Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation.
Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current "gold standard" for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. t.stahl@uke.de.
Tanja
T
Stahl
Biotype Diagnostic GmbH, 01109 Dresden, Germany. c.rothe@biotype.de.
Caroline
C
Rothe
Biotype Diagnostic GmbH, 01109 Dresden, Germany. m.boehme@biotype.de.
Manja U
MU
Böhme
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. aloisakohl@hotmail.com.
Aloisa
A
Kohl
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. n.kroeger@uke.de.
Nicolaus
N
Kröger
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. fehse@uke.de.
Boris
B
Fehse
D046528:Chimerism; D018380:Hematopoietic Stem Cell Transplantation; D006413:Hematopoietic System / Q000378:metabolism*; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014184:Transplantation, Homologous
allogeneic stem cell transplantation; chimerism; digital PCR; minimal residual disease