Performance Testing of a Semi‐Automatic Card Punch System, Using ...

J Forensic Sci, January 2015, Vol. 60, No. S1 doi: 10.1111/1556-4029.12622 Available online at: onlinelibrary.wiley.com

TECHNICAL NOTE CRIMINALISTICS

Samantha J. Ogden,1 M.Sc.; Jeffrey K. Horton,1 Ph.D.; Simon L. Stubbs,1 Ph.D.; and Peter J. Tatnell,1 Ph.D.

Performance Testing of a Semi-Automatic Card Punch System, Using Direct STR Profiling of DNA from Blood Samples on FTATM Cards

ABSTRACT: The 1.2 mm Electric Coring Tool (e-CoreTM) was developed to increase the throughput of FTATM sample collection cards used

during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlexTM 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTRTM IdentifilerTM Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch.

KEYWORDS: forensic science, DNA typing, collection cards, dried blood spots, STR profiling, polymerase chain reaction Collection cards have been used in human DNA sampling, processing, and long-term storage in forensic laboratories for many years (1,2). The e-Core semi-automatic electric handheld punching instrument is a laboratory tool designed for preparing punches from collection cards. It is considered to be an intermediate punching device between manual punching tools and automated high-throughput punching workstations. Many laboratories processing collection cards fall into the medium-to-high throughput category (3,4). The e-Core device may prove useful for increasing sample throughput for laboratories that have not yet entirely adopted automation. Chemically impregnated cards, such as FTA, have found extensive use in generating short tandem repeat (STR) profiles from human samples (1,2) and for the collection and analysis of nucleic acids from other organisms including plants (5,6). The DNA applied to a collection card is protected from degradation and can be stored at ambient temperature in a space efficient manner with the cards being amenable to automation (1,2,5,6). Indeed, recently Wong et al. (7) described a method for reducing the volume of PCR amplification reactions for DNA databasing using FTA Classic Cards. Thus, in combination with FTA cards, the e-Core instrument may be a useful alternative punching tool for a wide range of applications. The following study describes the use of the e-Core device in forensic applications, and the

1 GE Healthcare Life Sciences R&D, The Maynard Centre, Forest Farm, Whitchurch, Cardiff CF14 7YT, U.K. Received 18 Oct. 2013; and in revised form 9 Jan. 2014; accepted 20 Jan. 2014.

© 2014 American Academy of Forensic Sciences

performance was compared to that of a manual punching tool. The validation of the electric e-Core instrument with collection cards and STR profiling has not been reported elsewhere. Materials and Methods This study was designed to compare the performance of the e-Core punch system (GE Healthcare Life Sciences, Amersham Place, Little Chalfont, Buckinghamshire, UK, WB100052) with a manual punching system (Harris 1.2 mm manual micro-punch; GE Healthcare; WB100005) when punching discs from sample collection cards. Initial testing was carried out to test the operational performance of the e-Core device. Ninety-six 1.2-mm discs were taken from blank FTA Micro Cards (GE Healthcare) on which no sample had been applied, by three operators. Each disc was ejected into separate wells of 96-well plates. A successful punch was obtained when the FTA disc was transferred from card to target well. A combined success rate was calculated for all three operators based on the percentage of ejected discs transferred to target wells. Punch durability testing was carried out to test the robustness of the punch head of the e-Core device. To do this, 2000 1.2mm FTA discs were taken from blank cards, by three operators (total 6000 discs) using separate e-Core devices. Each punch operation was timed with a calibrated laboratory timer ensuring that the punching process took no longer than 1.5 sec. After 100 discs were excised, a microscopic examination (see below) was carried out on the one hundredth disc to determine that the appearance of the disc was comparable to those previously excised. If there was a defect in the disc (i.e., tearing, excessive S207

