophagus following photodynamic therapy

Background and aims:DNA ploidy abnormalities (aneuploidy/tetraploidy) measured by flow cytometry (FC) are strong predictors of future cancer development in untreated Barrett's oesophagus, independent of histology grade. Image cytometric DNA analysis (ICDA) is an optical technique allowing visualisation of abnormal nuclei that may be undertaken on archival tissue. Our aim was to determine the accuracy of ICDA vs FC, and evaluate DNA ploidy as a prognostic biomarker after histologically successful treatment with photodynamic therapy (PDT).Methods:Nuclei were extracted from 40 μm sections of paraffin-embedded biopsies and processed for ICDA at UCL and FC at UW using standardised protocols. Subsequently, DNA ploidy was evaluated by ICDA on a cohort of 30 patients clear of dysplasia 1 year after aminolaevulinic acid PDT for high-grade dysplasia (HGD). The results were correlated with long-term outcome.Results:In the comparative study, 93% (41 out of 44) of cases were classified identically. Errors occurred in the near-diploid region by ICDA and the tetraploid region by FC. In the cohort study, there were 13 cases of late relapse (7 cancer, 6 HGD) and 17 patients who remained free of dysplasia after a mean follow-up of 44 months. Aneuploidy post-PDT was highly predictive for recurrent HGD or cancer with a hazard ratio of 8.2 (1.8–37.8) (log-rank P=0.001).Conclusions:ICDA is accurate for the detection of DNA ploidy abnormalities when compared with FC. After histologically successful PDT, patients with residual aneuploidy are significantly more likely to develop HGD or cancer than those who become diploid. DNA ploidy by ICDA is a valuable prognostic biomarker after ablative therapy.

1073 Combination of Nuclear Textural Analysis and Image Cytometric DNA Analysis (ICDA) is More Accurate Than ICDA Alone for Risk Stratification in Barrett's Esophagus

G A A b st ra ct s higher than the mean intensity values for squamous (t=3.1, p<0.01) and gastric mucosa (t= 3.0, p<0.01). There was no statistically significant difference in the mean intensity values between squamous and gastric mucosa (t=0.4, p=0.71). Conclusions: We demonstrate selective binding of the novel fluorescence-labeled SNF peptide to Barrett's dysplasia over mucosal surface areas on the centimeter scale. These results are promising as a future means to target dysplasia in Barrett's esophagus on fluorescence endoscopy.

W1159 Elastic Scattering Spectroscopy is Highly Accurate for the Detection of High Grade Dysplasia and DNA Ploidy Arising in Barrett's Esophagus Using a Red Enhanced Spectrometer

Introduction Elastic scattering spectroscopy (ESS) is an emerging optical diagnostic technique which detects cellular abnormalities in vivo. In patients undergoing endoscopic surveillance for Barrett9s oesophagus (BE), we have demonstrated ESS is able to detect high-grade dysplasia (HGD) and DNA ploidy, both associated with significant increase in 5-year cancer risk (59% and 28%, respectively). The spectrometer used had a wavelength range of 350–800 nm, where there is significant haemoglobin absorption. We hypothesise that a red enhanced spectrometer, more sensitive in the range 450 nm–1000 nm, would detect more scattering data and may improve detection rates. The aim of this in vivo study was to assess the influence of the spectral range of the spectrometer on accuracy of diagnosis. Methods Spectral data for each spectrometer were collected at matched sites in vivo. A biopsy of the site was then taken from each site and histopathology reviewed. DNA ploidy was then assessed by image cytometric DNA analysis (ICDA) on these biopsies as previously described. A diagnostic algorithm of best fit was generated by principle component and linear discriminant analysis on the histology and DNA ploidy data. Results 49 patients were enrolled in the study, with 217 sites analysed. ESS using a red enhanced spectra was able to distinguish squamous and columnar tissue with comparable accuracy to a standard spectrometer (91% sensitivity and 91% specificity). When comparing HGD vs non-dysplastic tissue, there was also little difference between the two spectrometers. Sensitivity with the red enhanced spectrometer was 84%, with specificity of 83%, respectively. The standard spectrometer sensitivity was 86% and specificity of 83%. When combining aneuploidy+HGD vs normal columnar tissue, however, the red spectrometer was more accurate with sensitivity 90% and specificity 80%. The standard spectrometer sensitivity was 90% but specificity was 74%. Conclusion These data demonstrate ESS is highly accurate for the detection of BE and associated HGD and molecular abnormalities in vivo, whichever spectrometer is used. The red enhanced spectrometer is somewhat more accurate for the detection of DNA ploidy abnormalities than a standard spectrometer. We intend to test this spectrometer in a prospective equivalence study, comparing ESS with targeted detection of HGD and aneuploidy vs surveillance with quadrantic biopsy.

