[Chromosome banding analysis of peripheral blood lymphocytes stimulated with IL2 and CpG oligonucleotide DSP30 in patients with chronic lymphocytic leukemia]. / Cytogenetické vysetrení lymfocytu periferní krve po stimulaci IL2 a CpG oligonukleotidem DSP30 u pacientu s chronickou lymfocytární leukemií

BACKGROUND: Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics. PATIENTS AND METHODS: In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13. RESULTS: Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis. CONCLUSION: Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

The optimization of sample treatment for spectral karyotyping with applications for human tumour cells.

Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours--neuroblastoma and Wilms' tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods.

Application of a chromogenic medium and the PCR method for the rapid confirmation of L. monocytogenes in foodstuffs.

Detection of Listeria monocytogenes in foodstuffs by conventional cultivation methods carried out according to EN ISO guidelines is rather time-consuming. Therefore, two alternative methods were applied for rapid confirmation of L. monocytogenes in foodstuffs. Inoculum from liquid selective broth was plated on PALCAM and OXFORD agar and on chromogenic agar medium RAPID L. mono. Suspect colonies from PALCAM were confirmed according to EN ISO standards and by the multiplex PCR method. In total, 990 samples of foodstuffs were investigated and 63 strains of L. monocytogenes were isolated. The chromogenic medium RAPID L. mono provided results comparable to PCR, it is easier to handle and provides considerable financial savings.