title: Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction.
abstract: We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with r higher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful in guiding early implementation of additional treatments in hematopoietic stem cell transplantation.
authors.affiliation: Laboratoire Universitaire d'Immunologie, UPRES EA 1257-IFR 97, Faculté de Médecine, Rennes, France.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Alizadeh
authors.forename: Marc
authors.initials: M
authors.lastname: Bernard
authors.forename: Bruno
authors.initials: B
authors.lastname: Danic
authors.forename: Charly
authors.initials: C
authors.lastname: Dauriac
authors.forename: Brigitte
authors.initials: B
authors.lastname: Birebent
authors.forename: Christine
authors.initials: C
authors.lastname: Lapart
authors.forename: Thierry
authors.initials: T
authors.lastname: Lamy
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Le Prisé
authors.forename: Alain
authors.initials: A
authors.lastname: Beauplet
authors.forename: Dominique
authors.initials: D
authors.lastname: Bories
authors.forename: Gilbert
authors.initials: G
authors.lastname: Semana
authors.forename: Erwann
authors.initials: E
authors.lastname: Quelvennec
mesh_terms: D000293:Adolescent; D000328:Adult; D016026:Bone Marrow Transplantation / Q000592:standards; D015342:DNA Probes; D005260:Female; D005819:Genetic Markers; D006085:Graft Survival / Q000235:genetics*; D006410:Hematopoiesis / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D018183:Transplantation Chimera / Q000235:genetics*
chemical_list: D015342:DNA Probes; D005819:Genetic Markers