Optimization and validation of immunocytochemical detection of oestrogen receptors on cytospins prepared from fine needle aspiration (FNA) samples of breast cancer.

OBJECTIVE: To optimize and validate immunocytochemical (ICC) assessment of oestrogen receptors (ERs) on cytospins prepared from fine needle aspiration (FNA) samples. METHODS: Optimal conditions and variability in ICC detection of ERs were established on cytospins prepared from the human breast cancer cell line MCF-7. Protocols that yielded adequate results were further validated on 52 FNA samples of resected breast cancer tumours using analysis of concordance with the ER status, determined by standard immunohistochemistry on corresponding formalin-fixed, paraffin-embedded tissue (FFPET). On 37 diagnostic FNA samples, manual immunostaining with antibody 1D5 was compared with automated immunostaining with antibody 6F11. RESULTS: The highest percentage of ER-positive MCF-7 cells with lowest variability was obtained on methanol-fixed cytospins with or without microwave pre-treatment: 72 ± 5% and 75 ± 7%, respectively. Microwave pre-treatment was mandatory for Papanicolaou-stained cytospins in order to achieve between 63 ± 14% and 67 ± 9% of ER-positive MCF-7 cells. The concordance between ICC assessment of ERs on FNA samples and corresponding FFPET sections was complete for methanol-fixed cytospins (100%, kappa = 1) and adequate for Papanicolaou-stained cytospins (94%, kappa = 0.84) and Papanicolaou-stained smears (92%, kappa = 0.75). Complete agreement in ICC detection of ERs was obtained for manual immunostaining with antibody 1D5 and automated immunostaining with antibody 6F11. CONCLUSIONS: Methanol-fixed cytospins prepared from FNA samples ensure highly reliable ICC assessment of ERs, whereas Papanicolaou-stained cytospins or smears are conditionally suitable because of the small risk of false negative results.

Review of negative and low-grade cervical smears in women with invasive cervical cancer after the first 3 years of the national cervical screening programme in Slovenia.

OBJECTIVE: The purpose of the study was to perform a national review of negative, low-grade and inadequate smears reported during the latest screening period before cervical cancer diagnosis in 2006, after the first 3 years of the screening programme. METHODS: Among 162 new cervical cancer cases there were 47 (29%) without previous cytology, 47 (29%) with one high-grade smear prior to diagnosis and 68 (42.0%) with at least one previous negative, low-grade, atypical or inadequate smear 1-40 months before diagnosis. Of the latter 68 cases, 37 patients with 59 smears (together with 118 control slides) were included in the review as 31 had smears reported at laboratories no longer operating. Findings were related to the last cytology report before diagnosis as well as to histological type and stage of the cancer. RESULTS: In our study group, 19 (51%) of 37 patients had squamous cell carcinoma, 15 (41%) adenocarcinoma and 3 (8%) adenosquamous carcinoma, compared with 121 (75%), 26 (16%), 12 (7%), respectively, and 3 (2%) other types, for all carcinomas. Twenty-one of 37 women also had high-grade cytology prior to diagnosis of cancer. Women with previous cytology (with or without recent high-grade smears) were more likely to have stage I cancers than those without cytology (P < 0.0001). The expert group upgraded 17/33 smears in the patients with squamous carcinomas, which was more than in those with adeno- and adenosquamous carcinomas (5/24, P < 0.05). CONCLUSION: As expected, a higher proportion of smears preceding adenocarcinomas were true negative. Under-diagnosed smears were not related to cancer stage or last cytology report before diagnosis.

Expression of oestrogen receptor alpha during development of the skeleton in mice fetuses: immunohistochemical study.

