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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 2  |  Issue : 2  |  Page : 67-73

Identification of Colored Dyes that are Resistant to Fading on Exposure to Ethylene Oxide; Use with Indicating FTA™ Sample Collection Cards


R&D Bio-Research Tools, GE Healthcare Life Sciences, The Maynard Centre, Whitchurch, Cardiff, CF14 7YT, UK

Date of Web Publication16-Jun-2016

Correspondence Address:
Peter James Tatnell
R&D Bio-Research Tools, GE Healthcare Life Sciences, The Maynard Centre, Whitchurch, Cardiff, CF14 7YT
UK
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2349-5014.184191

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  Abstract 


Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as “DNA-free.” Recently, “DNA-free” status was described for HID-related products using ethylene oxide (EtO); this gas reduces the presence of amplifiable DNA and causes minimal interference to downstream HID-analytical methods. During sample collection, indicating cards, for example, Indicating FTA™ (GE Healthcare Life Sciences, UK), are used to collect and store buccal cell DNA. These cards contain a dye which changes color on application of a colorless sample. Generating “DNA-free” indicating cards using EtO should not impact the dyes' ability to indicate sample location or the efficacy of the card in downstream HID-analytical methods. This study was initiated to identify alternative dyes to those currently used with sample indicating collection cards. The most promising, dyes when applied to cellulose papers exhibited a uniform color distribution and excellent sample indicating properties even when mixed with chemicals associated with FTA™. When dyed cellulose papers were exposed to EtO, ultraviolet radiation, elevated temperature, and humidity, negligible fading or discoloration was observed. The presence of these dyes on cellulose papers did not interfere with direct short tandem repeat (STR) profiling. Allelic concordance, first pass success rate, and mean peak heights were comparable to samples applied to Indicating FTA. Biological samples applied to EtO-treated dyed cellulose papers and stored >1 month produced full STR profiles of sufficient quality to allow submission to DNA databases, confirming negligible interference from EtO treatment. These alternative sample indicating dyes resist EtO-mediated fading while fulfilling the Forensic Community's expectation for “DNA-free” with negligible impact on collection card performance.

Keywords: Ethylene oxide, forensic DNA analysis, identification of colored dyes, Indicating FTA sample collection cards, short tandem repeat profiling


How to cite this article:
Moran N, Tatnell PJ. Identification of Colored Dyes that are Resistant to Fading on Exposure to Ethylene Oxide; Use with Indicating FTA™ Sample Collection Cards. J Forensic Sci Med 2016;2:67-73

How to cite this URL:
Moran N, Tatnell PJ. Identification of Colored Dyes that are Resistant to Fading on Exposure to Ethylene Oxide; Use with Indicating FTA™ Sample Collection Cards. J Forensic Sci Med [serial online] 2016 [cited 2020 Dec 4];2:67-73. Available from: https://www.jfsmonline.com/text.asp?2016/2/2/67/184191




  Introduction Top


A growing expectation exists in the Forensic Community for “DNA-free” reagents, following a manufacturing incident resulting in contamination of HID products with DNA.[1],[2],[3] “DNA-free” status can be achieved by exposure to ethylene oxide (EtO).[4],[5] Indicating FTA sample collection cards (GE Healthcare, Life Sciences, UK) are used to store buccal cell DNA. These are impregnated with preservative chemicals and a dye that indicates the position of colorless cellular samples post application. Eventually, these cards will require “DNA-free” certification. Exposure to EtO should not compromise performance, especially the dyes' ability to indicate the location of colorless samples. Therefore, it is important to identify alternative sample indicating dyes that exhibit minimal discoloration on exposure to EtO.


  Materials and Methods Top


Experiments were performed according to the Scientific Working Group on DNA analysis methods.[6] All chemicals were purchased from Sigma-Aldrich, Fluka, or Fisher and were the highest purity available.

