By John Swansbury
A suite of key cytogenetic and FISH recommendations utilized by sleek medical laboratories within the genetic research of human malignancies. The book's sensible suggestion and techniques are compatible to be used at each point of craftsmanship, together with totally validated laboratories, yet with a sympathetic bias in the direction of a person contemplating constructing a brand new cytogenetics provider. the following the reader will locate not just user-friendly tutorials at the basics of human karyotypes and chromosome research, but additionally distinctive discussions on how laboratories might optimally improve their repertoire of services to incorporate such more moderen complementary thoughts as CGH, FISH, and M-FISH.
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Extra info for Cancer Cytogenetics. Methods and Protocols
Overnight; see Note 2). As described in Chapter 2, several cultures should be set up if there is sufficient material is available, because the cell cycle time is unpredictably affected by the disease. For this description of technique, it will be assumed that the supplied sample had enough cells for four cultures: 24-h, overnight colcemid, FdUr-blocked (1), and a PHA-stimulated culture to check the constitutional karyotype in case it becomes necessary to do so. In the author’s laboratory, it is also customary to set up a further culture, if there is sufficient material, blocked with excess thymidine as described in Chapter 9, and to have two cultures blocked with FdUr that are released at different times.
If possible, check a slide from each case before staining other slides, as the banding will vary according to the length of the chromosomes. 12. Leave the slides to dry overnight if possible before screening and analyzing the metaphases. Immersion oil and soft mounting medium tend to mix and produce a sticky compound that adheres to the microscope objective lens and makes it impossible to focus properly. Cytogenetic Techniques for Myeloid Disorders 55 13. When the microscopic analysis of each slide has been finished, place the slide face down on tissue to remove the immersion oil, as otherwise it may seep under the coverslip and leach the stain.
3. Generally just two slides are spread for each culture. Each slide should be labeled with the date, the patient’s ID, the type of culture, and a serial number. There are many ways of spreading the cell suspension on slides; three are described below. The author’s preference is to use wet slides. a. Using cold-frosted slides, kept in a freezer: Take a pair of slides from the freezer. Add two well-spaced drops of cell suspension to each slide. Wave the slides briefly in the warm moist air above a Bunsen burner flame adjusted to be clear but not fierce.