Dataset Information

Hematopoietic chimerism monitoring based on STRs: quantitative platform performance on sequential samples.

ABSTRACT: Hematopoietic stem cell transplantation (HSCT) creates a donor-recipient cellular chimerism in the patient, which is quantitatively assayed from peripheral blood based on STR-DNA. Since chimerism values often vary across a patient's samples, it is important to determine to what extent this variability reflects technical aspects of platform performance. This issue is systematically assessed in the current study for the first time. Using the SGM Plus multiplex PCR kit and ABI platform, the longitudinal performance of STR markers was quantitatively evaluated in two chimeric models with true values, and in patient samples (n >500 marker loci). Computation of percent chimerism for each marker, and mean (sample) percent chimerism, standard deviation, and coefficient of variance was performed by our ChimerTrack utility. In chimeric models with known values, individual markers exhibited an accuracy (observed/true) of 88-98%; replication precision was 92-100% true, with a mean error of 2%. Fragment size calling was greater than 99% accurate and precise. Patient results were comparable for markers, relaive to sample means. One source of technical variability in chimerism estimation was allelic differential amplification efficiency. The latter was influenced by signal amplitude, dye label, marker size, and allelic size interval. It can be concluded that long-term chimeric tracking is routinely feasible using this platform in conjunction with ChimerTrack software. Importantly, mean percent chimerism, for any sample, should closely approximate the true chimeric status, with a technical accuracy of 98%. Guidelines are presented for selecting an optimized marker profile.

SUBMITTER: Kristt D

PROVIDER: S-EPMC2291760 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Hematopoietic chimerism monitoring based on STRs: quantitative platform performance on sequential samples.

Kristt Don D Israeli Moshe M Narinski Ronit R Or Hagit H Yaniv I I Stein Jerry J Klein Tirza T

Journal of biomolecular techniques : JBT 20051201 4

Hematopoietic stem cell transplantation (HSCT) creates a donor-recipient cellular chimerism in the patient, which is quantitatively assayed from peripheral blood based on STR-DNA. Since chimerism values often vary across a patient's samples, it is important to determine to what extent this variability reflects technical aspects of platform performance. This issue is systematically assessed in the current study for the first time. Using the SGM Plus multiplex PCR kit and ABI platform, the longitu ...[more]

PMID: 16522860

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Project description:Allogenic hematopoietic stem cell transplantation (allo-HSCT) is a life-saving treatment for various hematological disorders. The success of allo-HSCT depends on the engraftment of donor cells and the elimination of recipient cells monitored through chimerism testing. We aimed to validate a next-generation sequencing (NGS)-based chimerism assay for engraftment monitoring and to emphasize the importance of including the most prevalent cell subsets in proficiency testing (PT) programs. We evaluated the analytical performance of NGS-based chimerism testing (AlloSeq-HCT and CareDx) with a panel of targeted 202 informative single-nucleotide polymorphisms (SNPs) (i.e., linearity and precision, analytical sensitivity and specificity, system accuracy, and reproducibility). We further compared the performance of our NGS panel with conventional short tandem repeat (STR) analysis in unfractionated whole blood and cell-subset-enriched CD3 and CD66. Our NGS-based chimerism monitoring assay has an impressive detection limit (0.3% host DNA) for minor alleles and analytical specificity (99.9%). Pearson's correlation between NGS- and STR-based chimerism monitoring showed a linear relationship with a slope of 0.8 and r = 0.973. The concordance of allo-HSCT patients using unfractionated whole blood, CD3, and CD66 was 0.95, 0.96, and 0.54, respectively. Utilization of CD3+ cell subsets for mixed chimerism detection yielded an average of 7.3 ± 7-fold higher donor percentage detection compared to their corresponding unfractionated whole blood samples. The accuracy of the NGS assay achieved a concordance of 98.6% on blinded external quality control STR samples. The reproducibility series showed near 100% concordance with respect to inter-assay, inter-tech, inter-instrument, cell flow kits, and AlloSeq-HCT software versions. Our study provided robust validation of NGS-based chimerism testing for accurate detection and monitoring of engraftment in allo-HSCT patients. By incorporating the cell subsets (CD3 and CD66), the sensitivity and accuracy of engraftment monitoring are significantly improved, making them an essential component of any PT program. Furthermore, the implementation of NGS-based chimerism testing shows potential to streamline high-volume transplant services and improve clinical outcomes by enabling early relapse detection and guiding timely interventions.

Project description:BackgroundIn-hospital therapeutic drug monitoring (TDM) requires a suitable quantification method for target drugs from the viewpoint of precision, throughput, and testing costs. We previously developed a practical HPLC-UV platform for quantification of serum levels of various drugs. In this report, the platform was effectively applied to the quantification of patient serum levels of five different drugs by clinical professionals in our hospital during their daily work.MethodsThe residual sera of patients receiving carbamazepine (CBZ), phenytoin (PHT), lamotrigine (LTG), vancomycin (VCM), or voriconazole (VRCZ) were used in the present clinical study. The quantification method for each drug consisted of rapid solid-phase extraction (SPE) of each drug in the patient serum, followed by optimized HPLC-UV analysis of the drug in the SPE eluate. Furthermore, patient serum levels of PHT, CBZ, and VCM were also measured by ligand-binding assay using a cobas® analyzer in our hospital, and those of LTG and VRCZ were measured by HPLC-MS/MS at an outsourced provider. Passing-Bablok regression analysis and Bland-Altman analysis were employed to analyze the agreement of drug levels in patient sera, which was separately quantified using two different methods-our HPLC-UV platform and the cobas analyzer, or HPLC-UV and HPLC-MS/MS.ResultsAll analytical conditions of the present method using our HPLC-UV platform were well optimized for each target drug quantification in the patient's serum, and the quantification method for each drug was fully validated for accuracy, precision and reproducibility. Furthermore, Passing-Bablok regression analysis and Bland-Altman analysis revealed that patient serum levels of PHT, CBZ, and VCM quantified by our HPLC-UV platform were closely correlated with those quantified by the cobas® analyzer, and the levels of LTG and VRCZ quantified by our HPLC-UV platform were also correlated with those quantified by HPLC-MS/MS.ConclusionsOur HPLC-UV platform can be performed without requiring special analytical techniques. This platform is expected to be used for the measurement of blood levels of multiple drugs for in-hospital routine TDM.

Dataset Information

Hematopoietic chimerism monitoring based on STRs: quantitative platform performance on sequential samples.

Publications

Hematopoietic chimerism monitoring based on STRs: quantitative platform performance on sequential samples.

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