Expression of the Fc-receptor for IgE (Fc epsilon RII, CD23) on alveolar macrophages in extrinsic allergic alveolitis.

Expression of the Fc receptor for IgE (Fc epsilon R) was analyzed on alveolar macrophages (AM) in 10 patients with extrinsic allergic alveolitis (EAA) compared with 10 patients with sarcoidosis and to 6 apparently healthy controls. By using the anti-Fc epsilon RII mAb M-L25 in immunocytochemistry experiments, we found that greater than 60% of AM in 10 of 10 patients with EAA were strongly positive, as evidenced by visual analysis in light microscopy and by cytometry. By contrast, no significant staining was detected in sarcoidosis or in controls with either method. Similar results were obtained when Fc epsilon R were identified with preformed immune complexes consisting of NIP-specific human/mouse chimeric IgE antibody plus NIP-ovalbumin. Furthermore, greater than 60% of AM in patients with EAA stained positive for IgE, demonstrating that endogenous IgE is bound to the AM. Our data suggest that IgE antibodies bound to Fc epsilon RII on AM may be involved in pathophysiology of extrinsic allergic alveolitis by activation of the AM after binding of allergen to the cell surface IgE. Furthermore, with the clearcut pattern of Fc epsilon RII expression in extrinsic allergic alveolitis it may be possible to use CD23 antibodies for differential diagnosis of inflammatory lung disease.

COX2 expression, angiogenesis, proliferation and survival in Barrett's cancer.

OBJECTIVES: To examine COX2 expression and its relation to angiogenesis, Ki67 and Bcl2 expression in Barrett's cancer. METHODS: Specimens from 48 R0-resected Barrett's adenocarcinoma were immunostained for cyclooxygenase 2 (COX2), CD 31 and alpha-sm actin to discriminate between mature and immature vessels, Mib-1 and Bcl2. COX2 staining, angiogenesis, Ki67 expression and Bcl2 expression were also measured. RESULTS: COX2 expression was increased in 25 of 48 cases. There was no significant correlation between COX2 expression and age, sex and tumor differentiation. A significant association was found between lymph node positive cases and elevated COX2 expression (p=0.008). The percentage of Ki67 positive cancer cells was 43.8% (range 15.4-67.5%) in the low COX2 group and 57.8% (range 12.0-84.6%) in the high COX2 group. The difference was statistically significant (p=0.046). The median neovascularisation coefficient in the low COX2 group was 11.68 (range 8.22-43.64) and 25.47 (range 8-38.3) in the high COX2 group. The difference was statistically significant (p=0.012). A significant difference in survival was observed between patients in the COX2 low category when compared with the COX2 high category (log-rank test p=0.013). CONCLUSIONS: Elevated COX2 expression is associated with lymph-node metastases and reduced survival in Barrett's cancer. This appears to be related to the induction of angiogenesis and proliferation.

Identification and in situ localization of the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor in the rat gastrointestinal tract: comparison with the IGF-I receptor.

In the present study we investigated pharmacological, biochemical, and immunological characteristics as well as the tissue distribution of the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor in the rat gastrointestinal tract, and compared the data with those from corresponding experiments for the IGF-I receptor. Competitive binding and affinity cross-linking studies with [125I]IGF-II, and [125I]IGF-I respectively, in rat jejunum yielded results analogous to those previously obtained for IGF-II/M6P and IGF-I receptors in intestinal epithelial membranes and other tissues. Furthermore, the IGF-II/M6P receptor antibody no. 3637 completely inhibited the association of [125I]IGF-II with receptor protein but nonimmune antibody did not, providing additional evidence for the presence of the IGF-II/M6P receptor in the rat gut. Also, analysis of the IGF-II/M6P receptor by immunoblotting using antiserum no. 3637 identified a specific band of mol wt 220.000 throughout the gastrointestinal tract with the highest content of immunoreactivity being present in colon and ileum. Autoradiographic mapping of the distribution of IGF-receptors in the rat gut showed that the expression of IGF-II/M6P receptors was in general 2-3 times greater than that of IGF-I receptors. IGF-II/M6P receptors were found 1) in greatest densities in colon and ileum, 2) more abundantly in the mucosa than in the muscularis propria, and 3) predominantly in the luminal part of the mucosal epithelial cells. Radioimmunocytochemistry employing anti-IGF-II/M6P receptor antibody no. 3637 and [125I]protein A demonstrated an IGF-II/M6P receptor distribution analogous to that shown by autoradiography with [125I]IGF-II). IGF-I receptors were present 1) in greatest densities in ileum and colon, 2) more abundantly in the muscularis propria than in the mucosa, and 3) within the mucosa in greater densities in the lamina propria than in the surface epithelium. For both receptor types densities were greater in crypt than in villous epithelial cells. We conclude: 1) the presence of IGF-II/M6P receptors throughout the rat gastrointestinal tract points to an important role for IGF-II in this organ, 2) the finding of different patterns of distribution for IGF-II/M6P and IGF-I receptors supports the concept of their different principal functions, 3) a high degree of expression of both receptor types in crypt epithelium suggests an essential role for both IGF receptors in the regulation of cell mitogenesis and growth.