Sequential pattern of ossification and expression of oestrogen receptor alpha (ERalpha) during development of the skeleton in male and female mice fetuses was investigated. Twenty-seven mice fetuses of gestational age between 14.5 and 18.5 days post coitum (p.c.) were examined by haematoxylin-eosin and toluidine blue staining to determine the ossification. The presence of ERalpha was detected by immunostaining using ERalpha-specific antibodies. Ossification centres were determined in fetuses of 14.5 days p.c. of both sexes in the base of skull, ribs and front limbs, while in the mandible ossification was observed only in female fetuses at that age. ERalpha was found in all investigated tissues in which the occurrence of ossification centres was determined. ERalpha was first detected in some tissues involved in ossification at 14.5 days p.c. in fetuses of both genders. There were some minor gender differences in the pattern of ERalpha expression. ERalpha was localized in the metatarsal chondrogenic condensations at 14.5 days p.c. and in phalangeal chondrocytes at 17.5 and 18.5 days p.c. only in females. ERalpha-positive osteogenic cells at 14.5 days p.c. in the mandible were seen only in females. At 16.5 days p.c. male but not female fetuses expressed ERalpha in the vertebrae. Our findings support the view that ERalpha protein is found in the tissues that undergo bone formation and that ERalpha expression in these tissues shows only minor gender differences in mice fetuses.

Efficient electrotransfection into canine muscle.

Two different types of electroporation protocols have been developed for efficient electrotransfer of plasmid DNA into skeletal muscle of experimental animals. At first, only low voltage electric pulses have been used, but lately, a combination of high and low voltage pulses has been suggested as more efficient. Up to date, in dogs, this type of electroporation protocol has never been used for muscle targeted plasmid DNA electrotransfection. In this study, we used two different DNA plasmids, one encoding green fluorescent protein and one encoding human interleukin-12. Five different electroporation protocols were evaluated. Three of them featured different combinations of high and low voltage pulses, and two were performed with delivery of low voltage pulses only. Our study shows that combination of 1 high voltage pulse (600 V/cm, 100 mus), followed by 4 low voltage pulses (80 V/cm, 100 ms, 1 Hz) yielded in the same transfection efficiency as the standard trains of low voltage pulses. However, this protocol is performed quicker and, thus, more suitable for potential use in clinical practice. In addition, it yielded in detectable systemic expression of human interleukin-12. Electrotransfer of either of the plasmids was associated with only mild and transitory local side effects, without clinically detectable systemic side effects. The results indicate that electrotransfection is a feasible, effective, and safe method for muscle targeted gene therapy in dogs, which could have potential for clinical applications in veterinary medicine of small animals.

Ultrasonographically and stereotactically guided fine-needle aspiration cytology of non-palpable breast lesions: cyto-histological correlation.

OBJECTIVE: To determine the diagnostic accuracy of ultrasonographically (US) and stereotactically guided fine needle aspiration cytology (FNAC) for the diagnosis of non-palpable breast lesions. PATIENTS AND METHODS: From January 2002 to December 2004, 470 women with 478 mammographically detected non-palpable breast lesions had US or stereotactically guided FNAC of the breast lesion. Subsequent histological evaluation of the same lesion was performed at the Institute of Oncology Ljubljana, Slovenia. The correlation between the original cytological and histological diagnosis was assessed and the sensitivity, specificity, positive predictive value and negative predictive value were calculated. RESULTS: Among US-guided FNACs, 144 (53.5%) were histologically verified benign lesions and 125 (46.5%) were carcinomas. Cytological diagnoses were: true positive (TP) in 63 cases (50.4%), suspicious in 35 (28%), false negative (FN) in eight (6.4%), and in 19 (15.2%) cases, the material was inadequate for diagnosis. In the stereotactically guided FNAC group, there were 209 women with 209 lesions, with 95 (45%) histologically proven carcinomas and 114 (55%) benign lesions. Cytological diagnoses were TP in 49 (51.6%) cases, true suspicious in 21 (22.1%), FN in nine (9.5%), and in 16 (16.8%) cases, the material was not adequate for the diagnosis. CONCLUSION: The sensitivity of 88.7% and specificity of 98.6% for US-guided FNAC and 84.5% and 100% for stereotactically guided FNAC, respectively, suggest that clinicians can rely upon cytological diagnosis for planning further management of women with non-palpable breast lesions.

