Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms.
Analysis of changes in recipient and donor hemopoietic cell origin is extremely useful to monitor the effect of stem cell transplantation (SCT) and sequential adoptive immunotherapy by donor lymphocyte infusions (DLI). We developed a sensitive and accurate method to quantify the percentage of recipient and donor cells by real-time PCR using single nucleotide polymorphisms (SNPs) as markers. Allele-specific PCR of seven SNPs resulted in specific markers for donor or recipient in 97% of HLA-identical sibling pairs. Both, recipient- and donor-derived hemopoietic cells can be simultaneously analyzed in 67% sibling pairs. We expect this can be increased to approximately 99% by developing three additional SNP-PCR. Serial dilution of SNP-positive DNA into either SNP-negative DNA or water revealed a detection limit of 0.1-0.01% depending on the amount of input DNA and start C(t) of the used SNP-PCR. Application of our real-time SNP-PCR method for a CML patient treated by allogeneic SCT and DLI demonstrated its feasibility to follow donor T-cell chimerism and early detection of residual and recurrent autologous hemopoiesis in response to treatment. This detailed monitoring of the genetic origin of hemopoietic cells, in particular immune effector cells and target cells after SCT and DLI, may substantially contribute to understanding of the mechanisms that play a role in the success of treatment.
Maas
F
F
Central Hematology Laboratory, University Medical Center St Radboud, Nijmegen, The Netherlands.
Schaap
N
N
Kolen
S
S
Zoetbrood
A
A
Buño
I
I
Dolstra
H
H
de Witte
T
T
Schattenberg
A
A
van de Wiel-van Kemenade
E
E
D000483:Alleles; D001773:Blood Cells / Q000166:cytology; D005240:Feasibility Studies; D018380:Hematopoietic Stem Cell Transplantation / Q000592:standards*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000628:therapy; D017710:Lymphocyte Transfusion; D022423:Myeloid Cells / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000166:cytology; D018183:Transplantation Chimera; D014184:Transplantation, Homologous / Q000592:standards