Rapid DNA Systems

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Roald M. Tiggelaar	--	4141	2023-02-09 11:42:23	\|
2	format correction	Dean Liu	Meta information modification	4141	2023-02-10 04:12:14	\|

This entry is adapted from the peer-reviewed paper 10.3390/s23031075

The generation of STR profiles currently requires highly skilled geneticists and dedicated laboratories. Therefore, there is an urgent need for fast and reliable DNA devices for analysis directly at the crime scene. A few systems became commercially available for integrated forensic DNA analysis to be used in the field, which are fully integrated platforms that can generate STR profiles from (reference) samples within 2 hours. In the following a distinction is made between 'rapid DNA analysis' - defined as using a rapid DNA instrument without human intervention - and 'modified rapid DNA analysis'' which is defined as using a rapid DNA instrument in combination with human interpretation of the DNA analyses results.

rapid DNA analysis RapidHIT ANDE ParaDNA

1. ParaDNA

The ParaDNA Intelligence Test System has been developed by LGC (Laboratory of the Government Chemist, Teddington, UK). ParaDNA screens five STR loci (D3S1358, D16S539, D8S1179, D18S51, and TH01) and the sex marker Amelogenin. The time-to-result of the ParaDNA system is 75 min, and this system is based on HyBeacons technology and melts curve analysis. These beacons are made of short DNA sequences with one or more fluorescent dyes, which will emit light when the complementary DNA is attached to the beacon. The system consists of a screening unit, a sample collector, and a test kit. The collector is used similarly to a swab; its head consists of four plastic tips (used for sampling of (potential) traces). Post to sample collection, the head of the collector is inserted into a 4-well reaction plate.

The ParaDNA system is a so-called ‘Level 1 DNA Screening Solution’, which implies that it meets the following criteria: (1) the test must be a DNA test, (2) it should provide the police with a more robust and reliable decision making tool, (3) should speed up early intelligence, (4) is easy-to-use by non-scientific personnel, and (5) the test result must be interpretable by minimally trained personnel. Li et al. validated the ParaDNA test with mock evidence samples, according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. They used blood on glass, on Flinders Technology Association (FTA) cards and on denim, saliva on glass, buccal swabs, drink bottles, smoked cigarettes, semen on glass and carpet, and touch DNA on clothing and on a mobile phone. Both the blood and saliva samples had a success rate of 100%. When using saliva on swabs, the success rate dropped to 80%. Semen samples and touch DNA had a success rate of 60–75% (depending on the analysis software) and 14%, respectively ^[1]. Tribble et al. tested the ParaDNA Screening System with seminal samples, which profiles D16S539, TH01, and Amelogenin loci. They tested serial dilutions of semen, and at 2 nL of semen in each well, the assay showed a drop in Amelogenin. At 0.2 nL, the presence of DNA was no longer detected ^[2]. Ball et al. tested 381 DNA samples, and only 0.15% was discordant. Dropout only occurred in samples with a heterozygote imbalance or in cases of high stutter ^[3]. Donachie et al. concluded that the ParaDNA system could be used as a presumptive test for samples with medium to high template DNA present, such as blood, saliva, and semen stains. For low template samples, such as touch DNA (e.g., single fingerprints), the success rate was too low ^[4]. Dawnay et al. report results with the ParaDNA screening system as obtained during the UK Police pilots’ phases III and IV (in which validation of the future operating procedure of this system in police hands by various Police Forces took place). A profile was defined as successful when 14 or more alleles were determined. For blood, the success rate was 100% in both phases. When using saliva as a sample the success rates were 88.9% and 82% for phases III and IV, respectively. Touch samples scored relatively low, viz. 33.3% in phase III and 65.2% in phase IV ^[5]. They determined that upon using this system for touch DNA samples, 28/34 samples would result in a successful STR profile, while without this technique it would be 42/83. Therefore they state that the ParaDNA system will increase profiling success rates, reduce backlogs and result in cost savings while noting that it is only a presumptive test and cannot replace existing STR profiling ^[6]. Dawnay et al. also performed field tests to determine the impact of sample degradation and inhibition when using human remains as a sample. They valued the possibility of performing rapid processing in the field with the ParaDNA system. However, further optimization is required concerning the collection process in the case of human remains ^[7]. Using 4 ng of more DNA (corresponding to 1 ng per well), Blackman et al. showed that more than 65% of the samples provided a full profile. A ‘usable’ (≥ 7 alleles detected) profile could be generated with 250 pg of DNA in more than 80% of the samples by using the ParaDNA system ^[8].