A thresholding method for automatic cell image segmentation.

An algorithm for automatic segmentation of PAP-stained cell images and its digital implementation is described. First, the image is filtered in order to eliminate the granularily and small objects in the image which may upset the segmentation procedure. In a second step, information on gradient and compactness is extracted from the filtered image and stored in three histograms as functions of the extinction. From these histograms, two extinction thresholds are computed. These thresholds are suitable to separate the nucleus from the cytoplasm, and the cytoplasm from the background in the filtered image. Masks are determined in this way, and finally used to analyse the nucleus and the cytoplasm in the original image.

Expression of CD14 correlates with lung function impairment in pulmonary sarcoidosis.

CD14 expression on alveolar macrophages (AM) was studied in patients with sarcoidosis using immunocytochemistry and cytometric analysis. Compared with healthy control donors, patients had elevated percentages of CD14-positive AM (22 percent vs 34 percent), and the antigen density was threefold higher (92 vs 297 channels). Furthermore, soluble serum CD14 (ssCD14) was significantly elevated in patients with sarcoidosis with an average of 5.3 +/- 1.6 mg/L vs 3.2 +/- 0.7 mg/L in healthy control subjects. Follow-up of one patient, whose lung function test results improved during therapy with corticosteroids, revealed a concomitant decrease of CD14 staining on AM and of ssCD14. Statistical analysis revealed a negative correlation between CD14 expression on AM and PO2 at rest (p = 0.0005), and after labor (p = 0.02). Levels of ssCD14 gave a positive correlation to reduction of Dco (p = 0.006) and VC (p = 0.05). These data suggest that CD14 expression is related to severity of disease and that it may be useful for monitoring in sarcoidosis.

Cell type-specific induction of O6-alkylguanine-DNA alkyltransferase mRNA expression in rat liver during regeneration, inflammation and preneoplasia.

PURPOSE AND METHODS: To investigate the potential role of an aberrant cellular DNA repair in target cells during malignant transformation we studied cell type-specific mRNA expression of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (O6-AGT) in normal and regenerating rat liver, chronic hepatitis and preneoplastic liver lesions by in situ hybridization and semiautomatic image analysis. RESULTS: O6-AGT mRNA expression was found to be four to five times higher in hepatocytes than in nonparenchymal cells. A 1.9-fold increase in O6-AGT mRNA was observed after partial hepatectomy. Intraperitoneal injection of diethylnitrosamine led to a 1.3-fold and 2.6-fold rise in periportal and perivenous hepatocytes, respectively. Ethylnitrosourea produced an enhancement of mRNA levels up to 1.6-fold in hepatocytes without regional differences. In megalocytic hepatocytes of Long-Evans Cinnamon rats with chronic hepatitis, a 4.4-fold mRNA induction was found. In small preneoplastic lesions induced after chronic diethylnitrosamine-exposure, O6-AGT mRNA expression was identical to that of adjacent normal tissue. Intermediate and large lesions revealed 1.5- to 1.6-fold higher mRNA levels. CONCLUSIONS: These results suggest an induction of O6-AGT mRNA expression in hepatocellular target tissue under conditions of increased carcinogen sensitivity . The O6-AGT expression in early preneoplastic lesions did not differ from normal surrounding liver tissue, thus excluding the possibility that progression of preneoplasia in rat liver is associated with a deficient mRNA expression of this DNA repair protein. On the contrary, enhanced O6-AGT mRNA expression in more advanced foci and early neoplastic nodules may confer a selective advantage upon early malignant hepatocytes with regard to further tumor progression.

