Go to Top




Project ID: PN-III-P2-2.1-PED-2016-1932


Funding contract: 252PED ⁄ 2017 (UEFISCDI)


Funding amount:  475.000,00 lei


Period: 08/2017-12/2018



Project title: Assessment of RNA chimerism by the droplet digital PCR procedure to diagnose the outcome of the hematopoietic stem cell transplant



Cristina Niculite, PhD Gisela Gaina, PhD Sevinci Pop, PhD Stefania Rogozea

Valeriu Cismasiu, PhD (project coordinator)


Final Report/ Scientific Report




In this project we plan to validate both in vitro and in vivo a new method for chimerism assessment with several layers of novelty: the target is RNA rather than genomic DNA; the targeted RNA is highly expressed in population of interest, i.e. graft/host hematopoietic stem and progenitor cells (HSPCs) and myeloid cells; the assay evaluates the polymorphism transferred to RNA; the polymorphism is quantified with the droplet digital PCR technology (ddPCR).


Project Description

The main aim of the project is to set up a method of chimerism assesment with better sensitivity and specificity than the current clinically approved assays.   During the last four decades, bone marrow (BM), periferal blood (PB) or cord blood (CB) transplant have become a major procedure for many hematopoietic  disorders,  both  malignant  and  nonmalignant,  in  children  and  in  adult  patients. Chimerism represent the ratio of recipient-origin cells to donor-origin cells and is considered the best way to measure the treatment success. Thus, the chimerism analysis is critical for detecting early signs of graft rejection, preventing relapse of the underlying disease, and monitoring of minimal residual disease (MRD) (ref 1, 2).

Historicaly, the first chimerism assays were developed by cytogenetics, red cell phenotyping, restriction fragment length polymorphism analysis (RFLP) and fluorescence in situ hybridization of sex chromosomes (X/Y FISH) (ref 3). However, these methods are time consuming and not always applicable. For instance, although the X/Y FISH has the sensitivity (detection limit) close to 1%, the procedure is restricted to sex-mismatched transplants. In addition, most of the methods cannot acchieve sensitivity bellow 1%. Sensitivity is a critical parameter since lower values correlate with early diagnostic of the treatment output and consequently allow a more efficient treatment adjustment to the patient status. The major breakthrough for the investigation of chimerism came when polymerase chain reaction (PCR) technique was developed. The PCR-based analyses were initialy performed by amplification of variable number of tandem repeats (VNTR) or by the characterization of short tandem repeat (STR) markers (ref 4). Most recently, the real-time (fluorescent/quantitative) PCR assays aiming at the amplification of ‘single-nucleotide polymorphisms’ (SNPs) were established (SNP-qPCR). SNPs are biallelic variants that differ from each other only at a single nucleotide and occur on average every 1.3 kb in the human genome (ref 5). In 2002, group of dr. Erwann Quelvennec reported a set of 11 biallelic SNPs for chimerism analysis using real-time PCR (ref 6). The important finding of this study is that the SNP-qPCR detection limit of the minor cell population is 0.1% and thus the sensitivity is higher than in case of STR-PCR (ref 6). Independent groups have demonstrated the advantages of qPCR for chimerism assesment (ref 7, 8). Clinically relevant for patient monitoring, the qPCR assay has a significantly reduced number of post-tansplant days required for mixed chimerism detection (MCD). For instance, one study report a median number of 31 versus 120 post-treatment days until MCD comparing SNP-qPCR with STR-PCR, respectively (ref 9). Similar rezults have been published by another group: SNP-qPCR vs. STR-PCR = 31 vs. 71, when the total bone marrow has been used as input material (ref 10). Although, the SNP-qPCR method represent a step forward due to increased sensitivity, the fluorescent STR-PCR is still the prefered assay for chimerism assesment. The major bottleneck of SNP-qPCR is the reduced specificity (i.e. quantification accuracy) that favor a significant rate for false- positive data (ref 9, 10). The standard deviation in case of SNP-qPCR is arround 20% while the STR- PCR has a value close to 5% (ref 9, 10). The qPCR specificity is highly dependent of DNA quality and of the PCR efficiency for a given SNP.

Project objectives are:

– to demonstrate that the diagnostic chimerism could be measured by quantification of the graft specific RNA expression and by the RNA polymorphism quantification.

– to establish the RNA-SNPs useful to distinguish bettween the patient and donor derived RNA molecules for genes that are highly expressed in both graft and host HSPCs;

– to set up droplet digital PCR (ddPCR) for RNA chimerism assesment.




1) Liesveld JL & Rothberg PG. Mixed chimerism in SCT: conflict or peaceful coexistence? Bone Marrow Transplant 2008; 42: 297–310.

2) Dubovsky J et al. Kinetics of chimerism during the early post-transplant period in pediatric patients with malignant and non-malignant hematologic disorders: implications for timely detection of engraftment, graft failure and rejection. Leukemia. 1999; 13: 60–69.

3) Thiede C, Bornhauser M & Ehninger G. Strategies and clinical implications of chimerism diagnostics after allogeneic hematopoietic stem cell transplantation. Acta Haematol 2004; 112: 16−23.

4) Khan F, Agarwal A & Agarwal S. Significance of chimerism in heamtopoietic stem cell transplantation: new variations on an old theme. Bone Marrow Transplant 2004; 34: 1−12.

5) Sachidanandam R et al. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 2001; 409: 928−933.

6) Alizadeh M  et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002; 99: 4618−4625

7) Senitzer D, Zhou Y, Gaidulis L & Lazarak K. Chimerism detection in hematopoietic cell transplant recipients by real-time quantitative PCR (QPCR) vs. short tandem repeat (STR) analysis. Biol Blood Marrow Transplant 2009; 15: 138.

8) Frankfurt O, Zitzner JR & Tambur AR. Real-time qPCR for chimerism assessment in allogeneic hematopoietic stem cell transplants from unrelated adult and double umbilical cord blood. Hum Immunol. 2015; 76: 155-60.

9) Koldehoff M et al. Quantitative analysis of chimerism after allogeneic stem cell transplantation by real-time polymerase chain reaction with single nucleotide polymorphisms, standard tandem repeats, and Y-chromosome-specific sequences. Am J Hematol. 2006; 81: 735–746.

10)  Willasch  AM  et  al.  Monitoring  of  hematopoietic  chimerism  after  transplantation  for pediatric myelodysplastic syndrome: real-time or conventional short tandem repeat PCR in peripheral blood or bone marrow? Biol Blood Marrow Transplant. 2014; 20: 1918-25.