In conclusion, the ParaDNA Screening System can be used for samples that contain a relatively high amount of DNA, such as blood, saliva, and semen. Since only 6 STRs (or less for some kits) are analyzed, the ParaDNA system can only be used for screening and indicative testing, which is a limitation of this system. Later on, the so-called ParaDNA Body Fluid ID Test became available. This test also uses of HyBeacon probes and is an RNA-based test to determine the type of body fluid (seminal fluid, sperm cells, blood, saliva, vaginal fluid, and menstrual blood). In 2013 ParaDNA was acquired by Life Technologies Corporation. However, the production of ParaDNA is currently discontinued.

2. RapidHIT

The RapidHIT ID (launched in 2013), a system from IntegenX (which is part of Thermo Fisher Scientific as of 2018), is a cartridge-based system and can create a full STR profile within 90 min. The GlobalFiler analysis results are acquired by integrating DNA extraction, PCR amplification, and CE in one machine. Prior to the RapidHIT system, IntegenX developed the Apollo 200 DNA HID System, which integrated cell lysis, DNA extraction, amplification, separation, and detection from buccal swabs or blood samples. Another predecessor was the RapidHIT 200, which was later on re-branded to the RapidHIT Human Identification System, whereby the lysis temperature was somewhat adapted. The RapidHIT 200 contains the same chemistry as the RapidHIT ID and can generate STR profiles within 2h. Thermo Fisher only sells the RapidHIT ID nowadays, which is an FBI NDIS-approved rapid DNA system. The ACE cartridge is used for the analysis of a single swab (e.g., a reference buccal swab), and the EXT cartridge is for the analysis of DNA extracts (evidence profile). Later on, there were two types of cartridges available: the ID ACE GlobalFiler and the RapidINTEL for oral cells and minute samples (e.g., blood stains), respectively. Although RapidINTEL was originally developed for the analysis of blood and saliva samples, it has also been validated for other tissue samples. Nowadays, the RapidHIT ID uses two chemistries/kit types: the GlobalFiler Express cartridge and the AmpFLSTR NGM SElect Express cartridge. GlobalFiler chemistry for buccal swabs (the ACE GlobalFiler Express Sample Cartridge) and for blood and saliva samples (the RapidINTEL GlobalFiler Express Sample Cartridge) can be ordered on the website of ThermoFisher. The AmpFLSTR chemistry is available as ACE NGM SElect Express Sample Cartridge to be used for buccal swabs. The EXT cartridges are not available under that name anymore. RapidHIT systems are evaluated extensively in the literature.