The 'angiogenic switch' in the progression from Barrett's metaplasia to esophageal adenocarcinoma.

AIMS: We investigated VEGF expression and neovascularisation in the metaplasia-dysplasia-carcinoma sequence of Barrett's esophagus and 47 shades of adenocarcinoma. METHOD: Slides of 27 cases of Barrett's metaplasia and high grade dysplasia were immunostained for VEGF, CD 31 and alpha-sm actin to discriminate between mature and immature vessels. VEGF stained slides were quantitatively evaluated measuring optical density with a computer based program. The neovascularisation coefficient was estimated with an interactive analytic computer program. RESULTS: The median VEGF expression increased from metaplasia to advanced carcinoma. VEGF expression and the neovascularisation coefficient reached statistical significance between Barrett's metaplasia and high grade dysplasia (p<0.001), but were not statistically different between high grade dysplasia and microinvasive carcinoma (p=0.421; p=0.146). Comparing microinvasive to advanced carcinoma the difference was significant for both parameters (p<0.001). CONCLUSIONS: Based on a quantitative computer based evaluation program, the present study suggests, that an angiogenic switch might exist and that it is an early event in the metaplasia-dysplasia-carcinoma sequence of Barrett's carcinoma. The neovascularisation phase in Barrett's carcinoma may precede tumour growth.

Cytometry in histological sections of colon carcinoma.

A short description of a project of cytometry in histological sections of colon carcinoma is given with emphasis on the methodical aspects. Possible strategies of cytometric measurement and problems related to it (focus, overlap, segmentation of objects) are described. The main effort concerns interactive selection of tumor cells and the segmentation in cases of densely distributed and overlapping nuclei. All other succeeding processing steps are performed fully automatically. The resulting quantitative features are stored together with the original images on an optical disk for further examinations and reexaminations, allowing the direct relation of feature values to visual image content. The evaluation of the features as well as their interpretation is only at the beginning. Especially the problem of relating section information with true 3-dimensional information is not described here and necessitates further research. In a first investigation only a few tumors without and with metastases were analyzed. The preliminary results correspond with findings of Kunze et al.

Nucleolus organizer regions (AgNORs) in ductal mammary carcinoma. Comparison with classifications and prognosis.

The relevance of silver-stained NORs for classifications and prognosis was investigated in breast tissue. Paraffin sections from 137 cases of invasive ductal breast carcinomas and 12 cases with non-tumorous ductus epithelium as controls were stained according to a modified technique and analysed. From the cancer cases follow-up data up to 10 years (45 to 165 months) and in addition clinical, histological and several DNA distribution parameters were available. The nuclei and the silver grains were measured by means of a semiautomatic image analysis system. Significant differences in AgNOR features were found between controls and diploid tumors (p < or = 0.001), diploid and aneuploid tumors (p < or = 0.001), Bloom-Richardson-gradings I, II, and III (p < or = 0.001), and between the tumor cells from patients developing metastases within 5 years and those without (p < or = 0.002). The prognostic significance of AgNORs was estimated using Cox regression analysis. Four AgNOR features were correlated significantly with survival time. In a multivariate approach offering all parameters available an AgNOR parameter (CV of relative area AgNORs) ranked at the third position beyond the SD of DNA distribution and pTNM-staging. Considering the metastases-free interval of patients the same AgNOR feature showed an independent prognostic validity.

Increased expression of the monocyte differentiation antigen CD14 in extrinsic allergic alveolitis.

Expression of the CD14 antigen was studied on alveolar macrophages in extrinsic allergic alveolitis (EAA), using immunocytochemistry and cytometry. Compared to control donors, EAA patients had higher percentages of My4 positive cells (40 versus 22%), and the antigen density was fourfold higher (410 versus 92 channels). Levels of soluble CD14 (sCD14) in serum were found to be increased in EAA patients with an average of 4.6 +/- 1.5 micrograms.ml-1 compared to 3.2 +/- 0.7 micrograms.ml-1 in controls. Follow-up of patients with antigen avoidance revealed a concomitant decrease of CD14 staining of alveolar macrophages (AMs) and of sCD14 in serum, whilst allergen exposure induces both parameters. These data are consistent with the concept that antigen contact upregulates CD14 expression on AMs in EAA, followed by shedding and increase of sCD14 in serum.

Stereological characteristics of the equine accessory nerve.

Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.

Estimation of the performance of an array-processor oriented system for automatic Pap smear analysis.

The rapid progress in computer technology makes possible the automatic analysis of thousands of cells on a slide in the field of automatic uterine cancer cytology. Our approach starts with the high-resolution scanning of visually selected and classified single cells determining the training set for discriminant analysis. On the basis of more than 15 morphologic and textural features measured, correct classification results of 95% are reached. Our aim is to speed up our image processing system by means of an Array-Processor, the model AP 120 B from Floating Point Systems. In this study a realistic time estimation of the scanning, segmentation, feature extraction and classification of about 40,000 cells on a slide is performed.

Discrimination of G1, S, and G2 cells using high-resolution TV-scanning and multivariate analysis methods.

Images of exponentially growing mouse L fibroblasts were used to test whether G1, S, and G2 cells could be discriminated by means of densitometric, geometric, textural and chromatin features. The cells were preincubated with FUdR and labeled with 3H-TdR. The definition of G1, S, and G2 was based on autoradiography used to define labeled S cells, and DNA content used to define unlabeled 2c (G1) and 4c (G2) cells. The methanol-fixed Feulgen-stained nuclei were scanned with a TV Plumbicon camera equipped with an array-processor system. Thirty-one nuclear features were calculated and stored together with the precise coordinates of each cell for later relocation and correlation with the autoradiographic data. The features were geometric, densitometric, textural and chromatin parameters of the Feulgen stained images. Feature evaluation and supervised learning were performed. When DNA content was excluded 14 textural and chromatin features were selected. By using these a correct classification of about 80% was achieved. Inclusion of DNA content and its correlates caused degeneration of performance, yielding 75% correct classification. DNA content alone gave 69% correct classification.

Malignancy associated changes in squamous epithelium of the head and neck region.

Subtle cellular changes are known to exist in normal host tissue adjacent to tumours. These are called malignancy associated changes (MAC). To get more insight into the degree of expression and local spread of such changes we performed high resolution image cytometry on visually normal intermediate cell nuclei in smears from patients with laryngeal or pharyngeal squamous cancer. The smears were taken from the tumour surface, from a border region of the tumour and from a distant unsuspicious buccal site. In addition buccal smears from healthy control persons were examined. In a pilot study smears from 12 cancer patients and 11 control persons and in a succeeding validation study 63 controls, 18 non-tumour patients and 25 cancer patients were investigated. In both studies the occurrence of MACs was demonstrated quantitatively. In cancer patients normal appearing intermediate cells from the three different sampling sites could be discriminated with 65% in the pilot study and with 53% correct classification in the validation study. In addition the influences of smoking behaviour and sex were investigated in the control group. Only in the latter case there was a significant difference between female and male with a 63% correct cell and 71% correct specimen classification.

Prognostic value of nucleolar organizer regions in neuroendocrine tumours of the lung.

The value of the number and size of the nucleolar organizer regions (NORs) as prognostic indicators in human neuroendocrine lung tumours was evaluated in a quantitative study of 57 cases, including 33 small cell carcinomas (SCLCs), 9 well-differentiated neuroendocrine carcinomas (WDNECs) and 15 "classic" carcinoids. NORs were visualized on paraffin sections by an argyrophilic technique (AgNOR) and measured by automatic image analysis. In each case, the mean number and area of AgNORs were evaluated; the results were compared with clinical follow-up and survival. AgNOR values for both number and area were significantly higher in SCLCs than in WDNECs and carcinoids. WDNECs had insignificantly higher AgNOR values than carcinoids. Among SCLCs, AgNOR values of the oat cell subtype and the intermediate cell subtype did not differ significantly. Regardless of the histological tumour type, AgNOR values strongly correlated with prognosis, with more and larger AgNORs indicating a more progressive clinical course. In the present study we demonstrate for the first time that the biological behaviour of neuroendocrine lung tumours is correlated with the number and size of AgNORs. Thus the measurement of AgNORs may serve as an additional prognostic indicator in these neoplasms, particularly in the separation of SCLCs from WDNECs with a more favourable prognosis.

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern.

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.

Prognostic value of quantitatively measured AgNORs in ductal mammary carcinoma.

