The Application of Growth Factor Stimulants to Improve Ctogenetic Analysis of Patients with Myeloproliferative and Myelodysplastic Syndromes.

Conventional cytogenetic methods rely on culturing bone marrow aspirates to obtain suitable and sufficient mitotic figures for G banded analysis. Samples from Myeloproliferative (MPD) and Myelodysplastic (MDS) patients often have increased failure rates due to reduced growth and poor morphology all of which hampers the conventional karyotyping investigation . The aim of this study was to investigate whether;

1. The quality and quantity of metaphase cells from MPD and MDS patients could be improved.

2. To assess whether the applied method(s) led to clonal selection.

Three culture systems using different growth factor stimulants were assessed alongside an un-stimulated control. These were Bone Marrow Growth Supplement (BMGS) containing IL-3, IL-6, G-CSF and CSF, 5637 conditioned medium (5637CM) containing IL-3, IL-6 and G-CSF and myeloid expansion medium (MEM) containing IL-3, IL-6, G-CSF, SCF and Flt-3. Samples were assessed qualitatively and quantitatively for mitotic index (MI = % dividing cells). Clonality was monitored using FISH, when suitable markers were present.

In 39 patients bone marrow samples chromosome morphology of mitotic figures was improved with all stimulated cultures. Variation was observed between patient samples. However the increase in MI was shown to be significant in all stimulated cultures at p<0.05. The most favourable results were obtained with 5637CM, producing increases in MI up to 14 fold (with a mean fold increase of 1.65). 5637CM significantly improved chromosome morphology and consequently banding quality. Examination of 10 patients’ cultures with FISH probes, supports the hypothesis that clonal expansion remains un-altered in stimulated cultures.

Conventionally, peripheral blood (PB) stimulated cultures are not used for cytogenetic analysis of MPD patients. However preliminary studies of six patients have shown that growth factor stimulation successfully produces high quality mitotic figures suitable for analysis by conventional and FISH methods.