A sensitivity level of 176 ng saliva DNA was reported by Jovanovich et al. for full concordant profiles and using 16 ng of DNA (on a swab), 57% of the alleles could still be detected. A 100% success rate was achieved for the positive controls (37 samples). Out of 250 buccal samples, 219 samples were fully in concordance with the PowerPlex 16 profile on the first pass ^[9]. Hennessy et al. assessed the sensitivity, accuracy, and genotype concordance of this RapidHIT system. Full profiles were obtained till 500 pg when DNA was added to the vials; down to 25 pg, 65% of the alleles were detected. In case DNA was added to swabs, a sensitivity of 100 ng was found. Moreover, when 5 ng DNA was loaded into the swabs, 52% of the alleles were detected. It has to be noted that all standards tested were in 100% concordance with the certified reference profiles. For 150 buccal swab samples, a 100% genotype concordance was obtained; the 13 Combined DNA Index System (CODIS) core loci were present on 94.7% of the samples ^[10]. Buscaino et al. could obtain complete profiles with 75 pg/µL for GlobalFiler Express and 50 pg/µL for AmpFlSTR NGM SElect Express and a total amount of input DNA (depending on the sample input volume) less than 250 pg. Five samples were run in triplicate, producing complete and concordant STR profiles, thus showing good reproducibility. The AmpFlSTR NGM SElect kit showed full concordance for all tested mock crime scene samples, while the GlobalFiler Express kit, 5/7 samples did not result in full concordance ^[11]. Martin et al. investigated the use of rapid DNA analysis on touch DNA samples. Samples such as cable ties, fabric, matchstick, and ziplock bags were analyzed. The cable ties and matchsticks were directly placed into the cartridge of the RapidHIT. Together with tape-lifted fabric, these samples showed the best results ^[12]. The RapidHIT was extensively evaluated by LaRue et al. Among other things, they tested the variability, sensitivity, concordance, and reliability, and they performed a contamination study. The success rate using reference buccal swabs was comparable to standard methods. Full profiles were obtained with an input of 200 ng of DNA, while with 10 ng, more than 10% allelic drop-out was observed, resulting in partial profiles ^[13].

2.1. RapidHIT 200

Thong et al. compared the performance of the RapidHIT 200 System (in combination with the GlobalFiler Xpress Cartridge) with the standard protocol. They tested swabs with blood, semen, and buccal samples, blood-stained FTA punches, tissues, bone marrow, fingernails, and cigarette butts. Overall, the RapidHIT 200 System performed well compared to standard laboratory protocols. It was recommended by Thong et al. to perform more experiments with more challenging DNA samples, such as degraded samples, samples containing inhibitors, mixtures, and samples with a small amount of DNA ^[14]. Shackleton et al. investigated the performance of the RapidHIT200 with crime stain type samples on the RapidHIT 200, which contain various (and compared to buccal swab samples lower) input amounts of DNA. Extending the sample work-up time (DNA bead binding time or lysis time) had a positive effect on the results, but this negatively affected the time-to-result: i.e., up to 30–60 min extra time was needed ^[15]. Shackleton et al. validated the RapidHIT 200 further for accreditation and used it in the UK. Sensitivity down to 4.5 ng (750 cells) on a swab was obtained, while for lower cell loads, allelic drop-out was observed. They further demonstrated that the AmpFlSTR NGMSeE kit, in combination with the RapidHIT 200, is a reliable method for obtaining reference profiles from a buccal swab ^[16]. The RapidHIT 200/GlobalFiler Express system (with RH200/GFE cartridge kits) was evaluated by Date-Chong et al. The built-in quality flags in the software appeared, among others, in cases of intra-locus imbalance and inconclusive homozygote allele. Of all reference buccal samples, 50% passed the criteria of generating a full profile without raising any quality flag, which would require manual editing. With manual data review, the success rate is increased to 91% for obtaining a full genotype at each locus ^[17]. Holland et al. also evaluated RapidHIT 200 for automated human identification of single source samples. A total of 85 buccal swabs were analyzed, and 100% concordance was obtained with known DNA profile information. When using the software of the instrument for data analysis, 95.8% provided full profiles in the reproducibility study ^[18]. The RapidHIT 200 was explored by Verheij et al. with different types of samples. They concluded that the system is very suitable for buccal swabs. Moreover, saliva, semen, skin, and hair samples were tested, but profiling success rates were variable, and with lower input samples, profiling artifacts were present. The blood samples tested presumably contained inhibitors since no profile could be obtained ^[19]. Only 74% of the buccal swab samples that were tested by Mogensen et al. on a RapidHIT 200 resulted in a correct STR profile. The working ranges in order to generate full profiles were found to be 900–1200 ng. Lower and higher amounts of DNA resulted in allelic drop-out ^[20].

