REDUCTION IN SIGNAL OVERLAP RESULTS IN INCREASED FISH EFFICIENCY - IMPLICATIONS FOR PREIMPLANTATION GENETIC DIAGNOSIS

Background: In the absence of mosaicism, one of the problems of preimp lantation genetic diagnosis with FISH is the occurrence of false-negat ive hybridization results. It has been hypothesized that missing signa ls are produced by spatial overlap of signals. Methods and Results: To investigate the relation among cell density, signal overlap, and hybr idization signal detection, 371 blastomeres and 4556 lymphocytes were fixed in different cellular concentrations and analyzed by FISH using probes for chromosomes X, Y, and 18, and their nuclear diameters and F ISH results scored. The results showed that the lower the diameter of fixed nuclei, the higher the number of signal overlaps and missing sig nals. The minimum number of missing signals was obtained when. lymphoc yte and blastomere nuclei had 40 or more microns in diameter after fix ation and FISH. Since blastomeres were fixed individually, results wit h blastomeres were invariably better than with lymphocytes.