M1103 Comparison of Image Cytometric DNA Analysis of Brush Cytology Specimens Versus Formalin Fixed Tissue in Barrett's Esophagus

M1103 Comparison of Image Cytometric DNA Analysis of Brush Cytology Specimens Versus Formalin Fixed Tissue in Barrett's Esophagus

PWE-074 Combination of nuclear textural analysis and image cytometric DNA analysis is more accurate than image cytometric DNA ploidy analysis alone for risk stratification in Barrett's oesophagus

Introduction Dysplasia is a marker of cancer risk in Barrett9s oesophagus (BE), but this risk is variable and diagnosis is subject to inter-observer variability. Cancer risk in dysplastic and non-dysplastic BE is increased when large scale genomic instability (DNA ploidy) is present, with published data showing the incidence of aneuploidy in patients with HGD is 67%. Nuclear textural analysis (Nucleotyping) is a new method that uses ultra-high-resolution digital images of stained nuclei to classify DNA content, chromatin structure and organisation both quantitatively and qualitatively. This information is derived from the same sections used for image cytometric DNA ploidy analysis (ICDA). Our aim was to evaluate nucleotyping as a prognostic biomarker in Barrett9s oesophagus, and compare with ICDA. Methods 120 patients with BE were studied. 60 patients had high-grade dysplasia or intramucosal cancer and went on to ablative therapy or surgery. A further 60 patients had specialised intestinal metaplasia only on two consecutive endoscopies with a median of 51 months follow-up (IQR=25–120 months). The two groups were then randomly assigned to a training set (30 HGD, 30 non-dysplastic) and a blinded test set. 40 micron sections were cut from formalin fixed paraffin embedded (FFPE) biopsies and a Feulgen stained nuclear monolayer was made as previously described for ICDA. Results Analysis of the training set demonstrated a DNA ploidy rate of 64% in the HGD group vs 0% in non-dysplastic. By comparison, nuclear textural analysis was abnormal in 71%. A correct classification rate (CCR) of 84% was achieved if the two features were combined. The test set was blindly analysed using the same textural features. DNA ploidy rate was 61% in the HGD group vs 0% in non-dysplastic group. Nucleotyping was again more accurate at 78%, and combining the two features gave a CCR of 84%. When all patients were analysed, sensitivity and specifity of combined nucleotyping with ICDA for detecting dysplasia was 75% and 94%, respectively. Conclusion Nucleotyping yields additional prognostic information vs image cytometry alone on the same nuclear monolayers. The textural features used to differentiate normal from dysplastic tissue were similar to those used in studies of other early cancers. These data demonstrate that nucleotyping is a promising additional test for separating at-risk groups in Barrett9s oesophagus. This work is supported by the CRUK Experimental Cancer Medicine Centre at University College London and the NIHR Comprehensive Biomedical Research Centre at UCLH.

DNA study of bladder papillary tumours chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in Fisher rats.

