Quantitative Chimerism Analysis Research Articles

Detection and Monitoring of Lineage-Specific Chimerism by Digital Droplet PCR-Based Testing of Deletion/Insertion Polymorphisms

Analysis of specific leukocyte subsets for post-transplantation monitoring of chimerism provides greater sensitivity and clinical informativeness on dynamic changes in donor- and recipient-derived cells. Limitations of the most commonly used approach to chimerism testing relying on PCR-based analysis of microsatellite markers prompted us to assess the applicability of digital droplet (dd) PCR amplification of deletion/insertion polymorphisms (DIPs) for lineage-specific chimerism testing in the related stem cell transplantation setting, where the identification of informative markers facilitating the discrimination between donor-derived and recipient-derived cells can be challenging. We analyzed 100 genetically related patient-donor pairs by ddPCR analysis using commercially available DIP kits including large sets of polymorphic markers. At least 1 informative marker was identified in all related pairs analyzed, and 2 or more discriminating markers were detected in the majority (82%) of instances. The achievable detection limit is dependent on the number of cells available for analysis and was as low as 0.1% in the presence of ≥20,000 leukocytes available for DNA extraction. Moreover, the reproducibility and accuracy of quantitative chimerism analysis compared favorably to highly optimized microsatellite assays. Thus, the use of ddPCR-based analysis of DIP markers is an attractive approach to lineage-specific monitoring of chimerism in any allogeneic transplantation setting.

Usefulness of the Quantitative Follow-up of Chimerism in Cell-Free Plasma DNA for the Prediction/Early Diagnosis of Complications After Hematopoietic Stem Cell Transplantation

AbstractAbstract 4146 IntroductionThe success of allogeneic stem cell transplantation (allo-SCT) relies on the result of the immune reactions between donor and recipient lymphohemopoietic systems. Chimerism quantification and monitoring post allo-SCT allows to estimate such immune reactions. In recent years, there has been a growing interest in the analysis of circulating cell-free nucleic acids in plasma. Although the ultimate origin of such nucleic acids is unknown, the most plausible hypothesis proposes cell disruption as the cause for the presence of nucleic acids in plasma. Analysis of chimerism in cell-free plasma DNA would complement the results of cell chimerism for a better prediction of different complications (engraftment failure, rejection, graft versus host disease…etc.). ObjectiveOur goal was to quantitatively monitor chimerism in cell-free plasma DNA and to evaluate its usefulness for the prediction/early diagnosis of complications after allo-SCT that would favour early therapeutic intervention. Patients and MethodsThe study comprised 270 plasma samples from 29 allo-SCT patients. With a median follow up of 45,8 months (14,6– 74,7).Whole blood (WB) (10 mL) was collected in EDTA containing tubes and was processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600g for 10 min and cell-free plasma DNA was extracted according to the blood and body fluid protocol of the QIAamp DNA Blood Mini Kit (Qiagen). Quantitative chimerism analysis was performed by microsatellite PCR (STR; AmpFlSTR SGM plus, Applied Biosystems) in whole blood (WB), T-cells (CD3+) purified using immunomagnetic means (Miltenyi Biotec) and cell-free plasma DNA.Chimerism was monitored at regular intervals post-transplant (+30,+60, +90, +180, +365). Sensitivity of the technique is 1% in WB and cell-free plasma DNA, and 5% in T-cells (derived from a 95% purity of enriched samples). ResultsDuring the first year of allo-SCT, the number of patients with mixed chimerism in cell-free plasma DNA was higher than in WB and T-cells (Table 1). Cell-free plasma DNA mixed chimerism was observed for a longer timer after transplant than “cellular” mixed chimerism, with a median of 105 vs. 40 and 27 days for cell-free plasma DNA, T-cells and WB, respectively.Chimerism analysis revealed two groups of patients, one with mixed chimerism in both cell-free plasma DNA and “cellular DNA”, in which the percentage of recipient plasma DNA ranged between 10–30%, probably mostly derived from hematopoietic cells. The other group presented mixed chimerism in cell-free plasma DNA but complete “cellular” chimerism (100% donor) in which cells the percentage of recipient plasma DNA was lower than in the former group (1–10%).A greater number of patients with graft versus host disease (GVHD),mainly acute but also chronic, presented mixed chimerism in cell-free plasma DNA and complete chimerism in WB and T-cells (Table 2). The presence of recipient cell-free plasma DNA could be derived by cell disruption in GVHD target organs. DiscussionQuantitative follow-up of chimerism in cell-free plasma DNA, does not reproduce the results of “cellular” chimerism, as reported previously, and therefore, could not replace it for post-SCT follow-up. On the other hand, chimerism analysis in cell-free plasma DNA could be combined with the conventional follow-up of “cellular” chimerism for an improved diagnosis of life-threatening complications such as acute and chronic GVHD.Table 1Percentage of patients in mixed chimerism during the first year post-allo-SCT and time to CC in whole blood, T-cells and cell free plasma DNA.Days post-allo-SCTDays until CC (median)+30+60+90+180+365WB3524204027T cells48282011040Plasma8372662410105Table 2Pacients with mixed chimerism in whole bllod, T-cells and cell free plasma DNA in the moment of aGVHD and cGVHD developed.acute GVHDchronic GVHDTotal15/29 (51%)15/29 (51%)WB2/15 (13,3%)0/15 (0%)T cells4/15 (26,6%)2/15 (13,3%)Plasma11/15 (73,4%)7/12 (58,3%) Disclosures:No relevant conflicts of interest to declare.

