Sara Galimberti, Serena Balducci, Francesca Guerrini, Marzia Del Re, Rossella Cacciola

Diagnostics (Basel); 2022 May 24; 12(6):1305. doi: 10.3390/diagnostics12061305.

Abstract

Digital droplet PCR (ddPCR) is a recent version of quantitative PCR (QT-PCR), useful for measuring gene expression, doing clonality assays and detecting hot spot mutations. In respect of QT-PCR, ddPCR is more sensitive, does not need any reference curve and can quantify one quarter of samples already defined as "positive but not quantifiable". In the IgH and TCR clonality assessment, ddPCR recapitulates the allele-specific oligonucleotide PCR (ASO-PCR), being not adapt for detecting clonal evolution, that, on the contrary, does not represent a pitfall for the next generation sequencing (NGS) technique. Differently from NGS, ddPCR is not able to sequence the whole gene, but it is useful, cheaper, and less time-consuming when hot spot mutations are the targets, such as occurs with IDH1, IDH2, NPM1 in acute leukemias or T315I mutation in Philadelphia-positive leukemias or JAK2 in chronic myeloproliferative neoplasms. Further versions of ddPCR, that combine different primers/probes fluorescences and concentrations, allow measuring up to four targets in the same PCR reaction, sparing material, time, and money. ddPCR is also useful for quantitating BCR-ABL1 fusion gene, WT1 expression, donor chimerism, and minimal residual disease, so helping physicians to realize that "patient-tailored therapy" that is the aim of the modern hematology.

See https://pubmed.ncbi.nlm.nih.gov/35741115/

Figure 2

Multiplex assay based on the amplitude of the amplifiers. (a) the different targets are detected by probes labeled with the same fluorochrome (FAM or HEX) but used at different concentrations. This strategy allows to quantify four targets within a single reaction (A, B, C, D). (b) Targets A and B have relative concentrations of 100% and 50% of FAM-labeled probe, respectively, while C and D have relative concentrations of 100% and 50% of HEX-labeled probe. In the 2D plot, 16 possible clusters are generated: clusters that contain only one target, clusters that simultaneously contain two targets and possibly clusters that contain three targets.