6th ESACP Congress, Heidelberg, April 7-11, 1999

A101
LASER SCANNING CYTOMETRY FOR FISH AND CELL CYCLE ANALYSIS
März H, Baumgartner A, Hambsch J, Nüsse M, Schmid T, Schneider P, Zotz R, Tarnok A

Heart Center Leipzig GMBH - University of Leipzig, Pediatric Cardiology, Leipzig, Germany

The Laser Scanning Cytometer (LSC) enables multicolor fluorescence and light scatter measurements on a slide featuring relocation of single cells for further investigation and the ability to confirm and capture investigated cells via a CCD camera.
The use of flow cytometry (FCM) is limited in certain cases of FISH assays. While investigating sperm of human and mice aneuploid chromosomes are identified due to the amounts of FISH spots. Since FCM only detects integrated fluorescence signals of single cells, it cannot provide the resolution which is therefore demanded while the LSC can do. Due to its technique, the Laser Scanning Cytometer enables to detect immobilised cell specimen on a slide. Therefore, it provides a distinguishable distribution of FISH spots on sperm which is applied for an automated scoring technique.
In relation to time-consuming manual scoring using a fluorescence microscope, the LSC enabled to determine more than 15,000 sperm in about 30 minutes. Furthermore, we took advantage of another LSC feature, automatic cell cycle analysis. This feature is directly supported by the software but due to the relocation after the measurement it is also possible to validate the correlated cells preventing false positive events in calculation of the apoptosis index. In the next future, we are aiming to establish the LSC for our clinical diagnostic facilities for neonates undergoing heart surgery. Therefore, we hope to reduce the demanded blood sample volume from 400 µl to 5-10 µl. For the moment, we succeeded to minimize the needed volume to approximately 100 µl.