Enzootic nasal adenocarcinoma of sheep in Slovenia.

SUMMARY Clinicopathological and electron microscopical findings of eight cases of enzootic nasal adenocarcinoma of sheep, diagnosed solely in one big flock in Slovenia between years 2001 and 2003 are described. All affected sheep were female, their mean age was 4.5 +/- 1.5 years and they either belonged to the Istrian pramenka breed (five sheep) or were crossbreeds (three sheep). Tumours that arose from the ethmoid area of the nasal cavity were unilateral in six cases (75%) and bilateral in two cases (25%). All tumours were classified as adenocarcinomas by histopathological examination and they displayed either a combination of tubular and papillary growth or less often solely tubular proliferation. No metastases were detected in regional lymph nodes, brain or other organs. Electron microscopical studies performed on the reprocessed paraffin-embedded tissues revealed the presence of the virus-like particles with an average diameter between 70 and 90 nm.

The reliability and accuracy of intraoperative imprint cytology of sentinel lymph nodes in breast cancer.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is a new component of the surgical treatment of breast cancer that accurately predicts axillary status. In this study the authors evaluated the accuracy of intraoperative imprint cytology (IC) in comparison with definitive histologic evaluation of SLN in breast cancer patients. METHODS: A total 413 women with breast carcinoma and clinically negative axillary nodes underwent breast surgery and SLN biopsy. Mapping of SLN involved injection of (99m)Technecium labelled human albumin nanocolloid particles and Patent Blue dye. At the Department of Pathology, SLNs were bisected along its major axis. Both halves were imprinted 2-4 times on the slides and immediate staining with Hemacolor (Merck Germany) was performed for intraoperative examination. Imprint node negative women underwent no further surgery, while node positive women proceeded to full axillary clearance. Histological analysis of the SLN involved serial sectioning of the whole node with H&E and immunostaining for cytokeratin. RESULTS: Definitive histology revealed metastases (pN+) in 159/413 patients (38.5%): 69 (16.7%) macro metastases, 57 (13.8%) micro metastases, and 33 (8%) women with only isolated IHC positive cells or positive cell groups smaller than 0.2 mm (pNO sn+). The other 254 women had negative SLN biopsy. Imprint cytology detected 54/69 macro metastases, and 4/57 micro metastases. In the group with negative SLN (254), 2 cases were ''false positives''. CONCLUSIONS: Imprint of SLN biopsy can identify a negative axilla with high accuracy (specificity 99.2%). Overall sensitivity is only 36.5%, but macrometastases are detected in 77% which is important for performing ALDN in one session with operation of primary tumour.

An attempt to estimate students' workload.

Following the recent introduction of the European Credit Transfer System (ECTS) into several European university programs, a new interest has developed in determining students' workload. ECTS credits are numerical values describing the student workload required to complete course units; ECTS has the potential to facilitate comparison and create transparency between institutional curricula. ECTS credits are frequently listed alongside institutional credits in course outlines and module summaries. Measuring student workload has been difficult; to a large extent, estimates are based only upon anecdotal and casual information. To gather more systematic information, we asked students at the Veterinary Faculty, University of Ljubljana, to estimate the actual total workload they committed to fulfill their coursework obligations for specific subjects in the veterinary degree program by reporting their attendance at defined contact hours and their estimated time for outside study, including the time required for examinations and other activities. Students also reported the final grades they received for these subjects. The results show that certain courses require much more work than others, independent of credit unit assignment. Generally, the courses with more contact hours tend also to demand more independent work; the best predictor of both actual student workload and student success is the amount of contact time in which they participate. The data failed to show any strong connection between students' total workload and grades they received; rather, they showed some evidence that regular presence at contact hours was the most positive influence on grades. Less frequent presence at lectures tended to indicate less time spent on independent study. It was also found that pre-clinical and clinical courses tended to require more work from students than other, more general subjects. While the present study does not provide conclusive evidence, it does indicate the need for further inquiry into the nature of the relationship between teaching and learning in higher education and for evaluation of the benefits (or otherwise) of more "self-directed" study.