To examine DNA abnormalities in bladder papillary tumours induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in female rats, using image cytometric DNA analysis and cytogenetics. Thirty female rats were exposed to BBN in their drinking water for 20 weeks. One group of 10 animals served as controls. The animals exposed to BBN were killed at a rate of two per week, with the bladder being collected under aseptic conditions and those tumours with exophytic growth removed. The nuclear DNA content of the tumours was evaluated using image cytometric analysis. In two rats part of the tumour pieces was stipulated for culturing. Cytogenetic analysis was performed on at least 30 cells from each cell population and on both tumours. Papillary carcinomas were classified as low grade and high grade. DNA ploidy studies were carried out on 28 low-grade and 21 high-grade papillary carcinomas. Histograms obtained by image analysis showed that a normal urothelium was diploid; 28.6% and 100% of low-and high-grade papillary carcinomas were aneuploid respectively. Both tumours used for cell culture showed multiple numerical and structural chromosome alterations and several marker chromosomes. Image cytometric DNA analysis proved to be a good and reliable method for examining DNA alterations in papillary bladder carcinomas. The present findings establish that the DNA content is statistically different between low-grade and high-grade papillary carcinomas and that deviation from the diploid number is markedly higher in the high-grade ones. In addition, the occurrence of marker chromosomes seems to be related to the aggressiveness of the tumour.

Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia

To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis. Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with monoclonal antibodies MIB-1 for Ki67 and PAb 1801 for p53, in addition to the conventional histologic examination for dysplasia. Nuclei of fresh biopsy material were enzymatically and mechanically isolated, and the DNA content was determined with image cytometry after Feulgen staining. DNA grading of malignancy was assessed according to Boecking to determine the variability of DNA values noted around the normal diploid peak. Further indices measured included the aneuploid rate, and the 5c-, 7c- and 9c-exceeding rate. The histological examination did not demonstrate dysplasia; while MIB-1 (basal) showed a positive reaction in 8/15 achalasia specimens, p53 was negative in all specimens. Image cytometric DNA analysis detected aneuploidy in 4/15 (26.7%) specimens. Samples from 15 patients with squamous cell carcinoma as well as specimens obtained exclusively 2 cm proximal to the tumor served as reference tests. All carcinomas (15/15) as well as 9 of the peritumoral samples (9/15) were aneuploid. The comparison of biopsies from achalasia patients with peritumoral and carcinoma specimens revealed statistically significant differences regarding the aneuploid rate (diploid: P < 0.0001; tetraploid: P = 0.001), grading of malignancy according to Boecking (P < 0.0001) and the 5c- (P < 0.0001), 7c- (P < 0.0001), and 9c- (P = 0.0001) exceeding rate with progredient DNA alterations in the respective order. The finding that DNA aneuploidy was identified by image cytometry in esophageal specimens of patients with achalasia, which may be due to specific chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool.

DNA IMAGE CYTOMETRY ON BIOPSIES CAN HELP THE DETECTION OF LOCALIZED GLEASON 3+3 PROSTATE CANCERS

In spite of classifications based on digital rectal examination, prostate specific antigen (PSA), transrectal echography and histological analysis, 20% to 40% of operated prostate adenocarcinomas are not yet organ confined. New diagnostic features to predict extracapsular invasion before treatment are needed to avoid surgical extraction within positive margins. A retrospective study was performed for 74 prostate adenocarcinomas with Gleason 3+3. A total of 54 organ confined tumors (T1T2) at digital rectal examination were compared with 20 nonorgan confined tumors (T3T4). Image cytometric DNA analysis was performed on prostate initial biopsies. DNA ploidy results were compared in both groups for values of PSA greater than 15, less than 15 and less than 10 ng/ml. For a PSA rate less than 15 ng/ml, 83.8% of T1T2 were diploid vs 33% T3T4 (p = 0.042). For a PSA rate less than 10 ng/ml, 96% of T1T2 were diploid vs 33% T3T4 (p = 0.025). DNA ploidy appears to be an interesting feature at diagnosis. When in doubt about the localized character of a tumor, DNA ploidy it makes it possible to predict a nonorgan confined tumor. Gleason 3+3 prostate cancers that are organ confined at digital rectal examination are more often diploid than T3T4 tumors. The prognostic interest in DNA ploidy is more reserved because of its correlation with PSA level.

Image cytometric DNA analysis and proliferating cell nuclear antigen (PCNA) expression in transitional cell carcinoma of the bladder.