2.2. RapidHit ID

The use of blood samples in combination with the RapidINTEL cartridge and the RapidHIT ID System was investigated by Chen et al. They also concluded that FTA cards are the preferred method for storage. Urea and melanin cause inhibition resulting in signal decrease and allelic drop-outs. Using 0.5 µL of blood or 7 ng of genomic DNA resulted in a concordance rate of 100% ^[21]. Murakami et al. evaluated the performance of the RapidHIT ID (with ACE and INTEL cartridges) with samples from postmortem bodies at different stages of decomposition. Moreover, nail clipping samples were collected and analyzed. A detection rate of 100% was obtained with the latter, even after 10 years of storage, as long as the nail clippings were pretreated with dithiothreitol and TNE (Tris, NaCl, and EDTA) buffer. For blood stains, the recommended storage method is using filter paper at room temperature ^[22]. A success rate of 72% was reported by Wiley et al. upon testing the RapidHIT ID (cartridge with GlobalFiler Express primer set and master mix). After manual interpretation of first-pass inconclusive results, the success rate could be increased up to 90%. While coffee, smoking tobacco, and chewing tobacco did not influence the result, the swab type used is of importance since it can affect the typing success rate ^[23]. The RapidHIT system was used by Gangano et al. to demonstrate the usability of the system with blood and saliva samples. They concluded that 88% of the samples (blood and saliva) generated DNA profiles that were classified as qualitatively sufficient enough to be able to search against a database. Moreover, a reanalysis study was performed where the swabs were recovered from the cartridge after analysis on the RapidHIT. Reanalysis of these samples on the RapidHIT demonstrated that conserving these samples is possible with an average of 92% of the original full loci calls ^[24]. Upon use of the GlobalFiler Express kit, Salceda et al. found a sensitivity of 12,500–200,000 cells/swab, while for 6250 cells, only 51.6% of the alleles could be detected. Five control samples were tested with three RapidHIT ID machines, and all 15 profiles were complete and concordant. The authors claim that this system showed 100% concordance in comparison with conventional methods ^[25]. Ward et al. used the RapidHIT ID system and RapidINTEL sample cartridges to evaluate the performance with mixtures and touch DNA samples. The mixtures were artificially constructed from raw DNA as well as from body fluids. While the profiles from the simulated DNA mixtures on the RapidHIT showed the expected mixture proportions, the peak heights were lower compared to results when using a standard laboratory workflow. For the mock casework, mixtures, blood, saliva, or both blood and saliva were used. A combination of saliva and blood led to low levels in the profile of the saliva contributor. The sensitivity of the RapidHIT is lower than a standard laboratory workflow, which affects the results when touch samples are analyzed. Many of the analyzed touch DNA samples produced very low-level DNA profiles ^[26]. Cihlar et al. validated the RapidHIT ID system with the ACE GlobalFiler Express sample cartridges in combination with reference buccal swabs. Among others, they performed a concordance, contamination, sensitivity, reprocessing, and mixture study. The data generated showed that the workflow of RapidHIT generates reliable and reproducible DNA profiles. A first-pass success rate of 92% was obtained with the reference buccal swabs. However, Cihlar et al. also advise using a trained forensic analyst to increase the pass rate success ^[27].

In conclusion, the RapidHIT 200 provides good results in the case of single-source reference samples, especially when manual evaluation is performed. A limitation of the system is the inability to quantify the amount of DNA that is added to the PCR step. The RapidHIT 200 was later on replaced with the RapidHIT ID, which also shows good results for reference buccal samples when combined with the ACE cartridge. Wiley et al. and Cihlar et al. recommend performing a manual evaluation of the generated DNA profiles, especially for case samples ^[23]^[27].