Analysis of routine cytopathologic reports in 1,598 histologically verified benign breast lesions.

This retrospective study was designed to evaluate the accuracy of cytopathologic diagnosis and of correct classification of benign breast diseases. A total of 1,598 FNABs were identified to have met the study criteria; of these, 1,258 (78.7%) cases were cytologically benign, 88 (5.5%) suspicious, 3 (0.18%) false-positive, and in 249 (15.6%) cases an inadequate sample was obtained. A specific diagnosis was made in 847/1,258 (67.3%) cases; the other 411 were diagnosed as benign NOS. Out of 847 specific FNABs diagnoses, 451 were fibroadenomas, 27 phyllodes tumors, 289 fibrocystic diseases, 4 proliferative fibrocystic diseases, 38 papillomas, 22 fat necrosis, 9 mastitis, 1 pseudolymphoma, 2 lipomas, 2 duct ecstasies, and 2 atheromas. In our study group the cytopathologic diagnosis of benign breast diseases excluding unsatisfactory aspirates was correct in 93%. Specific diagnosis was correct on average in 50% of cases, only in FA was its accuracy over 60%; in adequately sampled tumor, the predictive value of FA was 86.2%.

Cytopathology in Slovenia.

Cytopathology started in Slovenia in the early 50s with exfoliative cytology, while fine needle aspiration biopsy (FNAB) was introduced some 10 years later. Today cytopathology is a well accepted diagnostic method in Slovenia and there are currently 20 cytopathological laboratories and 17 cytopathologists. The number of specimens examined in 2001 was 26 230 FNABs, 13 355 exfoliative non cervical and 323 888 cervical smears. FNABs are performed by cytopathologists, by clinical doctors and by radiologists. So far only the cytopathologists have a supervised training period in performing biopsies. In future the same requirement will be obligatory for non pathologists. In four laboratories immunocytochemistry is used as an ancillary technique to morphology and one laboratory is using also flow cytometry for immunophenotyping of lymphomas. The classification system used in Slovenia for reporting the findings in cervical cytology is a combination of Papanicolaou's classification and assessment of dyskariosis. In spite of a long tradition in opportunistic screening for cervical cancer (Cca) an organised screening programme was started late. A four year pilot study, which included one third of the women population of Slovenia, was concluded in 2002 and an organised screening programme was introduced to the whole country in 2003. The incidence rate of Cca in Slovenia has been rising slowly since 1994 and it reached 19.6/100 000 in the year 2000. The mortality rate has remained roughly constant at 5-7/100 000 for the last 20 years. During the last few years quality assurance measures have been taken for improving the performance in cervical cytology.

Role of imprint cytology in the intraoperative evaluation of sentinel lymph nodes for malignant melanoma.

Controversy exists over the utility of different methods for intra-operative sentinel lymph node (SLN) evaluation in patients with malignant melanoma (MM). The aim of this study was to evaluate the role of intra-operative imprint cytology (IC) in patients with MM. 215 SLNs from 99 patients with MM were examined by IC and results compared with the results of permanent sections. 24 patients had MM deposits in their SLNs and this was confirmed by histological examination. Intraoperative IC was positive in 11 of these patients (46% sensitivity). In addition, there were three false-positive IC diagnoses (79% positive predictive value); one of these was due to contamination during the sectioning of the SLN. The specificity and the negative predictive values of the IC were 96 and 85%, respectively. IC is a valuable method of intra-operative SLN evaluation which can spare approximately half of the patients with clinically occult regional metastases from a second surgical procedure. However, special care must be taken to avoid false-positive results due to contamination.

Apoptosis and proliferation during seasonal testis regression in the brown hare (Lepus europaeus L.).

