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Blood, 15 June 2002, Vol. 99, No. 12, pp. 4618-4625
TRANSPLANTATION
Quantitative assessment of hematopoietic chimerism after bone
marrow transplantation by real-time quantitative polymerase chain
reaction
Mehdi Alizadeh,
Marc Bernard,
Bruno Danic,
Charly Dauriac,
Brigitte Birebent,
Christine Lapart,
Thierry Lamy,
Pierre-Yves Le Prisé,
Alain Beauplet,
Dominique Bories,
Gilbert Semana, and
Erwann Quelvennec
From the Laboratoire Universitaire d'Immunologie
(UPRES EA 1257-IFR 97), Faculté de Médecine, Rennes,
France; Etablissement Français du Sang Bretagne, Rennes, France;
Service d'Hématologie clinique, Centre Hospitalier
Régional et Universitaire, Rennes, France; and Laboratoire
d'Hématologie, Hopital Henri Mondor, Créteil, France.
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.
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