Myeloid Leukemia

Chimerism Analysis 293
Table 2
Commonly Encountered Problems in Chimerism Calculation and Recommended
Measures
Possible cause Recommended measure
Extra signals:
Contamination with extraneous DNA Use appropriate precautions and controls
(aerosol-resistant pipette tips)
DNA input too high Decrease amount of template DNA or
reduce the number of Polymerase chain
reaction (PCR) cycles
Sample not completely denatured Heat sample to 93°C for 3 min before
capillary electrophoresis
Faint or no signals:
Impure DNA: Presence of inhibitors Test different extraction methods
combined with filtration of DNA
through a spin column
Insufficient template DNA Increase amount of DNA template
Primer concentration too low Increase primer concentration
Incorrect PCR program Check the PCR program
Wrong MgCl 2 concentration Test different concentrations of MgCl 2
25. Rubber cement must be completely dry to avoid leakage of probe solution. The
incubation is performed in darkness to avoid fading of fluorescence.
26. Hybridization for 4 h is sufficient, but may be carried out overnight or even over
the weekend (for convenience) without any negative effect.
27. Under the storage conditions indicated, the fluorescence remains stable for years,
thus permitting analysis of the slides at any later time point.
28. The reported average sensitivity in detecting minor (in most instances recipientderived)
cell populations using different microsatellite markers ranges from 1 to
5%. The sensitivity may to a large extent depend on the size (i.e., amplicon length)
of the informative recipient allele(s), the allelic constellation, and the number of
alleles co-amplified. In practice, however, some markers from the panels used at
individual centers tend to provide higher sensitivity than others and are therefore
used preferentially.
Acknowledgments
Supported by the Österreichische Kinderkrebshilfe. The authors wish to acknowledge
the contribution of the following colleagues from the CCRI,
Vienna, Austria: Dieter Printz and Gerhard Fritsch (flow-sorting), Margit
König (FISH analysis), Helga Daxberger and Sandra Preuner (STR-PCR and
capillary electrophoresis).