3. ANDE

Before the ANDE systems became commercially available in their current format, it was developed and tested by NetBio. In 2013 Tan et al. reported on a fully integrated system for the automated generation of DNA profiles ^[28]. With the so-called BioChipSet cassette, a total of 5 buccal swabs could be analyzed simultaneously. The system integrated lysis, purification, amplification, CE separation, and detection. The generated profile involves 16 loci and is generated in 84 min. The PCR mixture is present in the cartridge in lyophilized format. A full CODIS profile was obtained with 85 of the 100 buccal samples analyzed. The other samples resulted in a partial profile (5) or no profile (10). Blocked channels were the cause of failure, resulting in no amplification or failing electrophoresis. In order to be able to generate profiles from lower amounts of DNA, a Low DNA Content BioChipSet cartridge was developed. In order to generate profiles from a minor amount of DNA, the chemistry was adapted, and a purification module was integrated into the cartridge, while the system (both analysis and read-out) remained identical to a great extent. Full profiles could be generated with 1 µL or more blood as an input sample in the sensitivity study. A study was carried out with eight laboratories (including NetBio), which tested over 2300 swabs in total with the DNAscan/ANDE Rapid DNA Analysis System to be compliant with the FBI’s Quality Assurance Standards and the NDIS Operational Procedures. Concordant results were obtained for the reference buccal swabs, including automated data analysis and an accuracy allele calling rate of 99.998%. In 2012, DNAScan was the first system released by ANDE (‘Accelerated Nuclear DNA Equipment’), which was later renamed to ANDE 4C (4-color). The system integrates the steps of DNA extraction, STR amplification, separation by electrophoresis, and detection and performs them all within 90 min. The A-Chip for the ANDE 6C (6-color), which can process up to five buccal samples, was previously known as the BioChipSet cassette. The so-called I-chip is specifically designed for low-template DNA samples and can process up to four samples simultaneously. The ANDE 6C system uses a Bode SecureSwab2 with an integrated RFID chip for sample tracking in the swab cap and is able to analyze 27 loci. The ANDE 6C received NDIS approval in 2018.

In addition to DVI, trafficking in persons is also a situation in which rapid DNA results are crucial. Palmbach et al. investigated whether the ANDE system (at that time still part of NetBio) was suitable for this purpose. A success rate of 95% was obtained for the reference profiles; the other 5% resulted in partial profiles. The other samples consisted of various objects, such as cigarette butts, condoms, plastic bottles, and straws. Overall, a success rate of 71% was obtained, consisting of 23%, 42%, and 6% for full profiles, partial profiles, and mixed profiles, respectively ^[29]. Moreno et al. evaluated the first version, the DNAscan, which generated a profile from CODIS 13 loci. Without human review, an overall success rate of 75% was achieved from reference buccal swabs ^[30]. The developmental validation of the DNAscan/ANDE system was carried out by Della Manna et al. using a BioChipSet Cassette. Over 2000 buccal swabs were analyzed, which resulted in over 99.998% concordant alleles ^[31]. The California wildfires in 2018 were the motivation for Gin et al. to use rapid DNA for victim identification. Among others, blood, as well as liver, brain, and muscle tissue, were used for DNA profiling. In 90% of the cases, the ANDE (no type mentioned) led to the identification and was even the primary identification modality ^[32].

3.1. ANDE 4C

The ANDE (probably 4C) system was used by Hinton et al. for the analysis of reference samples (buccal swabs), samples from a controlled setting, and from an uncontrolled environment. In combination with the A-Chip, 19 of the 22 buccal swabs resulted in a complete profile. For the controlled setting, the I-Chip was used, which provided a full profile for 7 of the 12 samples. The I-Chip was also used within military exercise, i.e., the uncontrolled environment. Only 7 out of 44 samples could generate a full profile from a single contributor. The other samples resulted in no DNA profile (25), partial DNA profile (9), and DNA mixtures (3) ^[33].