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loss of fibers, or an abnormal shape), the following nine discs were also checked. If all of these 10 FTA discs exhibited an abnormal appearance, this was then classed as a defect, implying that the punch head was not excising the FTA discs correctly and therefore required changing. This process of checking every one hundredth disc, during the durability testing process, was repeated 20 times from the total of 2000 discs from each of the three operators (overall total of 6000 discs). Disc images were captured on an INCell Analyzer 2000 (GE Healthcare Life Sciences, Amersham Place, Little Chalfont, Buckinghamshire, UK) using a 49 objective and data analyzed using INCell Investigator Software version 1.9, generating diameter sizes and surface areas for discs derived from the e-Core and the manual punch systems. In addition, the internal diameter size of the punch head was determined for all the punching devices used during this study using calibrated pin gages (Vermont Gages, Swanton, VT). When inspected microscopically, all FTA discs appeared circular and equivalent in appearance, indicating no significant deterioration in the e-Core punch head shape over the course of the study. Sample Preparation Dried blood spots for sample collection purposes were prepared as follows. Human whole blood, from two donors (75 lL), was spotted on to FTA Micro Cards, allowed to dry at ambient temperature for 3 h, and stored in a desiccator before use. FTA cards were sampled within 1 week of spotting. These defined storage conditions were used to carefully control the environment for card storage to ensure evaluation of punch performance and not that of the card. Dried blood spots were prepared to simulate sample collection on FTA for human identification purposes. Short Tandem Repeat Profiling To demonstrate agreement between the e-Core and the manual micro-punch with human short tandem repeat (STR) profiling, an analysis was carried out using discs taken with both punches from dried blood spots on FTA Micro Cards. Sixty-seven discs, in duplicate, were taken by three different operators (giving a total of 402 punches) from separate blood-spotted FTA Micro Cards, prepared as above, with blood from two donors, using both the e-Core device (with a 1.2-mm punch head) and a 1.2mm manual micro-punch. Cards were punched at the center of the sample spot to reduce variability. Direct STR profiling was carried out on all 402 punches using a PowerPlex 18D System (Promega, Southampton, U.K.) over 26 amplification cycles, following the kit manufacturer’s instructions. Thermal cycling conditions were as follows: 96°C for 2 min, then: 94°C for 10 sec, 60°C for 1 min for 26 cycles, then: 60°C for 20 min, 4°C soak. The resulting PCR products were analyzed on an ABITM 3130xl Genetic Analyzer capillary electrophoresis system. The electrophoresis run cocktail mix was prepared using 1.0 lL of CC5 (Promega) internal lane standard plus 10.0 lL HiDi formamide per sample. Eleven microliters of run cocktail mix was added to 1.0 lL of sample. Instrument settings were as follows: injection time, 5 sec; injection and run voltages, 3 kV; run time, 1500 sec with a G5 dye set. Results were evaluated with GeneMapperTM ID v3.2 software (Life Technologies, Paisley, U.K.). The STR profiles generated from FTA Micro Cards taken with each of the two sample punch devices were compared. For

the study, peak height thresholds were set at 200 relative fluorescence units (RFU) for both heterozygous peaks and homozygous peaks to represent values comparable for uploading profiles to National DNA databases. Additionally, more stringent thresholds were also set at peak heights of 500 RFU for heterozygous peaks and 1000 RFU for homozygous peaks for high quality STR profiling purposes. Profiles exhibiting peak heights below both of these thresholds were considered to represent partial profiles. These higher quality peak height values are those typically observed in our laboratory when engaged in profiling studies using the PowerPlex 18D System. Our laboratory is following the guidelines from the Scientific Working Group on DNA Analysis Methods (8), with the application of appropriate controls to generate STR profiles and assess contamination. Contamination Analysis Contamination testing was undertaken to demonstrate that taking a blank FTA disc between sampling of dried blood spots was sufficient for cleaning the punch heads, ensuring that no DNA carryover occurred during the punching process. This assessment was carried out in the following manner; sampling was performed by three operators who first took a 1.2-mm disc (Punch A) from the center of a blood spot using the e-Core device. Second, each operator took a “cleaning” disc (Punch B) from either the outer unsampled section of card or a separate blank FTA card. The “cleaning” disc was discarded. Finally, each operator took a disc (Punch C) from a separate blank FTA card. This process was repeated 84 times by each operator. Punches A and C (total of 504 punches) were subjected to direct STR profiling using an Applied Biosystems AmpflSTRTM IdentifilerTM Direct PCR Amplification Kit (Life Technologies) over 26 amplification cycles, following the kit manufacturer’s instructions. Thermal cycling conditions were as follows: 95°C for 11 min, then: 94°C for 20 sec, 59°C for 2 min, and 72°C for 1 min for 26 cycles, then: 60°C for 25 min, 4°C soak. The capillary electrophoresis unit employed to evaluate the STR amplicons was an Applied Biosystems 3130xl Genetic Analyzer. The electrophoresis run cocktail mix was prepared using 0.3 lL of Liz 500 (Life Technologies) internal lane standard plus 8.7 lL HiDi formamide per sample. Nine microliters of run cocktail mix was added to 1.0 lL of sample. Instrument settings were as follows: injection time, 5 sec; injection and run voltages, 3 kV; run time, 1500 sec with a G5 dye set. Results were evaluated with GeneMapper ID v3.2 software, as above. The contamination study was carried out using the Applied Biosystems AmpflSTR Identifiler Direct PCR Amplification kits (as opposed to using PowerPlex 18D STR chemistry) to show compatibility of FTA discs generated by the e-Core device with two commercially available STR profiling kits. For the contamination study, the minimum peak interpretation thresholds were set at 75 RFU for heterozygous and 150 RFU for homozygous peaks for blood samples (Punch A), with minimum peak detection thresholds set at 50 RFU for blank samples (Punch C). None of the Punch B “cleaning” samples were analyzed. Peak height thresholds were set at these values following published guidelines (9) and in-house validated methods using our instrumentation with commercially available kits. Statistics Where appropriate, sampling regimes were stratified and sample numbers selected according to recognized methods (JMP 10.0.1; SAS Institute, Buckinghamshire, U.K.). Binomial confidence intervals were developed for numbers of observed defects.

