HID NIMBUS Presto QNA System Validation

The automated liquid handling robot includes a patented pipette tip technology that facilitates a tight seal for superior liquid measurement accuracy and precision without tip distortion and aerosol generation during tip pick-up and ejection, thus limiting cross contamination.

The checkerboard pattern in Figure 1, alternating high-level control single-donor DNA input samples with extraction reagent blanks, confirms that the HID NIMBUS Presto System does not introduce cross-contamination during automated extraction (PrepFiler BTA and PrepFiler 500-μL lysis protocols were tested).  

Figure 1. (Top panel) Sample input orientation in processing plate for each contamination study. Reagent blanks are indicated with pink shading and were placed in a checkerboard pattern around high-level control DNA (500 ng in PrepFiler 500 μL lysis protocol study 300 ng in PrepFiler BTA study,) input samples. (Middle panel) Electropherogram overlay of all 36 reagent blanks, displaying all dye channels, purified on the HID NIMBUS Presto System with PrepFiler 500 μL lysis protocol, then amplified with the GlobalFiler IQC PCR Amplification Kit (15 μL of eluate, 29 cycles). The Y-axis is scaled to 200 RFU, One Out-of-Marker-Range (OMR) doublet, one CE artifact spike and one in-bin peak (51 RFU) at an allele not shared with Donor DNA genotype is present above 50 RFU. Blue-channel peaks at far ends of either side of the EPG are the Internal Quality Control (IQC) peaks which are intrinsic to the kit. (Lower panel) Electropherogram overlay of all 36 reagent blanks, displaying all dye channels, purified on the HID NIMBUS Presto System with PrepFiler BTA protocol, then amplified with the GlobalFiler IQC PCR Amplification Kit (15 μL of eluate, 29 cycles). The Y-axis is scaled to 200 RFU, One CE artifact spike and one in-bin peak (64 RFU) at an allele not shared with Donor DNA genotype is present above 50 RFU. Blue-channel peaks at far ends of either side of the EPG are the Internal Quality Control (IQC) peaks which are intrinsic to the kit. Absence of cross-contamination during extraction process is evidenced by lack of peaks matching donor DNA genotype in any of the reagent blanks.

The HID NIMBUS Presto systems deliver DNA from challenging samples and are capable of generating full genetic profiles for use in forensic casework analysis. A variety of known and non-probative casework-type samples were used to evaluate the ability of the HID NIMBUS Presto System to extract inhibitor-free DNA of the highest quantity and quality possible from common human identification sample types; when noted, results from the same samples extracted with the AutoMate Express system for comparative purposes are shown (Figure 2).

Figure 3. 3500xL Genetic Analyzer electropherograms of mock case-type samples extracted on the HID NIMBUS Presto system unless otherwise noted and amplified with the GlobalFiler IQC PCR Amplification Kit showing an overlay of all dye channels. The Y-axis is adjusted individually per sample as indicated. From upper panel to lower: Swab of drink can rim extracted with PrepFiler 300-μL lysis protocol; swab of dried blood (2.5 μL) on surface extracted with PrepFiler 500-μL lysis protocol; 1 μL of dried saliva on tape extracted with PrepFiler BTA kit; 50 mg of slightly degraded bone sample extracted with PrepFiler BTA kit; 50 mg of slightly degraded bone sample extracted with PrepFiler BTA kit processed on AutoMate Express system.

The HID NIMBUS Presto systems combined with PrepFiler Automated chemistries are able to repeatedly and consistently isolate or extract actionable amounts of DNA from limited and challenging forensic sample types. Dilution series of blood and saliva spotted on swabs were processed with the PrepFiler 300-μL and 500-μL lysis protocols on the HID NIMBUS Presto System and on the AutoMate Express system with the standard PrepFiler protocol to assess comparability (Figure 3). Similar results were seen with PrepFiler BTA chemistry (data not shown). Internal Positive Control (IPC) CTs from the quantification assay indicated that all samples were free of inhibitory factors (data not shown).

Figure 3. DNA yields (ng/uL) in eluates (50 μL) obtained from each blood (right) and saliva (left) sample serial dilution point by standard PrepFiler - AutoMate Express protocol and the PrepFiler 300 μL and 500 μL lysis protocols on the HID NIMBUS Presto System. Inputs (spotted on swabs and dried) for each body fluid type ranged from 10 μL to 0.01 μL over a 1:3 dilution series. (NP is HID NIMBUS Presto; AE is AutoMate Express) (Y-axis is a logarithmic scale) (N = 4)

Since 2007, the Human Identification Professional Services (HPS) team has been helping customers successfully navigate the validation process required to bring new technologies into operation. HPS has completed over 700 validation and integration projects in more than 39 countries worldwide. With a dedicated team of validation specialists, each with forensic experience, HPS provides forensic laboratories with comprehensive validations and implementation support, both useful for bringing new technologies into operation. Using a consultative approach, the HPS team aims to design a service that meets most technical and budgetary needs. HPS has designed service offerings specifically for the HID NIMBUS systems and their associated kits.