ABSTRACT
Myelodysplastic syndromes (MDS) are clonal malignancies characterized by peripheral blood pancytopenia and signs of maturation disturbances of one or several cell lineages in bone marrow. MDS present as chimeras associating normal polyclonal and malignant monoclonal progenitors cells in various proportions. Numerous cytogenetic abnormalities have been reported in MDS and can be detected by fluorescence in situ hybridization (FISH) on interphase cells. We have used this technique on methylcellulose cultured hematopoietic progenitors obtained from three patients suffering from MDS and exhibiting informative karyotypic features. Hematopoietic cells were cultured for 14 days, and individual clones (BFU-E, CFU-GM) were picked up and then cytocentrifuged for FISH analysis. We used centromeric probes realized and labeled in our laboratory by PCR to detect aneuploidies for chromosomes 7 and 11 in two patients. Furthermore, we could detect a 5q partial deletion on interphase cells from the third patient using a 5q31 specific probe visualized with the HNPP Fluorescent Detection Set from Boehringer Mannheim. In conclusion, FISH is a helpful method to detect malignant clones in hematopoietic progenitor cultures and hence to study the relative growth of normal vs. leukemic cells in MDS.
Subject(s)
Hematopoietic Stem Cells/pathology , In Situ Hybridization, Fluorescence/methods , Myelodysplastic Syndromes/diagnosis , Aged , Aneuploidy , Cells, Cultured , Centromere , Chromosome Aberrations/diagnosis , Chromosome Disorders , Female , Humans , Male , Methylcellulose , Middle AgedABSTRACT
The extended use of Fish with centromeric probes in many cytogenetic laboratories is often impaired by the cost of this technique. Polymerase Chain Reaction (PCR) constitutes a simple way to generate and label such centromeric probes at low cost. Two types of human DNA source can be used: 1--Somatic hybrid cell lines containing a unique human chromosome. The specific amplification of the human subset of alphoid DNA is realised with a primer pair specific for the consensus region of human alpha satellite sequence. 2--Total Human DNA. This time, a primer pair specific for the alpha satellite DNA of the chromosome of interest must be designed. These probes, labelled during the PCR reaction by direct incorporation of modified dUTP, are actually widely used in our laboratory, alone or mixed with other probes (chromosome painting or locus specific probes).
