Use of ubiquitous, highly heterozygous copy number variants and digital droplet polymerase chain reaction to monitor chimerism after allogeneic haematopoietic stem cell transplantation.
Chimerism analysis has an important role in the management of allogeneic hematopoietic stem cell transplantation. It informs response to disease relapse, graft rejection, and graft-versus-host disease. We have developed a method for chimerism analysis using ubiquitous copy number variation (CNV), which has the benefit of a "negative background" against which multiple independent informative markers are quantified using digital droplet polymerase chain reaction. A panel of up to 38 CNV markers with homozygous deletion frequencies of approximately 0.4-0.6 were used. Sensitivity, precision, reproducibility, and informativity were assessed. CNV chimerism results were compared against established fluorescence in situ hybridization, single nucleotide polymorphism, and short tandem repeat-based methods with excellent correlation. Using 30 ng of input DNA per well, the limit of detection was 0.05% chimerism and the limit of quantification was 0.5% chimerism. High informativity was seen with a median of four informative markers detectable per individual in 39 recipients and 43 donor genomes studied. The strength of this approach was exemplified in a multiple donor case involving four genomes (three related). The precision, sensitivity, and informativity of this approach recommend it for use in clinical practice.
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia; Department of Nephrology, Austin Health, Heidelberg, Victoria, Australia.
John B
JB
Whitlam
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Ling
L
Ling
Laboratory Services, Royal Children's Hospital, Parkville, Victoria, Australia.
Michael
M
Swain
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Tom
T
Harrington
Pathology West Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia.
Oksana
O
Mirochnik
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Ian
I
Brooks
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Sara
S
Cronin
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Jackie
J
Challis
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Vida
V
Petrovic
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Damien L
DL
Bruno
Children's Cancer Centre, Royal Children's Hospital, Parkville, Victoria, Australia.
Francoise
F
Mechinaud
Children's Cancer Centre, Royal Children's Hospital, Parkville, Victoria, Australia.
Rachel
R
Conyers
Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia; Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia. Electronic address: howard.slater@vcgs.org.au.
Howard
H
Slater
D064591:Allografts; D056915:DNA Copy Number Variations; D005260:Female; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018183:Transplantation Chimera / Q000235:genetics*