OGDEN ET AL.

The distributions of data were assessed using the Shapiro–Wilk test. Dependent upon distribution, test statistics were calculated using analysis of variance (ANOVA) or the Wilcoxon/Kruskal– Wallis test. Analysis of variance was used to compare normally distributed variables. Where the data were found, using the Shapiro–Wilk test, to have a non-normal distribution or to be substantially skewed, the nonparametric equivalent of ANOVA, the Wilcoxon/Kruskal–Wallis test was applied. Statistical significance was set at p < 0.05. Confidence intervals for mean values were set at 95%. Results and Discussion For punch performance testing, the overall success rate of the FTA disc reaching the target well was >97% from all three operators combined (n = 288). These losses are typically observed in our laboratory during a side-by-side comparison when also using manual punch systems and are associated with a variety of possible contributory factors such as static from related plastic ware and air movements. Indeed, such losses have been independently reported as a potential issue by Wong et al. (7). To determine punch head durability, a total of 6000 FTA discs were taken with the e-Core devices. For all 6000 punches taken by three operators with three different e-Core devices (2000 discs per operator), no excision took longer than 1.5 sec. When inspected microscopically, all discs appeared circular and equivalent in appearance (data not shown), indicating no significant change in the e-Core punch head shape or performance over the course of the study. This observation was supported by measurements that recorded disc area and diameter (see below). For STR profiling, the peak heights for all alleles following amplification of DNA and genetic analysis met the specifications and thresholds set for the study (Table 1). A general observation from these data was that the mean peak heights of alleles

TABLE 1––Mean peak height of loci for all samples prepared using the e-Core and the Harris manual micro-punch. Data were combined from the results from three different operators. Direct STR profiling was carried out using PowerPlex 18 D kits over 26 amplification cycles. The resulting PCR products were analyzed using an Applied Biosystems 3130xl Genetic Analyzer and GeneMapper ID v3.2 software. (Data show mean relative fluorescence units standard deviation, n = 6; two punches 9 three operators).

Locus Amelogenin CSF1PO D13S317 D16S539 D18S51 D19S433 D21S11 D2S1338 D3S1358 D5S818 D7S820 D8S1179 FGA Penta D Penta E TH01 TPOX vWA

e-Core Mean Peak Height (SD) 1970 5039 2783 4135 3589 2564 2917 1997 1879 1984 3440 2859 3613 4941 3262 1293 3181 2744

                 

416.5 311.5 412.7 1239.0 986.6 500.6 867.8 256.5 285.0 499.0 601.2 664.9 1612.0 577.0 583.5 89.6 535.4 667.4

Micro-Punch Mean Peak Height (SD) 1896 4050 2326 3285 3064 2063 2912 1635 1720 1664 2890 2510 3260 4040 2769 977 2521 2384

                 

488.4 381.9 101.3 1226.0 537.8 275.0 478.7 497.6 453.4 245.6 307.3 421.7 1478.0 361.1 341.4 138.3 366.6 426.1

Statistical significance was set at 0.05; ns, not significant.

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STR PROFILING FROM BLOOD SAMPLES ON FTA CARDS

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derived from discs generated by the e-Core device were marginally greater than the mean peak heights of the equivalent allele using the manual 1.2 mm Harris micro-punch. However, upon statistical analysis, only three loci (CSFIPO, Penta D, and THO1) were shown to be significantly different (p < 0.05; Table 1), while the mean peak heights from the remaining loci were not significantly different. It should be noted that there was a small but significant difference between disc sizes obtained using the e-Core and manual punch systems (see below). Additionally, there was no significant difference in peak heights between operators, irrespective of the device used (p > 0.2). This indicates that any difference in mean peak heights was not due to the operator. Punches derived from each of the two devices produced DNA profiles that were concordant (data not shown). These data indicate that the resolution of alleles, with a wide range of amplicon lengths, is resolved well using both punching devices. The area of each FTA disc was measured microscopically using the INCell Analyzer 2000 using a 49 objective and data analyzed using INCell Investigator Software. The mean area for discs derived from the e-Core punch was 1.52 9 106  1.42 9 105 lm2, compared with 1.42 9 106  1.50 9 105 lm2 for the manual punch (7% difference in disc area overall). These data indicated that there was a small but significant difference

FIG. 1––Punch images were captured on an INCell Analyzer 2000 using a 49 objective and data analyzed using INCell Investigator Software version 1.9, generating areas for discs derived from e-Core and manual micro-punch systems. Data are shown as mean lm 2  1 standard deviation, n = 20.

p Value ns