Droplet Digital PCR-Based Chimerism Analysis for Primary Immunodeficiency Diseases
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In the current study, we aimed to accurately evaluate donor/recipient or male/female chimerism in samples from patients who underwent hematopoietic stem cell transplantation (HSCT).
We designed the droplet digital polymerase chain reaction (ddPCR) for SRY and RPP30 to detect the male/female chimerism. We also developed mutation-specific ddPCR for four primary immunodeficiency diseases.
The accuracy of the male/female chimerism analysis using ddPCR was confirmed by comparing the results with those of conventional methods (fluorescence in situ hybridization and short tandem repeat-PCR) and evaluating dilution assays. In particular, we found that this method was useful for analyzing small samples. Thus, this method could be used with patient samples, especially to sorted leukocyte subpopulations, during the early post-transplant period. Four mutation-specific ddPCR accurately detected post-transplant chimerism.
ddPCR-based male/female chimerism analysis and mutation-specific ddPCR were useful for all HSCT, and these simple methods contribute to following the post-transplant chimerism, especially in disease-specific small leukocyte fractions.
KeywordsDroplet digital PCR chimerism severe combined immunodeficiency hematopoietic stem cell transplantation
Activated PI3K-delta syndrome type 1
Droplet digital PCR
Fluorescence in situ hybridization
Graft versus host diseases
Hematopoietic stem cell transplantation
Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked
Primary immunodeficiency diseases
Polymerase chain reaction
Reduced intensity conditioning
Severe combined immunodeficiency
Short tandem repeat
Variable number tandem repeat
X-linked lymphoproliferative syndrome type 1
We thank the patients and their parents as well as the doctors who provided the samples.
T.O. and Y.T. performed the experiments and wrote the manuscript. H.K. designed the study and wrote the manuscript. K.M-S., K.T., S.M., T-W.Y., M.Y., N.T., and Y.O performed the experiments. M.T., K.I., S.N., and T.M. provided critical discussion.
This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Ministry of Health, Labor, and Welfare of Japan.
Compliance with Ethical Standards
We obtained written informed consent from the parents of patients and healthy controls. The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics boards of Tokyo Medical and Dental University and National Defense Medical College.
Conflict of Interest
The authors declare that they have no conflict of interest.