Abstract

Introduction: The current gold standard for chimerism quantification after allo-stem cell transplantation (SCT) is based on the polymerase chain reaction of DNA short tandem repeats (STR-PCR). This methodology is sex-independent, highly informative and quantitatively accurate. However, this method has a moderate sensitivity (1–3%).

Objective: To test the usefulness of increasing the assay sensitivity by 3 different approaches in order to detect recipient residual cells.

Material and Methods: Two sets of 20 samples, belonging to artificial mixtures of male and female peripheral blood (PB) cells in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0) were prepared to test the assay sensitivity and reproducibility. Additionally 58 samples from 10 selected patients showing different clinical evolution post-SCT were retrospectively studied. Chimerism was analyzed on genomic DNA (purified QIAamp DNA blood kit, Qiagen) by multiplex STR-PCR (AmpFlSTR SGM Plus revealed on capillary electrophoresis in an ABI Prism 3100 automated DNA sequencer; Applied Biosystems). Three different approaches aimed to increase the sensitivity of STR-PCR were tested: (1) Increasing DNA concentration in the PCR (1x, 2.5x, 5x), (2) Increasing the concentration of the PCR product for the capillary electrophoresis (1x, 2.5x, 5x) and (3) Increasing the sample injection time into the capillary for the electrophoresis (10s, standard time, 30s and 50s).

Results: The standard STR-PCR showed a sensitivity of 1% in both sets of male-female PB artificial mixtures. Both approaches increasing DNA concentration in the PCR and the PCR product in the capillary electrophoresis did not achieved better sensitivity. Moreover, appropriate analysis of the samples was frequently hampered due to fluorescence saturation. However, longer injection times (30s, 50s) showed that currently underrepresented DNA fragments were conspicuously overestimated, giving a one-log increase in the sensitivity of the STR-PCR assay, which reached a 0.1%. This approach was used to re-analyze the 23 SCT patient samples previously identified as in complete chimerism by standard STR-PCR. The 0.1% sensitivity methodological approach allowed to detect residual/reappearing recipient cells in three of the 23 analyzed samples. Therefore this new approach would have allowed to anticipate the detection of graft rejection and disease relapse in 2/10 patients, 7 and 77 days respectively.

Conclusion: Long injection times (30s and 50s) in the capillary electrophoresis after STR-PCR result in a one log sensitivity increase (from 1% to 0.1%). This would allow earlier diagnosis of adverse events after SCT favoring a prompt introduction of therapeutic interventions.

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