Testes samples of 52 brown hares (Lepus europaeus L.), sacrificed between July and January, were subjected to immuno histochemical analysis. The terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method was applied to detect apoptosis; and antibodies to proliferating cell nuclear antigen (PCNA) were used to evaluate cell proliferation in the testes. In the seminiferous epithelium, the apoptotic processes were evident from August to early November with maximal values in September. Cell death in germ cells occurs predominantly during the prophase of the first meiotic division. In July, and from mid-November onwards, only the occasional TUNEL-positive cells can be seen. The proliferation of germ cells continues during the testis regression phase. The average number of PCNA-positive cells decreases slightly from September onwards and rises again in mid-November.

Giant muscle fibres in pigs with different Ryr1 genotype.

This study examined the frequency, morphological and immunohistochemical characteristics of the giant fibres in the longissimus muscle of local Krsko polje pigs with different Ryr1 genotypes. Giant fibres were round-shaped and had significantly increased cross-sectional area compared with normal muscle fibres. Only fast-twitch glycolytic fibres were affected, usually showing enhanced succinate dehydrogenase activity. On the ultrastructural level, the dilation of the sarcoplasmic reticulum, swelling of mitochondria and destruction of myofilaments was observed. The incidence of giant fibres was the highest in Ryr1 dimutant pigs (Ryr1 nn), which also exhibited lower muscle pH1 than heterozygous (Ryr1 Nn) or pigs with the wild Ryr1 gene (Ryr1 NN). However, the giant fibres were also present in pigs free of Ryr1 gene mutation. Our results suggest that the giant fibre syndrome depends mostly upon the rate and intensity of early post-mortem glycolysis, which results in acidity of muscle tissue. We suppose that the giant fibre formation is a result of excessive intracellular lactate accumulation in some fast-twitch glycolytic fibres. This process could also explain the ultrastructural alterations and the consequent changes in the oxidative enzymes and myofibrillar ATPase staining pattern observed in our and some previous studies.

Bioconcentration of lipophilic organochlorines in ovine dentine.

The bioconcentration of lipophilic organochlorines in ovine dentine in comparison to adipose tissue was examined. Sheep were given a single dose (0.2-1.4 mg/kg body wt) of individual polychlorobiphenyl (PCB) congeners (tetrachlorobiphenyls IUPAC Nos. 54 and 80, and hexachlorobiphenyls IUPAC Nos. 155 and 169) and organochlorine pesticides 1,1-bis(4-chlorophenyl)-2,2-dichloroethene) (4,4'-DDE) and hexachlorobenzene (HCB). They were killed 2 months after the dose and teeth and adipose tissue were collected. For the extraction of organochlorines, dentine was treated ultrasonically with sulfuric acid and hexane. The concentration of organochlorines in dentine and adipose tissue was determined by high-resolution gas chromatography with electron-capture detection. The bioconcentration of 'planar' compounds (PCB-169, HCB) in dentine, compared to adipose tissue, was lower than that of 'non-planar' (PCB-155, 4,4'-DDE). Ratios of the non-planar to planar compounds PCB-155/-169 and 4,4'-DDE/HCB in dentine were 1.4 and 7.2, and in adipose tissue 0.5 and 2.4, respectively. The dentine:adipose tissue bioconcentration ratios (on a lipid basis) of PCB-54, -80, -155, -169, 4,4'-DDE and HCB were 47, 16, 0.3, 0.1, 3.2 and 1.0, respectively. The results indicate that the bioconcentration of organochlorines in different tissues cannot be based on lipid content only. It is suggested that the physicochemical properties of individual organochlorines, i.e. lipophilicity (K(ow)), diffusivity, metabolism and tissue-specific interactions, play a part in the different bioconcentrations of individual organochlorine pollutants in dentine compared to adipose tissue.

Dopamine D2 receptor mRNA measured in serial sections of the rat anterior pituitary.

