Myeloid Leukemia

288 Lion and Watzinger
from the donor. In some instances, no cell material is available from the allograft
recipient before transplantation. To identify an appropriate genetic marker for
the monitoring of chimerism, assessment of the patient’s genotype is necessary,
but PB can no longer be used due to the presence of donor cells. We have tested
cell material from different sources to identify patient-specific genetic fingerprints.
Epithelial cells derived from the oral mucosa or from the skin may contain
donor leukocytes. In fact, buccal swabs were demonstrated to contain granulocytes
of donor origin already during the first days after SCT, before they were
detected in PB (15, and our own unpublished observations). The same applies to
cells isolated from urinary sediment. Hair provides a reliable source of endogenous
DNA, but may not be available in patients after several courses of chemotherapy.
Nail clippings were found to be an ideal source of patient DNA in this
setting. They are readily available in all patients, and sufficient quantities of goodquality
DNA adequate for PCR genotyping can be obtained from approx 5 mg
nail material. Usually, clippings of fingernails from both hands yield 20–50 mg
of material, providing 10–25 µg of DNA by using the procedure indicated.
9. The amount of cells within individual fractions isolated by flow-sorting ranges
mostly between 2000 and 10,000, but may occasionally be as low as a few hundred.
A technique permitting efficient DNA extraction from small cell numbers
is therefore required. We try to obtain 4000 cells per cell fraction in order to have
sufficient amounts of DNA for PCR analysis. The use of a modified protocol for
the Qiagen DNA Blood Mini Kit provides very pure DNA, but the yields are
relatively low, albeit sufficient for subsequent PCR. The DNA extraction based
on cell lysis and proteinase K treatment is simpler, faster, and cheaper. The DNA
yields are virtually quantitative, as determined by real-time PCR analysis of control
genes, but the quality (purity) of DNA may not be adequate in all instances
(i.e., may not work equally well with all primer combinations).
10. The volume of elution buffer should be kept low in order to provide DNA concentrations
adequate for subsequent PCR analysis. The eluate can be re-applied
to the spin column in attempts to increase the DNA yield. Alternatively, if the
yields do not provide sufficient amounts of DNA for PCR analysis, addition of a
carrier to the cell lysate prior to application to the spin column may be warranted.
11. The employment of capillary electrophoresis instruments requires less hands-on
time than conventional gel electrophoresis due to the automated loading of
samples, electrophoresis, and measurement of fluorescence signals. However,
the capacity of devices with a single capillary and an average electrophoresis
time of 20–30 min per sample may be a limiting factor for sample throughput.
The efficiency can be improved by loading PCR products of different chimerism
assays onto the capillary and analyzing all fragments in the same run. This can be
done when primers for amplification of various microsatellite loci are labeled
with different dyes, thus permitting easy identification of the products after electrophoresis
(16). These considerations are certainly of relevance at major diagnostic
centers, where high sample throughput is required. Alternatively,
instruments with multiple capillaries can be used. Currently, the overall cost of