The proliferation rate as determined by Proliferating Cell Nuclear Antigen (PCNA) immunostaining and the DNA ploidy status as measured by static cytometry were studied in 70 transitional cell carcinomas of the bladder (TCCB) in relation to grade, stage, and recurrence. The follow-up period was 2 years. A significant difference was observed in PCNA expression among grades I, II, and III (P < 0.02), between superficial (pTa-pT1) and invasive (pT2-pT4) tumors (p < 0. 04), between recurring and non-recurring tumors (p < 0.001), and between tumors of the same grade with and without recurrence (p < 0. 05). A significant difference was also found in the ploidy pattern among grades I, II, and III (p = 0.002), and between superficial and invasive (p = 0.02) and recurring and non-recurring tumors (p < 0. 01). Finally, the recurrence status seems to be strongly influenced by the proliferation rate and ploidy of TCCB (p < 0.001). Our results suggest that the above-studied parameters may offer useful information on the biological behavior of TCCB.

腺様嚢胞癌の臨床病理学的研究 核ＤＮＡ量解析とｐ５３蛋白，ＷＡＦ１蛋白の免疫組織化学的検索

Adenoid cystic carcinoma (ACC) is generally classified into the following histologic types: tubular, cribriform, and solid. Numerous authors have attempted to correlate the histological characteristics of these tumors with their biological behavior.I retrospectively compared the image cytometric and immunohistochemical characteristics of formaldehyde-fixed and paraffin-embedded specimens obtaired from 24 cases. The specimens were also classified according to the histological grading system proposed by Szanto et al., the predominant pattern, and the presence or abscence of solid components. The nuclear DNA content of ACC on image cytometric analysis and the immunohistochemical characteristics of p53 protein and WAF 1 protein were studied as compared with clinicopathological findings. The following results were obtained:1) No correlation was found between the histological classification and clinicopathological findings. However, most patients with tumors characterized by solid components died of cancer-related causes.2) Tumors with a solid histological pattern and those with distant metastasis were more often associated with DNA aneuploidy, and these tumors had elevated levels of mean DNA index and %-over 4 c.3) No correlation was found between histological classification and p53 or WAF 1 protein expression. Lymph node metastasis was present in seven carcinomas, none of which expressed WAF 1 protein.4) Aneuploid carcinomas were related to p53 protein immunoreactivity. There were no relationship between DNA ploidy pattern and WAF 1 protein expression.5) There was a strong correlation between p53 and WAF 1 protein expression and most tumors that expressed p53 protein did not express WAF 1 protein.These results suggest that WAF 1 protein expression and image cytometric DNA analysis may be a useful index for assessing the malignant behavior of ACC. Although aneuploid tumors were associated with a solid histological pattern, there was no correlation between histological classification and p53 or WAF 1 protein expression.

Clinical course of metastatic parathyroid cancer.

In a retrospective study the clinical course of 40 patients with symptomatic persistent or recurrent parathyroid cancer was analyzed in order to assess the value of aggressive surgical intervention. Recurrence was diagnosed after a median period of 33 months (1-228 months). Twenty-two patients had locoregional disease, whereas 14 patients had both local and distant spread. The remaining four patients had distant spread. Patients with just locoregional disease were subject to one to nine reoperative procedures. The median survival time from the last operation was 39 months (1-204 months). Eight patients then had no evidence of disease, three were hypercalcemic and 9 of 11 had died of parathyroid cancer. Distant spread was demonstrated in 17 of 40 patients. Pulmonary metastases predominated (14 of 17). Surgical excision was performed in 9 of 14 cases. Of these nine, two patients had a subsequent disease-free interval of 36+ and 72+ months, respectively. One patient was reported hypercalcemic after 84 months, whereas five patients died of cancer between 4 and 60 months after their last surgical exploration. One patient was lost to follow-up. In all, 21 patients (53%) died of parathyroid cancer. Conspicuous nuclear atypia and frequent mitoses predominated. Image cytometric DNA analysis showed high rates for all three groups (median p90 = 80%, range 21-98%).