Candidate dyes

Color changes observed on indicating sample collection cards are primarily due to changes in pH caused by application of a biological sample. Color change however can also be attributed to the chromatographic displacement of the dye, in which sample application causes a capillary flow that carries the dye to the periphery and thus dilutes the dye centrally producing a surrounding chromatographic ring of increased dye concentration. Displacement was confirmed using Indicating-FTA, -FTA Elute, and -DMPK-C™ sample collection cards (GE Healthcare Life Sciences, UK). The areas corresponding to an aqueous sample exhibited a distinctive darker outline confirming the chromatographic movement of the dye. FTA and FTA Elute are impregnated with detergents and protein denaturants, respectively; DMPK-C lacks any specialized chemical coating. All three cards are impregnated with different propriety sample indicating dyes. An initial list of potential candidate dyes was produced (data not shown) including chromatographic, pH, and biological dyes that highlight cellular components. This list was subsequently reduced by eliminating dyes that were either toxic, would not give a suitable color, insoluble in water, possess a pH change not compatible with biological samples, exhibit poor chemical stability or were light sensitive.

Application of candidate dyes to cellulose papers

The remaining candidate dyes [Table 1] were dissolved in 0.1 M Tris-HCl pH 8.0, (typically 0.02–0.1% [w/v]) the actual amounts dissolved were related to the color intensity required. Dyes were applied to cellulose papers together with the chemicals associated with FTA and FTA Elute sample collection cards. Dyes were also applied to cellulose papers lacking any specialized chemical coating (uncoated). The pH of solutions was adjusted to compensate for any effect associated with the FTA or FTA Elute chemicals, thus maintaining the appropriate color. Cellulose papers (4 cm × 6 cm) were dipped into each dye solution and dried.
Table 1: Performance of alternative sample indicating dyes applied to cellulose-papers

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Exposure to ethylene oxide, ultraviolet radiation, temperature, and humidity

EtO is used to sterilize medical devices and has recently been shown to achieve “DNA-free” status by minimizing the amount of amplifiable DNA associated with HID-related products.[4] EtO probably generates “DNA-free” status by either cleaving the DNA strand or facilitating the generation of apurinic sites which disrupt the activity of DNA polymerases.[7],[8]

Dyed cellulose papers were forwarded to Synergy Health Sterilization, Swindon, UK, and exposed to EtO. Cycle 1: preconditioning, 42.0–45.0°C for 12 h, humidity of 64.5–72.8%; EtO exposure, 43.7–46.5°C for 4 h at 480 mbar and EtO de-gassing, 42.2–46.2°C for 12 h. Cycle 2: preconditioning, 43.6–47.7°C for 12 h, humidity of 64.7–72.8%; EtO exposure, 44.1–46.7°C for 4 h at 480 mbar and EtO de-gassing, 43.0–46.2°C for 12 h. To assess dye stability to temperature and humidity, dyed cellulose papers were exposed to 25°C, 60% humidity (real time; [THRT]) and 40°C, 75% humidity (accelerated conditions; [THAC]) for 4 weeks. After exposure to EtO, UV, and THRT and THAC, the appearance of the dyed cellulose papers was compared to non-exposed controls.

Indicating the location of biological samples

HeLa cells (~8000) were transferred to a foam applicator and applied to dyed cellulose papers using mild pressure for ~10 s and allowed to dry. This mimics the collection and transfer of buccal cells to sample collection cards during HID workflows. Indicating versions of FTA, FTA Elute, and DMPK-C cards were used as controls. Dyes considered to have performed well were subjected to quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analyses. Dried samples were routinely stored for 1 month prior to subsequent analysis.

Inhibition in quantitative polymerase chain reaction

To test if candidate alternative dyes interfere with DNA amplification reactions, portions of dyed cellulose papers impregnated with HeLa cells were extracted using 1.2 mm Harris™ Uni-Core Punch (GE Healthcare Life Sciences, UK) and added directly to qPCR reactions in a 96-well MicroAmp PCR plate (in triplicate). Indicating FTA was used as a control. Quantifiler ® Human DNA Quantification Kit was used according to the manufacturer's instructions and qPCR was performed using a 7900HT Fast Real-Time PCR System (both Life Technologies, Paisley, UK).

