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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the effective methods for the treatment of hematological malignancies and immunodeficiency diseases. The success of transplantation and the implantation of donor cells in the recipient, ie the formation of chimeras related. Donor chimerism (DC) refers to the phenomenon that donor-derived hematopoietic stem cells exist in the recipient after allo-HSCT. Dynamic detection of chimerism after transplantation is particularly important for judging the effect of implantation and implementing early clinical intervention.
Introduction to chimeric detection methods
Detection of donor cell chimerism (DC) by fluorescent-labeled multiplex PCR amplification of short tandem repeats (PCR-STR) combined with capillary electrophoresis is currently the gold standard for chimeric detection [1], but its sensitivity is only 1-5%. The patient’s cell ratio cannot be detected after reaching full chimerism, and is currently only suitable for conventional chimeric (large chimeric) clinical tests.
Microchimerism refers to a state in which a patient or a donor accounts for less than 1% in a chimera, and is carried out using a micro-chimeric detection technique based on fluorescent PCR. Microchimeric detection utilizes the Indel (insertion deletion) polymorphism between donors and recipients to find differential sites, and the ΔΔCt value amplified by fluorescence PCR (experienced when the fluorescent signal in each reaction tube reaches the set domain value) Cycle number) to calculate the donor (or patient) cell proportion [2]. The sensitivity of micro-chimeric detection based on real-time PCR is high (up to 10-4), but the Ct value is affected by the amplification efficiency in the fluorescent PCR reaction, so it is impossible to distinguish the specimen with little change (generally less than one order of magnitude). Therefore, the current fluorescence PCR instrument can only perform micro-chimeric detection, and is not suitable for clinical detection of conventional chimeric (large chimeric).
The concept of digital PCR was proposed in the 1990s by dividing a PCR reaction into multiple reactions. The template molecule is divided into thousands of water droplets or oil droplets, and the PCR reaction is converted into a digital signal to quantitatively output the signal. Digital PCR has the advantages of high sensitivity, high accuracy and high tolerance. Compared with fluorescence PCR, it does not depend on Ct value and standard curve, and is not affected by primer and enzyme amplification efficiency, and achieves absolute quantitative detection [3]. Digital PCR has been widely used in tumor detection sites and drug resistance site mutation detection, fusion gene detection, gene expression, copy number variation and virus quantitative detection.
Digital PCR chimeric detection advantage
In recent years, digital PCR has also been widely used in chimeric detection [3-10]. The detection principle is similar to that of microchimerism. The Indel (insertion deletion) polymorphism between donors and recipients is used to find the difference sites. Absolute quantification of digital PCR by differential sites. Compared with PCR-STR, its advantages are mainly high sensitivity and low sample input. Compared with fluorescence PCR, micro-chimerism has the advantage of being unaffected by amplification efficiency and has good reproducibility. It is suitable for clinical use of conventional chimerism (large chimerism). Detection (Table 1).
Table 1: Comparison of three chimeric detection methods
Digital PCR chimeric clinical application
Micro-transplantation; cord blood-assisted transplantation [7];
Organ transplantation [8];
Conventional chimerization is required for small sample sizes: for example, Treg cells after sorting; CD34+ cells [9];
Patient chimerism was monitored routinely after complete chimerism, and recurrence was predicted earlier [10].
references:
[1] Nollet F, Billiet J, Selleslag D, et al. Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing [J]. Bone marrow transplantation, 2001, 28(5): 511.
[2] Frankfurt O, Zitzner JR, Tambur A R. Real-time qPCR for chimerism assessment in allogeneic hematopoietic stem cell transplants from unrelated adult and double umbilical cord blood [J]. Human immunology, 2015, 76(2-3): 155-160.
[3] Goh S K, Musafer A, Witkowski T, et al. Comparison of 3 methodologies for genotyping of small deletion and insertion polymorphisms [J]. Clinical chemistry, 2016, 62(7): 1012-1019.
[4] George D, Czech J, John B, et al. Detection and quantification of chimerism by droplet digital PCR [J]. Chimerism, 2013, 4(3): 102-108.
[5] Santurtún A, Riancho JA, Arozamena J, et al. Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis [J]. International journal of legal medicine, 2017, 131(1): 67 -72.
[5] Stahl T, Rothe C, Böhme M, et al. Digital PCR panel for sensitive hematopoietic chimerism quantification after allogeneic stem cell transplantation [J]. International journal of molecular sciences, 2016, 17(9): 1515.
[7] Kliman D, Castellano-Gonzalez G, Withers B, et al. Ultra-sensitive droplet digital PCR for the assessment of microchimerism in cellular therapies [J]. Biology of Blood and Marrow Transplantation, 2018, 24(5): 1069 -1078.
[8] Schütz E, Fischer A, Beck J, et al. Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: a prospective, observational, multicenter cohort study [J]. PLoS medicine, 2017, 14(4): e1002286.
[9] Okano T, Tsujita Y, Kanegane H, et al. Droplet digital PCR-based chimerism analysis for primary immunodeficiency diseases [J]. Journal of clinical immunology, 2018, 38(3): 300-306.
[10] Valero-Garcia J, del Carmen Gonzalez-Espinosa M, Barrios M, et al. Earlier relapse detection after allogeneic haematopoietic stem cell transplantation by chimerism assays: Digital PCR versus quantitative real-time PCR of insertion/deletion polymorphisms [J] PloS one, 2019, 14(2): e0212708.
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