Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation

Stahl, Tanja; Böhme, Manja U.; Kröger, Nicolaus; Fehse, Boris

doi:10.1016/j.exphem.2015.02.006

Cited by 66 publications

(62 citation statements)

References 23 publications

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“…To assess vector-copy numbers in the 21 GL261 clones, we carried out duplex digital PCRs using the QX100 Droplet Digital PCR System (Bio-Rad) essentially as described, 43 with the addition of 3 mM (final concentration) MgCl 2 into the reaction mix, an elongation time of 120 s, a ramping rate of 2 C/s, and 40 cycles total. Two independent duplex PCRs were designed that detect sequences within either the 5 0 SIN-LTR or the internal SFFV promoter of the LeGO vector in conjunction with a reference (REF) gene (murine erythropoietin receptor).…”

Section: Digital Pcr For Copy-number Analysismentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

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Intratumoral heterogeneity has been identified as one of the strongest drivers of treatment resistance and tumor recurrence. Therefore, investigating the complex clonal architecture of tumors over time has become a major challenge in cancer research. We developed a new fluorescent "optical barcoding" technique that allows fast tracking, identification, and quantification of live cell clones in vitro and in vivo using flow cytometry (FC). We optically barcoded two cell lines derived from malignant glioma, an exemplary heterogeneous brain tumor. In agreement with mathematical combinatorics, we demonstrate that up to 41 clones can unambiguously be marked using six fluorescent proteins and a maximum of three colors per clone. We show that optical barcoding facilitates sensitive, precise, rapid, and inexpensive analysis of clonal composition kinetics of heterogeneous cell populations by FC. We further assessed the quantitative contribution of multiple clones to glioblastoma growth in vivo and we highlight the potential to recover individual viable cell clones by fluorescence-activated cell sorting. In summary, we demonstrate that optical barcoding is a powerful technique for clonal cell tracking in vitro and in vivo, rendering this approach a potent tool for studying the heterogeneity of complex tissues, in particular, cancer.

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Section: Digital Pcr For Copy-number Analysismentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

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“…5, 6 However, new PCR strategies for chimerism quantification have dramatically increased technical sensitivity with current methods enabling the detection of chimerism below 0.1%. 7, 8, 9 Therefore, applying these methods for chimerism quantification in peripheral blood (PB) may reduce the need for BM analysis. The influence of sample source on the sensitivity of chimerism analysis in the context of highly sensitive quantification methods, however, is not well substantiated.…”

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confidence: 99%

Systematic comparison of donor chimerism in peripheral blood and bone marrow after hematopoietic stem cell transplantation

et al. 2017

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“…This approach has a distinctly higher sensitivity than STR analysis. Problems occurred because of the lower accuracy in mixed chimerism . Recently, the ddPCR was introduced, which uses the InDel markers as well, but the high sensitivity of InDel‐based detection is linked with a high accuracy in mixed chimerisms by partitioning the reaction in multiple droplets (Table ).…”

Section: Discussionmentioning

confidence: 99%

“…Insertion/Deletion polymorphism (InDel) analysis by quantitative real‐time PCR (qPCR) represents an alternative technique. It has been evaluated for chimerism analysis after alloHSCT and demonstrated high sensitivity, but there are concerns about its inherent limit of accuracy in mixed chimerism . As a new approach, InDel analysis by digital droplet PCR (ddPCR) showed high sensitivity, accuracy, and reproducibility in chimerism monitoring .…”

Section: Introductionmentioning

confidence: 99%

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Digital droplet PCR‐based chimerism analysis for monitoring of hematopoietic engraftment after allogeneic stem cell transplantation

Mika

Baraniskin

Ladigan

et al. 2019

Int J Lab Hematology

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Introduction Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative approach for multiple hematologic diseases. The success of alloHSCT is evaluated by analyzing the proportion of living donor cells in blood and bone marrow samples of the recipient (chimerism analysis). To monitor the engrafted cells, donor's individual genetic markers are analyzed in peripheral blood and bone marrow samples, usually by using short tandem repeat (STR) analysis. An alternative method to measure chimerism is based on insertion and deletion markers (InDels) analyzed by digital droplet PCR (ddPCR); however, this approach is rarely evaluated in clinical practice. Methods In this study, we examined the usefulness of ddPCR‐based chimerism analysis against the standard STR analysis in samples around day+30 after alloHSCT in clinical practice using peripheral blood and bone marrow samples. Results The median absolute difference between ddPCR and STR analysis was 0.55% points for bone marrow chimerisms and 0.25% points for peripheral blood chimerisms, respectively, including variation in the range of maximum 2% for both methods. The results of every single sample gave the same clinical message. Conclusion According to our data, chimerism analysis by ddPCR has an excellent correlation with STR‐based analyses. Due to its fast and easy applicability, the ddPCR technique is suitable for chimerism monitoring in clinical practice.

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Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation

Cited by 66 publications

References 23 publications

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

Systematic comparison of donor chimerism in peripheral blood and bone marrow after hematopoietic stem cell transplantation

Digital droplet PCR‐based chimerism analysis for monitoring of hematopoietic engraftment after allogeneic stem cell transplantation

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