Dopamine D2 receptors (D2-Rs) on lactotrophs in the pituitary gland are targets for dopamine to inhibit prolactin synthesis and release. The aim of our study was to examine if subpopulations of cells in the anterior pituitary that respond differently to dopamine show different pattern of D2-R mRNA expression. Therefore, we have used quantitative in situ hybridization technique to study the localisation of D2-R mRNA in the rat adenohypophysis. Pituitary tissue was obtained from mature and 18 days old rats. Riboprobe was transcribed from rat pituitary cDNA clone encoding D2-R and hybridized in situ with the serial sections of the pituitaries. Our results show that, although the anterior lobe of the pituitary gland contains a variety of cell types distributed in clusters, D2-R mRNA is relatively evenly distributed through the adenohypophysis. Level of expression of D2-R mRNA in the pituitary is slightly higher in mature than in young rats.

Growth dynamics of lipizzan horses and their comparison to other horse breeds.

A typical body format of Slovenian Lipizzan horse was investigated. The study included 6 foals (5 colts and 1 filly) at the Lipica stud farm. They were measured from birth to twenty-seven months and again at forty-four months of age. Measurements included body length, chest circumference, withers height and body mass. All those measurements were statistically evaluated and compared to some other horse breeds to determine the similarity of the growth dynamics of those horse breeds. It was concluded that the parameters of the head and neck reached their full growth at the age of 27 months and also exceeded the values of their parents. The same results were obtained with the length and heights of the body (body length, withers height, etc.) and with the parameters of the front and hind legs. However the girth width, hip width and some other parameters had not yet reached their full growth at 27 months. We observed different growth dynamics with the body weight. The parameter continued to grow after 27 months of age. The same growth dynamics also seem to apply to other investigated horse breeds although the actual measurements were slightly different.

Sampling, sampling errors and specimen preparation.

To obtain an adequate cervical smear for making a correct cytologic diagnosis, smear taking, laboratory handling and interpretation must be optimal. Many people are involved, and only by a combined effort of all links can this target be seriously approached: the smear takers will have to be open minded about technical improvements and read the morphologic descriptions cautiously; in the laboratory, cytotechnicians and physicians will have to challenge themselves and each other. It is mandatory to discard specimens that do not meet general standards of adequacy. At present a host of new techniques are being implemented. It is not feasible for all laboratories to be engaged in testing these new methods, but we are all requested to follow the development the best we can and switch to new ways when justified. Our working conditions are very different; therefore, it is our professional responsibility and plight to respond at the right time. So far the conclusion is that the conventional Pap smear is the international standard of care for the diagnosis of cervical cancer precursers in cancer screening programs. Certainly, this may change within a very short time. Liquid-based techniques, and in particular HPV technologies, are just around the corner.

Tissue distribution of planar and non-planar chlorobiphenyls, 4,4'-DDE and hexachlorobenzene in sheep and lambs.

The bioconcentration and distribution pattern of individual PCB congeners (IUPAC Nos: -54, -80, -155 and -169) and organochlorine pesticides (HCB and 4,4'-DDE) in blood, adipose tissue, liver and brain were examined in sheep two months after administration and in their offspring continuously exposed during the two months lactation period. Analyses were performed by high resolution gas chromatography. The levels of individual organochlorines varied significantly between tissues; the tissue/blood ratio (on a fat basis) varied by two orders of magnitude for individual congeners. The bioconcentration of the toxic planar PCB-169 congener was the highest in the liver and the lowest in the brain of the sheep.

Malignant rhabdoid tumor of the liver diagnosed by fine needle aspiration cytology. A case report.

BACKGROUND: Primary malignant rhabdoid tumor (MRT) of the liver is a rare tumor of early infancy, with a grim prognosis. CASE: A 17-month-old female presented with a palpable mass in the upper right side of the abdomen. Ultrasonographically guided fine needle aspiration biopsy of the tumor contained malignant cells with medium-sized, vesicular nuclei; prominent nucleoli; and well-defined cytoplasm exhibiting paranuclear, dense inclusions. These inclusions reacted positively with cytokeratin and vimentin. Ultrastructural examination showed the presence of intermediate cytoplasmic filaments. CONCLUSION: Cytomorphologic and immunocytochemical characteristics permit the preoperative differentiation of MRT from other malignant tumors of childhood. That facilitates treatment planning and precludes unnecessary surgical intervention.