Tsubasa Okano1, Yuki Tsujita2, Hirokazu Kanegane3, Kanako Mitsui-Sekinaka2, Kay Tanita1, Satoshi Miyamoto1, Tzu-Wen Yeh1, Motoi Yamashita1, Naomi Terada1, Yumi Ogura2, Masatoshi Takagi4, Kohsuke Imai4, Shigeaki Nonoyama2, Tomohiro Morio1. 1. Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. 2. Department of Pediatrics, National Defense Medical College, Saitama, Japan. 3. Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. hkanegane.ped@tmd.ac.jp. 4. Department of Community Pediatrics, Perinatal and Maternal Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Abstract
OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Entities:
Keywords:
Droplet digital PCR; chimerism; hematopoietic stem cell transplantation; severe combined immunodeficiency
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