Proper technique for preparing slides is considered essential for both classical cytogenetics (banding) and molecular cytogenetic procedures (fluorescence in situ hybridization "FISH", comparative genomic hybridization "CGH", and multicolor karyotyping "M-FISH"). Much was written over the years about these procedures Different protocols were specifically designed for use with particular types of biologic material or cells (amniocytes, lymphocytes, solid tumors, leukemias) or for particular purposes (G-, C-, Q-, R-banding, FISH, CGH, M-FISH). Various instruments designed to aid the harvesting process were described and some are available from different vendors.
It is beyond the purpose of this report to discuss all these procedures, but in order to help the newcomer in the molecular cytogenetic field to better understand the technical aspects discussed throughout the manuscript, a brief outline of the procedural cytogenetic steps is presented. (1) cell culture: although cell culture methods vary significantly with the tissue of origin (amniotic fluid, chorionic villi, fetal tissues, peripheral blood, bone marrow, solid tumors, cell lines of various origins), the final goal is to achieve cell growth and division, ultimately leading to a good mitotic index,. (2) harvesting: mitotic spindle formation is blocked, usually by adding colcemide to the culture, and the cell division is stopped at the metaphase level. Cells are subjected to hypotonic treatment, which increases their volume, and disrupts the cell membrane of the red blood cells allowing their removal. A fixative solution is added to the cell suspension to preserve the cells in their "swollen" state and to remove the water, thus "hardening" the biologic material. The common fixative (3:1 methanol:acetic acid) removes lipids and alters/denatures proteins thus making the cell membrane remnant very fragile, which is important for subsequent chromosome spreading. (3)slide preparation: drops of cell suspension are placed on a slide, and allowed to dry in a controlled fashion, leading to chromosome spreading. (4) aging: slides are subjected to dry heat and/or ethanol, in order to denature the proteins, to remove water and fixative from the preparations, and to enhance the adherence of the material to the glass. (5a)banding: spreaded chromosomes are stained/banded, which allows their visualization and identification at the microscope, or (5b) pre-treatment, denaturing and hybridization: steps to which the slide is subjected during various FISH protocols. Careful adjustment of these steps allows optimal hybridization efficiency and fluorescent signals.
Every cytogenetic laboratory has inherited or further developed and refined its own slide preparation procedure(s), using different fixatives, buffers, drying temperatures, and/or various humidity conditions. It is interesting to notice that, with all this variation in technical procedures, most slide-preparation methods provide reproducibly good results. However, as the cytogenetic world is so accustomed to, people working in many laboratories are often talking about "bad" days for slide preparations, in which chromosomes don’t spread or are shiny or become surrounded by cytoplasmic residua, making them less likely to band properly. In most of these cases, the main culprit is the variation in the atmospheric humidity but a bad hypotonic solution or distilled water or an old fixative can also negatively influence a harvesting/slide preparation day.
Our report describes a simple and reproducible approach to slide preparation and chromosome spreading, which works in all climate conditions and is atmospheric-humidity independent. The novelty of the procedure is that it takes advantage of the processes taking place during drying of the cell suspension, as observed at the microscope. Proper use of hot steam vapors and a gradient of temperature for drying, allows good spreading of all types of cellular suspensions tested. G-banding and FISH images are provided for comparison with "classical" procedures.