Short tandem repeat analysis of biological samples

HeLa cells applied to dyed cellulose papers were used to test the compatibility of candidate alternative dyes with direct STR profiling. Indicating FTA cards were used as a control. STR reactions were performed using the AmpFLSTR ® Identifiler ® PCR Amplification Kit (Life Technologies, Paisley, UK) according to the manufacturer's instructions. Punches (1.2 mm) were added directly to STR reactions in triplicate. A 28-cycle PCR was performed using a GeneAmp ® PCR System 9700, and resultant STR products were detected using a 3130xl Genetic Analyzer (Life Technologies, Paisley, UK) and analyzed using GeneMapper™ v3.2 software (Life Technologies, Paisley, UK). STR profiles that exhibited full, partial, or no profiles, average peak heights, and first pass success rates were determined as defined by Ogden et al., 2015.[9]

Statistical analysis

The distribution of data was assessed using the Shapiro–Wilk test. For normally distributed data, statistics were calculated using analysis of variance; for nonparametric data, statistics were calculated using Wilcoxon/Kruskal–Wallis test. Statistical significance was set at P < 0.05. Confidence intervals for mean values were set at 95%. All statistical analyses were performed using the JMP 11.1.1 Statistical Software (SAS Institute Inc., Marlow, UK).


  Results Top


A summary of results is described in [Table 1].

Exposure to ethylene oxide, ultraviolet radiation, temperature, and humidity

On exposure to EtO, the majority of the alternative dyes displayed good color stability demonstrating little discoloration when applied to uncoated and FTA-coated cellulose papers. In contrast, all dyes when applied to cellulose papers coated with chemicals associated with FTA Elute exhibited almost complete fading ([Figure 1] and [Figure 2] show the performance of alizarin red and chlorophenol red dyed cellulose papers, respectively). Several dyes exhibited a color change on both uncoated papers and chemically-coated papers probably as a reflection of a pH change mediated by EtO. Bromophenol blue and bromoxylenol blue exhibited a green coloration after exposure. m-Cresol purple changed from blue to yellow and chlorophenol red changed from purple to red. Cresol red and rosolic acid when applied to uncoated cellulose papers changed from red to yellow and white, respectively. It is appreciated that any dye that changes color due to exposure to EtO does not necessarily discount the dye from functioning as a sample indicator providing the change does not compromise the ability of the dye to indicate the position of an applied sample. Chlorophenol red is used as the dye with Indicating FTA Elute [10] and changes color from purple to yellow over a pH range 4.8–6.7. FTA Elute is not traditionally used in forensics workflows, but it is routinely used in situations when purified DNA is required.
Figure 1: Composite image of uncoated, FTA–, and FTA Elute–chemically coated cellulose papers dyed using alizarin red (1 mM). Papers were exposed to ethylene oxide, ultraviolet, and elevated temperature/ humidity (25°C/60% THRT or 40°C/75% THAC). Minimal discoloration was observed

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Figure 2: Composite image of uncoated, FTA–, and FTA Elute–chemically coated cellulose papers dyed using chlorophenol red (5 mM). Papers were exposed to ethylene oxide, ultraviolet, and elevated temperature/ humidity (25°C/60% THRT or 40°C/75% THAC). Significant discoloration was observed

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To simulate exposure to sunlight, dyed cellulose papers were subjected to accelerated UV exposure. Chlorophenol red is known to fade slightly during prolonged exposure to natural light and therefore, represents a control to assess the performance of alternative dyes. Under accelerated UV conditions, chlorophenol red faded significantly when applied to noncoated and FTA-coated cellulose papers [Figure 2]. In the presence of FTA Elute chemicals, the dye discolored to dark red. Therefore, candidate dyes that do fade or discolor less during accelerated UV exposure probably retain their color significantly longer when exposed to natural sunlight.

