Myeloid Leukemia

Chimerism Analysis 287
4. Allow the cement to dry completely at room temperature in darkness (see Note 25).
5. For denaturation of target DNA and probes, place the slide(s) on a heating plate
at 73°C for 5 min.
6. For hybridization, transfer the slide(s) to a humid chamber within an incubator at
37°C for 4–48 h (see Note 26).
3.4.4. Stringency Washes and Counterstaining of Cells
1. Carefully remove the rubber cement and cover slip(s) from the slide(s). Avoid
scratching.
2. Incubate the slide(s) in 0.25X SSC at 72°C for 2 min.
3. Incubate the slide(s) in washing solution at room temperature for approx 5 min
(allows cooling of slide(s) to room temperature).
4. Rinse slide(s) with deionized water.
5. Counterstain cells by adding 15 µL Vectashield mounting medium per slide
(across all chambers).
6. Apply a glass cover slip to the slide.
7. Store in darkness at 4°C until analysis (see Note 27).
3.4.5. Analysis by Fluorescence Microscopy
Whenever possible, evaluate at least 500 nuclei to calculate the percentage
of male/female cells in the sample with adequate precision (see Fig. 3 and
Note 28).
4. Notes
1. The main advantages of automated fluorescence-based detection of microsatellite
markers over the use of conventional polyacrylamide gel electrophoresis (PAGE)
include greater precision and easier performance of quantitative analysis, reduced
manual handling of PCR products, and higher sensitivity.
2. Use, e.g., overhead transparency cut to appropriate size.
3. Use, e.g., a metal box with lid containing wet filter paper.
4. We use a total of 4 × 106 nucleated cells (NC) per patient sample for cell sorting.
The total blood volume required is therefore based on the WBC count.
5. The incubation at 4°C minimizes nonspecific staining by blocking nonspecific
antigen-binding regions.
6. If the subsequent DNA extraction is performed by proteinase K lysis, the cells
should be collected in a small amount of buffer, e.g., 50 µL Tris, as indicated, in
order to avoid the need for a DNA-concentrating step.
7. Capillary electrophoresis-based product analysis seems to be sensitive to variations
in DNA template quality. Therefore, it is necessary to use isolation protocols
yielding high-quality DNA in order to obtain reproducible results and
satisfactory sensitivity. DNA isolation kits (e.g., from Qiagen) have proven to be
adequate for this application.
8. Initial genotyping for the detection of informative STR loci is usually performed
using peripheral blood (PB) from the recipient and PB or bone marrow (BM)