The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one negative (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.

Digital PCR, a state-of-the-art nucleic acid quantification technique, works by spreading the target material across a large number of partitions. The average number of molecules per partition is estimated using Poisson statistics, and then converted into concentration by dividing by partition volume. In this standard approach, identical partition sizing is assumed. Violations of this assumption result in underestimation of target quantity, when using Poisson modeling, especially at higher concentrations. The Poisson-Plus Model accommodates for this underestimation, if statistics of the volume variation are well characterized. The volume variation was measured on the chip array based QuantStudio 3D Digital PCR System using the ROX fluorescence level as a proxy for effective load volume per through-hole. Monte Carlo simulations demonstrate the efficacy of the proposed correction. Empirical measurement of model parameters characterizing the effective load volume on QuantStudio 3D Digital PCR chips is presented. The model was used to analyze digital PCR experiments and showed improved accuracy in quantification. At the higher concentrations, the modeling must take effective fill volume variation into account to produce accurate estimates. The extent of the difference from the standard to the new modeling is positively correlated to the extent of fill volume variation in the effective load of your reactions.

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T-cell-dependent humoral immune response is one of the more complex immunological events in the biological immune system, involving interaction of B cells with antigen (Ag) and their proliferation, differentiation and subsequent secretion of antibody (Ab). Inspired by these immunological principles, a Multilevel Immune Learning Algorithm (MILA) is proposed for novel pattern recognition. This paper describes the detailed background of MILA, and outlines its main features in different phases: Initialization phase, Recognition phase, Evolutionary phase and Response phase. Different test problems are studied and experimented with MILA for performance evaluation. The results show MILA is flexible and efficient in detecting anomalies and novel patterns.

Abstract. The biological immune system is an intricate network of specialized tissues, organs, cells, and chemical molecules. T-cell-dependent humoral immune response is one of the complex immunological events, involving interaction of B cells with antigens (Ag) and their proliferation, differentiation and subsequent secretion of antibodies (Ab). Inspired by these immunological principles, we proposed a Multilevel Immune Learning Algorithm (MILA) for novel pattern recognition. It incorporates multiple detection schema, clonal expansion and dynamic detector generation mechanisms in a single framework. Different test problems are studied and experimented with MILA for performance evaluation. Preliminary results show that MILA is flexible and efficient in detecting anomalies and novelties in data patterns.