When applied to chemically-coated and uncoated cellulose papers, alizarin red and alcian blue 8GX demonstrated excellent color stability to UV exposure. When applied to cellulose papers coated with FTA Elute chemicals, both dyes exhibited only slight discoloration. Allura red AC and indigo carmine exhibited only slight fading irrespective of chemical coating. Methyl red, acid red 1, tartrazine, direct blue 71, direct red 23, acid red 4, chlorazol fast pink, crocein orange, methylene blue, orange G, and reactive blue 4 exhibited less fading than chlorophenol red (data not shown). After exposure to UV, all the above dyes were considered to retain sufficient color to function as sample indicators.

Mordant blue 9 and rhodamine B faded less than chlorophenol red when applied to uncoated cellulose papers but demonstrated complete fading when applied to chemically-coated papers (data not shown). Rhodamine B is fluorescent and will probably cause significant interference in downstream molecular applications. The UV color stability of the remaining dyes was considered either comparable to or worse than that exhibited by chlorophenol red and these were considered unsuitable as sample indicating dyes.

When exposed to elevated temperatures and humidity (THRT and THAC), none of the dyed cellulose papers faded or discolored significantly irrespective of the chemical coating ([Figure 1] and [Figure 2] shows the performance of alizarin red and chlorophenol red dyed papers, respectively, as an example). Ruthenium red was the only exception and faded significantly during THAC.

Indicating the location of biological samples

HeLa cells applied to dyed cellulose papers were used to test the ability of candidate dyes to function as sample indicators. Several dyes including allura red AC, tartrazine, direct blue 71, acid red 4, and chlorazol fast pink did not exhibit a color change and were considered compatible with the function of a sample indicating dye (data not shown). Several of these on sample application exhibited a darker outer chromatographic ring which disappeared after drying, making sample localization difficult. For the remaining dyed cellulose papers, areas that corresponded to HeLa cells were considered readily distinguishable from areas lacking cells.

Candidate dyes that did not fade or discolor after EtO exposure or UV irradiation were considered suitable as sample indicator dyes included alizarin red (1 mm), alcian blue (0.2 mm), indigo carmine (0.75 mM), methylene blue (1 mM), and reactive blue 4 (0.3 mM) [Figure 3].
Figure 3: Sample indicating performance. HeLa cells applied to cellulose papers (uncoated, left; FTA, middle; FTA Elute, right) and dyed using (a) alizarin red (1 mM), (b) methylene blue (1 mM), (c) reactive blue (0.3 mM), (d) alcian blue (0.2 mM), and (e) indigo carmine (0.75 mM)

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Potential inhibition in quantitative polymerase chain reaction reactions

Cellulose papers coated with alizarin red, alcian blue 8GX, indigo carmine, and methylene blue (at the stated concentrations) did not cause any significant inhibition to qPCR reactions as determined by the Quantifiler internal process control (IPC) [Table 2]. Comparable results were obtained when using Indicating FTA cards (P > 0.9973). Chlorophenol red (5 mM) and reactive blue 4 demonstrated comparable levels of interference (P > 0.73) that was significant greater than that exhibited by the other dyes (P< 0.02). Reducing the concentration of chlorophenol red and reactive blue 4 may reduce the observed IPC interference but may also compromise their ability to function as sample indicator dyes on solid supports. Comparable amplification efficiencies of the Quantifiler human telomerase catalytic subunit (TERT) subunit gene were observed irrespective of the dyed cellulose papers used. These were equivalent to those generated using Indicating FTA (Ct value; P > 0.37, quantity [ng/µl]; P > 0.17). Comparable results were obtained when an equivalent amount of each dye was added as a solution directly to the qPCR reactions (data not shown). These data suggest that some dyes interfere with qPCR but this may not disqualify their use as sample indicator dyes. During nondirect STR workflows, sample collection cards are routinely washed to remove inhibitory contaminants and chemicals prior to performing STR profiling.[11],[12] This washing step will also remove water soluble indicating dyes.
Table 2: Quantitative polymerase chain reaction using the Quantifiler Human DNA quantification kit and HeLa cells applied to FTA-coated cellulose papers dyed with alternative sample indicating dyes