3.2. ANDE 6C

Ragazzo et al. validated the ANDE 6C according to the ISO/IEC 17,025 standard. A total of 104 buccal swabs were analyzed with the ANDE system and with the traditional laboratory method. Of these 104 samples, three swabs gave no interpretable signals. A concordance rate of 99.96% was obtained by comparison of 2800 genotypes. With these results, the aforementioned standard was met ^[34]. While the ANDE 6C was originally designed for reference samples (i.e., buccal swabs), Manzella et al. tested the performance of this system with calcified and soft tissue samples. The success rate was 0%, 11%, and 50% for muscle tissue, ribs, and teeth, respectively ^[35]. Turingan et al. analyzed 1705 casework samples to analyze the performance of the ANDE 6C with the I-Chip to address the FBI’s Quality Assurance Standards. Among other things, they investigated specificity, sensitivity (including a limit of detection), reproducibility, mixtures, and a wide variety of sample types (e.g., dried blood, blood on various substrates, semen (neat and stains), and saliva). Their main conclusion was that the limit of detection is influenced by the type of sample, the condition (aging and degradation), and the substrate ^[36]. Six forensic and research laboratories worldwide took part in the validation study of the ANDE 6C of Carney et al. Over 99.99% concordant alleles were generated with the over 2000 analyzed samples (buccal swabs in combination with the A-Chip), without human review of the results ^[37]. Also, Grover et al. evaluated the ANDE 6C system and the A-Chip, using only buccal swab samples. Among others, they tested sensitivity, reproducibility, species specificity, and stability. Overall, they concluded that the system performs well and that the automated analysis results in accurate and concordant DNA profiles ^[38]. Manzella et al. used the ANDE 6C system to investigate the difference between the ANDE swab and a conventional cotton swab as a sampling method. Using both swabs for buccal samples resulted in a success rate of 33% and 88% for the conventional cotton swab and the ANDE swab, respectively ^[35]. Turringan et al. used the ANDE 6C to identify human remains. They used several tissue types, such as the buccal, brain, liver, and muscle. Up to 11 days of exposure, buccal swabs are the sample of choice. Moreover, bone and tooth samples provided good results for up to 1 year (which was the duration of the study). When the remains are refrigerated, all tissue types yield good results ^[39].

In conclusion, the ANDE systems (4C and 6C) work very well when buccal swabs are used as input, even without human intervention (i.e., rapid DNA analysis). While being able to have rapid DNA profiles in a situation such as DVI is extremely useful, these types of samples show a lower success rate compared to reference samples.

4. Other Systems

NEC developed a portable DNA analyzer that can perform DNA analysis within 25 min, with 5, 15, and 5 min for DNA extraction, PCR, and CE, respectively. The disposable cartridge contains 5 mm wells that are used as test tubes. The channels for fluid transfer serve as pipettes. The system weighs 32 kg and can analyze 16 loci. It must be noted, however, that the most recent publication using the NEC system dates from 2012.

MiDAS is a microfluidic system for rapid forensic DNA analysis. In contrast to other rapid DNA analysis systems with all reagents present in the cartridge, MiDAS requires manual preloading of all reagents onto the cartridge. Moreover, other manual steps are required in the operation of the system, such as the installation of the CE chip. The system consists of a total of five interdependent modules: (1) swab lysis, (2) DNA extraction, (3) PCR, (4) PCR product transfer, and (5) CE separation, including optical readout. Each module is discussed separately in various publications. The lysate was initially used as input, but after several improvements, the swab head could be used in combination with the swab sample lysis module. After insertion of the swab in the module, lysis occurs. Then, the lysate is transported to a chamber for purification, amplification, and detection. Hopwood et al. described a cartridge that integrates DNA extraction (from lysate), amplification, CE, and detection using laser-induced fluorescence. With this system, it is possible to obtain an STR profile within 4 h ^[40]. The PCR module is discussed by Estes et al. for the amplification of 17 STRs. This cartridge uses preloaded solid-phase reagents and paraffin valves for fluidic control. The electronic control components, the CE microchip, and the optical detection module are described by Hurth et al. ^[41].

5. Conclusion

The use of (modified) rapid DNA systems on the crime scene involves a trade-off between speed and performance. In general, rapid DNA systems are not as sensitive as conventional laboratory methods. While the speed and portability are extremely useful for, e.g., DVI cases, their performance (in terms of sensitivity and ability to use samples other than buccal swavs) has to be (further) improved to actually use these techniques on crime scenes. These systems work well when using single-source buccal swabs (i.e., reference samples), but for more complex samples the success rate (i.e., generating a full DNA profile) is too low.

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