Evaluation of the sensitivity of two recently developed STR multiplexes for the analysis of chimerism after haematopoietic stem cell transplantation

Forensic-oriented kits analysing short tandem repeat (STR) polymorphisms are widely used to determine the proportions of donor and recipient cells after haematopoietic stem cell transplantation. The sensitivity of this technology is crucial for the early detection of relapse and, in consequence, the adjustment of the treatment to enhance donor-origin haematopoiesis in transplant recipients. The objective of this study was to compare the performance of two recently developed STR multiplex kits, AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit (Applied Biosystems) and Investigator™ IDplex(®) (Qiagen), in the analysis of chimerism. Fifteen STR loci were amplified with both kits in 26 peripheral blood samples of transplantated patients showing chimerism. Peak amplitude threshold, detection limit (%DL), per cent donor chimerism and efficacy of each multiplex and STR were determined, and the results with both kits were compared. The %DL and the estimated per cent donor chimerism were similar with both kits. On the other hand, Identifiler(®) Plus kit allowed chimerism identification only in 24 (92%) of the 26 cases with chimerism detected by using the Investigator™ IDplex(®) when only 'type 5' allelic constellations (i.e. without potential interference by stutter peaks) were taken into account. However, IDplex(®) efficacy was somewhat lower than that of Identifiler Plus when only the most informative loci (D2S1338, D21S11, D18S51 and FGA) were considered. Therefore, although each system had some particular advantages and disadvantages, overall both STR multiplexes showed similar performance in qualitative and quantitative chimerism analysis.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

Real-time biallelic polymorphism–polymerase chain reaction for chimerism monitoring of hematopoietic stem cell transplantation relapsed patients

Background An accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP). Methods The allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients. Results Allele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003–0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127 ± 0.193% of recipient DNA. Conclusion We conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation.

Recent advances in quantitative chimerism analysis

Quantitative chimerism analysis is a diagnostic tool used to monitor engraftment kinetics after allogeneic stem cell transplantation. It reflects the proportion of recipient and donor genotypes and is based on the identification of genetic markers characteristic to a given transplant pair. Currently, PCR amplification of short tandem repeats and single-nucleotide polymorphism-specific quantitative real-time PCR are the most widely used techniques for this purpose. In this review, we will address advances as well as technology-specific imperfections, of both techniques that have emerged over the recent years. We will discuss new principles that may simplify assay design, and improve its robustness and reliability. A better chimerism assay could then guide clinical interventions and may, eventually, improve the outcome of allogeneic stem cell transplantation.

