Can novel methods replace the gold standard chimerism method after allogeneic hematopoietic stem cell transplantation?

After hematopoietic stem cell transplantation, chimerism assay is a useful approach to monitor the success of the transplant and to select the appropriate treatment strategy, such as donor leukocyte infusion or immunosuppressive drug dosage. Short tandem repeat PCR is the method that has been accepted as the gold standard for chimerism. However, it has not yet been sufficient to detect mixed chimerism in patients with minimal residual disease. Simultaneously, recent years have been marked by developing sensitive, high-throughput, and accurate molecular genetic assays. These novel methods have subsequently been adapted for the analysis of post-transplant chimerism. In this review, we discuss the technical features of both novel and conventional gold standard chimerism assays. We also discuss their advantages and disadvantages.

Evaluation of microfluidics-based droplet PCR combined with multiplex STR system in forensic science.

Because of its excellent monodispersity, high throughput, and low volume, microfluidics-based droplet PCR has become the core technology of digital PCR, next-generation sequencing, and other technology platforms. This study constructed a microfluidic water-in-oil droplet PCR system and amplified a commercially available forensic 22-plex short tandem repeat detection system. We analyzed the sensitivity, concordance, amplification efficiency of the droplet PCR, and influence factors of the above aspects. The droplet PCR showed high concordance with conventional bulk PCR and had high sensitivity as 0.125 ng. Furthermore, we observed the performance of droplet PCR in high-order mixed DNA. As the mixture ratios from 10:1 to 30:1, droplet PCR presented more mixture proportion (Mx) increased loci from 11 (57.89%) to 17 (89.47%). In the mixture ratios 20:1, 25:1, and 30:1, significant Mx differences between droplet PCR and bulk PCR were observed (p < 0.05). The results showed that the droplet PCR could improve the identification of the minor contributor's DNA in a two-person mixture and alleviate the imbalanced amplification problem. This study provides a reference and basis for the wide application of droplet PCR in forensic science.

Polymorphism of 12 STR loci included in the Investigator HD-plex kit in Polish population of Lodz region.

In this study Polish population data as well as efficiency parameters of 12 STR loci included in the Investigator HDplex set were presented. This set contains 9 systems not available in any other commercial multiplexes, ie.: D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325 and D21S2055. The evaluation was preformed based on DNA samples derived from 303 unrelated individuals living in Lodz region, central part of Poland. The obtained distribution of the genotypes is consistent with the assumptions of the Hardy and Weinberg equilibrium (HWE). It reflects properly genetic structure of the studied population compared with other populations of Europe and the world. It indicates the linkage equilibrium within the pairs of investigated loci, as well as with regard to other syntenic loci. The total value of the power of exclusion (PE) and the random match probability (MP) were respectively 0.99999988 and 5.2 × 10-18. Therefore the polymorphism of examined genetic markers within the Investigator HD-plex multiplex allows for a significant increase of the evidence value. Thus it constitutes an excellent tool for resolving difficult cases in the field of forensic genetics.

Strategies for the Design and Assessment of Y-Short Tandem Repeat Multiplexes for Forensic Use.

In order to improve the discriminatory potential, and hence the probative value, of Y-STR-based testing, the set of Y-chromosome STR loci available for forensic use needs to be expanded. We have designed and tested two novel Y-STR multiplexes for potential forensic casework use. In accord with the requirements of Y-chromosome multiplex analytical systems developed specifically for forensic casework use, we have tried to maximize the number of loci able to be co-amplified and minimize confounding female DNA artifacts while ensuring appropriate assay sensitivity (1-2 ng of input genomic DNA) and interlocus signal balance. This review will describe the strategies that we have developed that may provide guidance for the design and assessment of novel Y-STR multiplex systems. Novel Y-STR multiplexes developed for forensic use need to undergo a series of validation exercises that go beyond simply optimizing the PCR reaction conditions. Specifically, stringent performance checks on their efficacy need to be carried out using casework type specimens in order to determine potential confounding effects from female DNA.