Grading of follicular lymphoma in cytological samples.

OBJECTIVE: The treatment of follicular lymphoma (FL) depends on its grade. The current World Health Organization (WHO) 2008 Classification of Tumours of Haematopoietic and Lymphoid Tissues recommends the grading of FL on histological samples according to the Mann and Berard method, taking into consideration the number of centroblasts. There is no generally accepted method for the grading of FL in fine needle aspiration biopsy (FNAB) samples. The aim of the present study was to devise a grading system for FL in cytological samples. METHODS: Flow cytometry (FC) was performed on 60 FNAB samples of patients with primary FL. We assumed that FL cells larger than reactive T lymphocytes on FC histograms corresponded to centroblasts. The percentage of large cells was calculated and compared with histological grade, proliferative activity and number of centroblasts per high-power field (HPF) on histological slides, and with survival. RESULTS: The histological analysis of lymph nodes revealed 20 patients with high-grade and 40 patients with low-grade FL. The percentage of large cells in FNAB samples correlated significantly with histological grade (P = 0.02), MIB1 status (P < 0.001) and the number of centroblasts per HPF (P < 0.001). An age over 60 years and a percentage of large cells over 50% in FNAB samples were found to have a statistically significant impact on survival by univariate analysis (P = 0.001 and P = 0.006, respectively). CONCLUSIONS: The percentage of large lymphoma cells in FNAB samples of FL determined by FC can be used as a reliable method for FL grading, as it is comparable with the histological grading system.

Muscle gene electrotransfer is increased by the antioxidant tempol in mice.

Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.

Haemorrhagic cytology samples - how to get the best diagnostic results.

OBJECTIVE: To describe and evaluate the value of a simple filtration technique for use in processing haemorrhagic samples for cytomorphological evaluation and immunocytochemistry. METHODS: One hundred and sixty haemorrhagic cytological samples (133 FNAs, 27 effusions) received in our laboratory from August 2002 to September 2005 were included in this study. After preparing two smears for diagnostic evaluation, the residual sample was suspended in 2 ml of a cell medium prepared in our laboratory. These primary haemorrhagic suspensions were filtered through disposable nylon filter devices and the cells deposited on the upper membrane surface were transferred into 2 ml of fresh cell medium. From all three fractions - primary cell suspension, filter deposit and filtrate - cytospins were prepared and stained by Giemsa or Papanicolaou methods. Cytospins were examined under the microscope for the presence of diagnostic cells, red blood cells (RBCs) and debris. Additional cytospins for immunocytochemistry were prepared at the cytopathologist's request. RESULTS: RBCs and debris were successfully removed in 142 out of 160 haemorrhagic samples (88%) by using this new filtration technique. In all these cases the tumour cells were well presented and allowed substantially improved cytomorphological evaluation. Immunocytochemical staining was performed on 112 filtered samples with three different markers per case on average. Filtration did not improve the quality of cytospins in 18/160 haemorrhagic samples, mostly attributable to insufficient numbers of diagnostic cells in the original samples. CONCLUSION: The presented filtration technique is very simple and quick. It substantially improves the quality of haemorrhagic samples for cytomorphological evaluation; moreover, the samples are well suited for multiple immunocytochemical stainings.

DNA ploidy of human granulosa cells from natural and stimulated in vitro fertilization cycles.

OBJECTIVE: To analyze and compare the DNA ploidy of granulosa cells from natural and gonadotropin-stimulated follicles obtained during IVF. DESIGN: Retrospective analysis of laboratory data. SETTING: University medical center. PATIENT(S): Seventy-three aspirates of dominant follicles from natural IVF cycles and 113 aspirates from gonadotropin-stimulated cycles were analyzed. INTERVENTION(S): Cytospins were prepared and stained by the Feulgen-thionine method. MAIN OUTCOME MEASURE(S): Image DNA analysis was performed on an automated high-resolution image cytometer. DNA content and the number of nuclei with DNA content >5c were measured. RESULT(S): All samples from natural and gonadotropin-stimulated follicles were found to be diploid. Single cells with DNA content >5c were found in follicular fluid samples of four women with natural IVF cycles and in samples of nine women with gonadotropin-stimulated cycles. CONCLUSION(S): DNA ploidy of granulosa cells from natural follicles has not been studied before. In natural samples, granulosa cells were only diploid, without euploid polyploidization. We were unable to confirm DNA aneuploidy of granulosa cells in gonadotropin-stimulated follicles of women undergoing IVF.

Hydrolysis profiles of formalin fixed paraffin-embedded tumors based on IOD (integrated optical density) and nuclear texture feature measurements.

The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin-embedded surgical samples, prepared as single cell suspensions for image cytometric measurements. The nuclear texture features along with the IOD (integrated optical density) of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature). Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study. IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy. Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment. Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor.