Myeloid Leukemia

Chimerism Analysis 289
capillary electrophoresis with fluorescence-based detection of PCR products is
considerably higher than conventional analysis using polyacrylamide gel electrophoresis.
12. The amount of template DNA in the PCR reactions can play a role in the achievable
sensitivity of the assay. Most investigators use 50–100 ng of template (corresponding
to approx 7.5–15 × 10 3 diploid human cells), a quantity readily
available when analyzing chimerism within the entire white blood cell population.
In such instances, it is feasible to reproducibly detect residual recipient cell
populations in the range of 1% (i.e., approx 100 cells). This level of sensitivity
may not be readily achievable, however, when specific white blood cell fractions
isolated by flow-sorting or by magnetic bead separation are investigated. In these
instances, only small cell numbers are available for DNA isolation, yielding no
more than 1–30 ng of DNA (corresponding to approx 150–4500 cells). Although
1% sensitivity (equivalent to detecting about 45 cells or less) can be reached even
in this experimental setting, it is more common to achieve sensitivities of approx
3–5%. Despite the slightly decreased sensitivity of assays using low cell numbers
as starting material, the overall sensitivity in detecting minor autologous cell
fractions within specifically enriched leukocyte populations is generally one to
two logs higher than chimerism analysis in whole white blood cell preparations.
13. Split peaks (see Fig. 4C): Several DNA polymerases can catalyze the addition of
a single nucleotide (usually adenosine) to the 3�-ends of double-stranded PCR
amplicons. This nontemplate addition leads to the generation of PCR products
that are one base pair longer than the actual target sequence. In order to avoid the
occurrence of split peaks that are one base apart due to inefficient nucleotide
addition, a terminal cycling step at 60°C for 45 min is included in the PCR profile.
This step provides the polymerase with extra time to complete nucleotide
addition to all double-stranded PCR products, thus usually preventing formation
of split peaks.
14. The injection parameters need to be adjusted according to the yield of the PCR
reaction and generally range between 5 and 15 s injection time and 1–6 kV. Usually,
samples are run using different parameters in order to achieve optimal sensitivity.
The peak height of the dominant alleles should be approx 5000 rfu to
permit detection of subdominant alleles at a sensitivity of approx 1%, because
the lower limit of detection (i.e., signal above noise) is approx 50 rfu. Quantitative
analysis is possible only if the detected peaks are not off-scale (indicated by
the apparatus). In some instances, very high peaks (saturated signals) are not
recognized and indicated by the software as being off-scale, and appear as double
peaks (tip of the peak is bent down) (see Fig. 4E).
15. A run time of 20 min is sufficient for PCR products up to approx 400 bp in size,
30 min are adequate for products up to approx 600 bp.
16. Quantification of the degree of mixed chimerism is often carried out relative to a
patient-specific standard curve established from serial dilutions of pre-transplant
recipient in donor DNA. For each patient, standard curves are produced for one
or more informative microsatellite markers. For quantitative analysis of donor