cIg-FISH optimization on fixed bone marrow cells
Multiple myeloma is frequently accompanied by different chromosomal abnormalities. Some of them are recurring aberrations and as such have an important diagnostic and prognostic value. They are difficult to be determined by conventional cytogenetics due to low mitotic index of plasmacytoma cells. Therefore, abnormalities that result in poor survival of afflicted patients, e.g. t(4;14)(p16;q32), t(14;16)(q32;q23), chromosome 13 abnormalieties, are usually determined by interfase FISH. However, bone marrow infiltration by plasmocytoma cells can vary considerably depending on type and progress of multiple myeloma. Due to inability of FISH to distinguish between plasmocytoma and the rest of bone marrow cells, the results can be well below the correct value.
cIg-FISH is a combination of immunolabeling of cell cytoplasm Ig light chain (l or k) and FISH. Different approaches have been proposed to merge both procedures into a single one. Majority of them is based on cytospin preparation of fresh cells from a bone marrow aspirate. For everyday routine molecular cytogenetics this approach is inconvenient due to additional work.
To overcome described drawbacks, we decided to optimize cIg-FISH on already fixed cells previously cultivated and prepared for cytogenetic analysis. The influence of cell aging, formaldehyde fixation and sequence of procedures - FISH and immunolabeling - were studied. Additionally, formamide denaturation and washing conditions were optimized. Clearly visible plasmocytoma cells with evidently colored cytoplasm combined with intensive probe signals were obtained under optimized conditions. We were able to determine proportion of plasmocytoma cells and the proportion of targeted chromosomal abbreviation on the labeled cells.