Argyrophilic proteins of nucleolar organizer regions (AgNOR proteins) were stained by a modified silver staining technique. Paraffin sections from 137 cases of invasive ductal carcinoma of the breast were investigated. The nuclei and AgNOR dots were quantitatively measured by means of a semiautomatic image analysis system. Follow-up data for about 120 months (mean, 102 +/- 47) and clinical/histologic and DNA distribution parameters were available for all patients. The prognostic significance of AgNOR parameters was tested by Cox regression analysis. Four AgNOR features showed a significant univariate correlation with survival time. In multivariate analysis offering all the available parameters, one AgNOR parameter (coefficient of variation of relative AgNOR area) ranked at the third position beyond the standard deviation of DNA distribution and pTNM/staging. When considering the distant recurrence-free survival of patients, the same AgNOR feature provided significant additional prognostic information. Performing survival analysis for the prognostically defined subgroup of pTNM/stage I patients and offering all DNA and AgNOR features the same AgNOR parameter yielded the highest prognostic validity. These results show that quantitatively measured AgNORs yield prognostic factors in breast cancer.

Discrimination of esophageal dysplasia with progression and nonprogression. High-resolution image analysis for surrogate end point biomarkers.

OBJECTIVE: To detect textural nuclear features correlated with nonprogression and progression in esophageal dysplasia. STUDY DESIGN: Asymptomatic adults from Heshun Commune, Linxian County, China were examined with a balloon sampler in 1983 fifty cases of moderate esophageal dysplasia and 68 cases of mild were selected for study. By means of an Axiomat microscope equipped with a TV camera, 100 visually normal intermediate squamous cell nuclei per specimen were randomly measured from routinely Papanicolaou-stained slides. RESULTS: Of 50 esophageal moderate dysplasia cases, 24 and 7 progressed to carcinoma within three and nine years, respectively. The other 19 cases remained stable or regressed to normal and were used as the control group. By means of chromatin features, correct specimen classification rates of 79.2% (19/24), 73.7% (14/19), 85.5% (6/7) and 84.2% (16/19) were achieved, respectively (P < .001). Of 68 cases classified as mild dysplasia, 16, 13 and 12 progressed to carcinoma within three, five and nine years, respectively. The other 27 cases remained stable or regressed to normal and were used as the control group. The correct specimen classification rates were 93.8% (15/16), 88.9% (24/27), 69.2% (9/13), 74.1% (20/27), 83.3% (10/12) and 77.8% (21/27), respectively, using chromatin features of the nuclei (P < .001). CONCLUSION: In this study, nuclear chromatin features measured by high-resolution image analysis could sufficiently well forecast the outcome of precancerous lesions and discriminate precancerous lesions with progression and nonprogression. It also can be employed as surrogate end point biomarkers in clinical chemoprevention trials. Stoichiometric staining and standard preparations should increase the correct classification rates in further studies.

Prognostic value of MIB-1 in neuroendocrine tumours of the lung.

The spectrum of neuroendocrine lung tumours ranges from highly aggressive small cell carcinomas (SCLC) to carcinoid tumours (CD) of low malignant potential. Between these two extremes, the 'well-differentiated neuroendocrine carcinomas' (WDNEC) form a transitional group with uncertain biological behaviour. This study investigated the prognostic value of the proliferation marker MIB-1 (paraffin Ki-67) in 59 neuroendocrine lung tumours (32 SCLC, 13 WDNEC, 14 CD) by immunostaining of routinely processed paraffin sections. Morphometric evaluation was done by semi-automatic image analysis. The results were compared with survival data (mean follow-up: 42 months). The proliferation rates of the tumours as determined by MIB-1 immunoreactivity (MIB-1-PR) were significantly different between the tumour types (SCLC > WDNEC > CD) and showed a strong inverse correlation with survival time. In CD, the percentage of MIB-1-labelled nuclei never exceeded 1.1 per cent; higher values would therefore favour the diagnosis of WDNEC over that of CD. Among WDNEC, MIB-1 was able to differentiate a subgroup with excellent prognosis (MIB-1-PR: 0.3-3.4 per cent) from another subgroup with a death rate of 50 per cent (MIB-1-PR: 7.3-20.3 per cent). Within each tumour type, all patients without distant metastases at diagnosis survived when MIB-1-PR was < or = 9.4 per cent, suggesting a potential threshold for prognosis. Although the status of metastases are complementary prognostic indicators and are best used in combination to characterize the biological behaviour of neuroendocrine lung tumours.

Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution.

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.