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Short tandem repeat analysis of biological samples applied to dyed cellulose papers

During STR profiling, all dyed cellulose papers produced full profiles exhibiting concordant allelic designations with mean peak heights comparable to HeLa cells applied to Indicating FTA (P > 0.43; [Figure 4]). The designated alleles are concordant with HeLa cells.[13] Peak heights were in excess of that required for uploading profiles to National DNA databases.[14] Chlorophenol red and reactive blue 4 dyed cellulose papers exhibited interference during the amplification of the IPC in qPCR; however, no comparable interference was observed in the amplification of HeLa cell-related STR products. The AmpFLSTR ® Identifiler PCR Amplification Kit used in this study has been developed to minimize the effects of inhibitory compounds [15] and therefore, may be more tolerant to potential inhibitory effects associated these two dyes. Chemically-coated Indicating FTA Elute cards and the associated dye chlorophenol red have also been successfully used in direct STR and qPCR workflows in combination with the PowerPlex 18D (Promega, Wisconsin, USA) and the amplification of HeLa cell HPV18 DNA sequences, respectively.[16] All the dyed cellulose papers generated first pass success rates of 100% and this was equivalent to that derived using Indicating FTA (data not shown).
Figure 4: Mean STR peak heights of HeLa cells applied to cellulose papers coated with FTA-chemicals and dyed with alternative sample indicator dyes. Chlorophenol red (5 mM) and Indicating FTA cards were used as controls. Mean RFU-values are derived n = 3 experiments

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Full STR profiles were also generated using HeLa cells applied to alizarin red (1 mM) dyed cellulose papers that had previously been exposed to EtO using the dual cycle described earlier. HeLa cells were applied and stored for 1 month prior to analysis [Figure 5]. This indicates that prior exposure of sample collection cards to EtO does not affect the subsequent amplification of nucleic acids derived from the applied biological sample.
Figure 5: Electrophoretogram of HeLa cell short tandem repeat products using alizarin red (1 mM) dyed cellulose papers exposed to ethylene oxide. D13S317 allele 13.3 is labeled as an off-ladder peak. Alizarin red artifacts (249 and 327 bp in the blue spectrum and 359 bp, colocating with D2S1338 loci in the green spectrum) are not visible at low alizarin Red concentrations

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  Discussion Top


Numerous dyes characterized as pH, chromatographic, or biological were considered as candidates for indicating the position of colorless biological samples on sample collection cards. Alternatives to those currently used for commercially-available sample indicating cards were selected based on solubility in water, color intensity, and stability to EtO, UV, temperature, humidity, and sample-indicating capabilities. Promising candidates were also evaluated using qPCR and STR profiling. Chlorophenol red is the current sample indicating dye associated with Indicating FTA Elute sample collection cards [10] and was used to compare the performance of candidate dyes. Several promising alternative candidate dyes were identified as alizarin red, alcian blue 8GX, indigo carmine, and methylene blue.

All exhibited a uniform color distribution and excellent sample-indicating properties when used to dye FTA-, FTA Elute-chemically coated, and non-coated cellulose papers. Alcian blue 8GX and methylene blue were classified as a biological dye but did not appear to function by indicating the location of specific cellular components, i.e., polysaccharides and nucleic acids, respectively; both probably function by chromatographic displacement of the dye by capillary flow.

On exposure to EtO, all dyes exhibited negligible fading, and the only exception was chlorophenol red. On UV exposure, no fading was observed by alizarin red and alcian blue 8GX. Minor fading was observed by indigo carmine and methylene blue, but this was significantly less than that observed for chlorophenol red and did not compromise sample-indicating properties.