Image cytometric DNA analysis in transitional cell carcinoma of the bladder

The current study was initiated to investigate measurable objective and reproducible characteristics that might have prognostic significance in bladder cancer. Tumor samples from 91 patients with primary transitional cell carcinoma (TCC) of the urinary bladder were studied by DNA image cytometry and cytogenetic analysis. Image cytometry is a more sensitive method of determining ploidy than flow cytometry, especially in tumors with a low number of aneuploid cells. There was a significant difference in survival between DNA image cytometry-determined diploid and nondiploid cases. The presence of nuclei with a high DNA content indicated poor prognosis. The 2C deviation index (2CDI) also was an indicator of survival. Image cytometry-determined factors also were found to be strong predictors of progression-free survival. In multivariate analysis, 2CDI was the only cytometric parameter with an independent but weak correlation with survival. In multivariate analysis, none of the cytometric parameters had an important contribution to prediction of progression-free survival. In superficial tumors (Ta and T1), 2CDI appeared to be the most important independent predictor of survival. With respect to progression-free survival, tumors with a high mitotic index proved to have a worse prognosis. Parameters determined by DNA image cytometry appear to be valuable in predicting survival and progression-free survival and may be useful in addition to the classic parameters of stage and grade, especially in superficial TCC.

False aneuploidy in benign tumors with a high lymphocyte content: A study of Warthin's tumor and benign thymoma

The quality of results of flow cytometric DNA content analysis of formalin-fixed, paraffin-embedded tissue may be affected by a number of preanalytical variables. We performed flow cytometric DNA content analysis on two types of benign tumors to investigate the effect of a prominent lymphocytic component: Warthin's tumor (N = 20) and benign thymoma (N = 8). Malignant tumors (N = 23) were included as DNA aneuploid controls. All tissues studied were archival material processed using Hedley's technique either without prolonged rehydration in water (day 0 samples) or with 24- or 48-hour rehydration (day 1 and day 2 samples, respectively). Image cytometric DNA ploidy analysis was also performed on most cases. Eight cases (40%) of Warthin's tumor and five cases (63%) of benign thymoma showed either hyperdiploid peaks or marked asymmetry on the day 0 DNA histograms; nine of the malignant tumors were aneuploid. The DNA histogram abnormalities of the benign tumors could be gated out by excluding the lymphocyte nuclei. None of the DNA indices of the benign tumors corresponded with expected deviations based on published chromosomal studies. All of the DNA histogram abnormalities of the benign tumors disappeared and/or fused with the main peaks on the day 1 or day 2 samples, except for one case of benign thymoma. All the DNA aneuploid peaks on the malignant tumors persisted with prolonged rehydration. Image cytometric DNA analysis showed a diploid pattern in all benign tumors. We conclude that a high lymphocyte content may be a cause of false aneuploidy in these benign tumors. Furthermore, the degree of rehydration appears to be an important factor in achieving optimum fluorochrome staining of DNA.

Recurrent renal hyperparathyroidism and DNA analysis of autografted parathyroid tissue.

In spite of recent therapeutic advances, severe overt secondary hyperparathyroidism due to chronic renal failure necessitates parathyroidectomy and recurrent hyperparathyroidism is one of the most significant problems in this patient population. In the present study, the incidence of recurrent hyperparathyroidism was evaluated in relation to the histopathological features. Image cytometric DNA analysis was performed to estimate the proliferative potential of parathyroid tissue. The study comprised 248 patients who underwent parathyroidectomy from 1973 to 1991. The frequency of recurrent hyperparathyroidism after subtotal parathyroidectomy was 4 (21.1%) of 19 patients, the rate of graft-dependent recurrence after removal of residual parathyroid tissue with forearm autograft was 2 (50%) of 4 patients. The frequency of graft-dependent recurrence after total parathyroidectomy with forearm autograft was 16 (7.5%) of 212 patients. The frequency of recurrence was significantly higher (p less than 0.01) when nodular hyperplastic parathyroid tissue was autografted (17 of 68 patients, 25%) than when diffuse-hyperplastic tissue was grafted (1 of 105 patients, less than 1%). All 58 specimens subjected to image cytometric DNA analysis showed a diploid nuclear pattern cytometrically. However, the relative number of scattered cells that displayed cytometric nuclear DNA values outside the main diploid histogram peak was significantly greater (p less than 0.01) in nodular hyperplastic tissue before being autografted and in parathyroid tissue removed at re-operation performed for recurrence than in diffuse hyperplastic tissue. These clinical findings and results of DNA analysis clearly indicated that nodular hyperplastic parathyroid tissue has a higher growth potential, and it is concluded that to prevent graft-dependent recurrence, the nodular type of hyperplastic tissue should not be autografted.