QUANTITATIVE EVALUATION OF CHIMERISM STATUS FOLLOWING HAEMATOPOEITIC STEM CELL TRANSPLANTATION USING A MICROCHIP ELECTROPHORESIS SYSTEM

We aimed to establish a method for quantitative analysis of mixed haematopoietic chimerism based on microchip electrophoresis of selected molecular markers following PCR amplification for accurate monitoring of graft status post-transplantation. A 12-year-old girl withrelapsed acute lymphoblastic leukaemia who underwent allogeneic bone marrow transplantation had qualitative chimerism analysis using short tandem repeat markers at three time points following theprocedure. Her archived DNA samples were then used to test the ability to correlate her clinical course with changes in the quantity of donor chimerism at the different time points. Quantitative chimerism analysis was performed on the Agilent 2100 bioanalyser and donor-recipient ratios were calculated from generated electropherograms. Complete donor chimerism (98%) was demonstrated three weekspost- transplantation. Decreasing amount of donor chimerism to 24% was shown after three months and this concurred with clinical relapse. Following a second transplant, full donor chimerism was reestablishedwhere donor chimerism rose to 100%. High resolution microchip electrophoresis could be useful in predicting the occurrence of increasing recipient chimerism which may herald impending relapse in patients while the disease burden is still low. This investigational approach may provide useful information for clinicians to select appropriate intervention strategies to ensure successful transplantation.

The EuroChimerism Concept for a Standardized Approach to Chimerism Analysis Following Allogeneic Stem Cell Transplantation.

Transplantation of hematopoietic stem cells from related and unrelated donors is becoming an increasingly important approach in the successful treatment of different malignant and non-malignant disorders. There is thus growing demand for clinical diagnostic methodologies permitting the surveillance of chimerism during the post-transplant period. The techniques currently employed are rather heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from ten European countries have therefore performed a collaborative study supported by a grant from the European Commission, the EuroChimerism Concerted Action. The aim of this Concerted Action was the development of a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. A set of microsatellite markers, selected on the basis of their excellent performance in chimerism analysis at the reference centers involved, have been carefully evaluated and optimized for quantitative chimerism testing under standardized experimental conditions. A EuroChimerism panel designed to optimally meet the specific requirements of quantitative chimerism analysis was established utilizing 13 markers (D2S1360, D7S1517, D8S1132, D9S1118, D10S2325, D11S554, D12S1064, D12S391, D17S1290, D19S253, MYCL1, P450CYP19 and SE-33), which best satisfied the criteria of the EuroChimerism consortium. Individual microsatellites from the panel can be analyzed in multiplex reactions to facilitate the identification of one or more markers optimally suited for the monitoring of chimerism during the post-transplant period. Extensive testing of related individuals (>500 pairs) revealed that the EuroChimerism marker panel will provide at least two informative markers which meet the stringent criteria of eligibility defined by the EuroChimerism consortium (Watzinger et al., Leukemia 2006) in >99% of all patient/donor constellations. In addition to the outstanding informativeness of the marker panel, chimerism testing by singleplex assays permitted sensitive detection of residual cells of patient or donor origin at levels ranging between 0.8–1.6% in about 90% of instances. The assay also facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells in peripheral blood or bone marrow specimens and in specific cell lineages isolated by flow-sorting. The precision in determining the relative contribution of donor/recipient cell populations to mixed chimerism was high within the range between 10–90% donor- or recipient-derived cells, while there was a tendency to overestimate the percentage of the subdominant cell population within the range between 1–10%.Wide use of the EuroChimerism assay will provide a basis for international standardization of chimerism testing, which will ultimately contribute to improved clinical management of patients undergoing allogeneic stem cell transplantation.

Sensitive and quantitative chimerism analysis in AML patients with real-time PCR

Sensitive and quantitative chimerism analysis in AML patients with real-time PCR

Sensitive and quantitative chimerism analysis in all patients with real-time PCR

Sensitive and quantitative chimerism analysis in all patients with real-time PCR

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis.

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.