Indicating FTA sample collection cards are coated with free-radical scavengers that protect DNA from the effects of UV exposure.[16],[17],[18] In this study, the effect of the dye on the long-term stability of DNA was not investigated in detail; however, HeLa cells applied to the dyed cellulose papers and stored for ~1 month were shown to contain DNA that was of sufficient quality and purity to facilitate qPCR and STR analyses. Cellulose papers coated with alizarin red, indigo carmine, and methylene blue exhibited negligible inhibitory effects in qPCR and STR reactions. Slight interference in qPCR and STR reactions was observed by reactive blue 4 and alcian blue 8GX, respectively.

Variability in these data was observed during qPCR and STR analyses in terms of DNA concentrations and peak heights. This was attributed to known limitation of direct workflows, in which heterogeneously dispersed biological samples applied to solid supports are added directly to PCR-based amplification reactions.[19] The observed variation occurs because direct methods use only a portion of the dispersed sample. Comparable variability was observed from HeLa cells applied to Indicating FTA sample collection cards.

Alizarin red S when used at 20 mM to dye cellulose papers exhibited significant interference in qPCR and multiplex STR reactions (data not shown), for example, partial STR profiles were observed associated with a reduced yield of the higher molecular weight PCR products.

Using >4 mM alizarin red to coated cellulose papers resulted in the presence of dye-related artifacts in STR profiles. Three artifacts were observed corresponding to 249, 327, and 359 bp, respectively. The two lower molecular weight artifacts present in the blue spectrum are not within the Identifiler™ allelic ladder bin set, and when present above, the 50 RFU threshold setting was called as off-ladder peaks. The larger molecular weight artifact is located in the green spectrum and colocates with the D2S1338 loci. Reducing the alizarin red concentration decreased the size of the artifact significantly, at 1 mM; the peak height of all three artifacts was significantly below 10 RFU [Figure 4]. This is less than the threshold level required for uploading DNA profiles to the UK DNA database.[13] During standard nondirect HID workflows, forensic punches are washed prior to STR profiling, thereby removing any potential dye artifact. This was confirmed when cellulose papers dyed with decreasing concentration of alizarin red (20-1 mM) were washed using TE − 1 buffer in a nondirect STR workflow using the Identifiler STR kit (data not shown). Detailed inspection of the resultant electrophoretogram indicated that the dye artifacts were no longer visible. Artifacts have been reported previously with the AmpFLSTR Indentifiler PCR Amplification kit (Life Technologies, Paisley, UK).[20] These were described as being intermittent but occasionally reproducible. STR artifacts in the D21S11 locus (200–250 bp) of PowerPlex 16 HS kit (Promega Corp., Wisconsin, USA). have also been reported.[21] In this study, the D2S1338 artifact appears to be related to alizarin red, but kit-related artifacts cannot be discounted.

At lower alizarin red concentrations (<4 mM), full profiles were obtained comparable to those of controls. Alizarin red (1 mM) retained the ability to indicate the position of colorless sample applied to cellulose papers, produced good quality STR profiles exhibiting no artifacts. These data indicate that providing the concentration of the candidate dyes is sufficiently low, it has a negligible effect on PCR amplification efficiency and resultant STR profiles; however, the concentration must be such that it still allows the dye to function as an indicator of colorless biological samples.

Forensic regulatory standards, guidelines,[2],[3] and customer expectations place an onus on manufacturers to deliver HID-related consumable products that are certified as “DNA-free.” EtO gas is a sterilization technique for medical devices and has been demonstrated to minimize the presence of amplifiable DNA on forensic items while not affecting any downstream application such as STR profiling.[4] Indicating sample collection cards are used to store buccal cell DNA during HID workflows, and eventually, these products will also require “DNA-free” certification. An important aspect of exposing indicating sample collection cards to EtO is there should be no impact on performance, especially the ability of the dye to indicate the location of colorless samples and also card efficacy in downstream direct analytical methods. To address this, this study identified several alternative sample-indicating dyes that demonstrate excellent color stability to EtO, UV, temperature, and humidity while also exhibiting negligible interference in downstream HID-related workflows including qPCR and STR profiling. These alternative sample-indicating dyes are potentially able to fulfill the Forensic Community's requirement for DNA-free consumable products.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors are employed by GE Healthcare.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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