An image cytometric DNA-analysis in breast neoplasms: Parameters of DNA-aneuploidy and their relationship with conventional prognostic factors

In this prospective study, an image cytometric DNA-analysis was performed in 86 women with breast neoplasms (72 primary invasive carcinomas and 14 benign lesions). Four DNA ploidy parameters were analysed: histogram type (according to AUER classification), DNA-index, tumor cells with DNA content above the 5n limit and DNA malignancy grade (DNA-MG, calculation according to Böcking). Their correlations with well established prognostic factors in breast carcinomas (tumor size, lymph node status, histologic grade, hormone receptor content) were studied. All but one benign lesions were diploid (13/14 cases), whereas the majority of the primary invasive breast carcinomas were aneuploid (58/72 cases). A predominance of carcinomas with a percentage of cells superior or equal to 1% with DNA content above the 5n limit was observed (54 cases out of 58). Most of the aneuploid tumors had a histogram type III or IV (53 cases) or a high DNA-index (50 cases). Of these 58 aneuploid cases, only 26 tumors had a DNA-MG superior to 1. Interestingly, 26 tumors had the 4 criteria of aneuploidy, 19 had 3 and 9 had 2 and only 4 tumors had one parameter. The DNA-MG was significantly related to hormonal receptors (p less than 0.001) and tumor size (p less than 0.01). The histogram types (Auer classification) and the DNA content above the 5n limit were correlated with histologic grade (SBR or SBRM) (p less than 0.02). Concerning the DNA-index no correlation was observed with well established prognostic factors. On the other hand no significant correlation was found between these new biologic variables and lymph node status.(ABSTRACT TRUNCATED AT 250 WORDS)

Image cytometric DNA analysis in smear specimens of non-small cell lung cancer

Image cytometric DNA analysis in smear specimens of non-small cell lung cancer

Image cytometric DNA analysis of neuroblastoma. Correlation with conventional prognostic factors

Image cytometric DNA analysis of neuroblastoma. Correlation with conventional prognostic factors

Towards a quick image cytometric DNA analysis in acute lymphoid leukemia

Biology of the CellVolume 73, Issue 2-3 p. 27a-27a TOWARDS A QUICK IMAGE CYTOMETRIC DNA ANALYSIS IN ACUTE LYMPHOID LEUKEMIA Souchier Catherine, Souchier Catherine Cytologie analytique, 8 av Rockefeller, 69373 LYON, FRANCESearch for more papers by this authorAnne Manel Marie, Anne Manel Marie Cytologie analytique, 8 av Rockefeller, 69373 LYON, FRANCESearch for more papers by this authorPaul Bryon André, Paul Bryon André Cytologie analytique, 8 av Rockefeller, 69373 LYON, FRANCESearch for more papers by this author Souchier Catherine, Souchier Catherine Cytologie analytique, 8 av Rockefeller, 69373 LYON, FRANCESearch for more papers by this authorAnne Manel Marie, Anne Manel Marie Cytologie analytique, 8 av Rockefeller, 69373 LYON, FRANCESearch for more papers by this authorPaul Bryon André, Paul Bryon André Cytologie analytique, 8 av Rockefeller, 69373 LYON, FRANCESearch for more papers by this author First published: 1991 https://doi.org/10.1016/0248-4900(91)90173-KAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume73, Issue2-31991Pages 27a-27a RelatedInformation