Quantitative and qualitative study of STR DNA from ethanol and formalin fixed tissues

Forensic Science International 262 (2016) 18–29

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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint

Quantitative and qualitative study of STR DNA from ethanol and formalin fixed tissues Maryam Alqaydi, Reena Roy * The Pennsylvania State University, Forensic Science Program, Eberly College of Science, University Park, PA 16802, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 July 2015 Received in revised form 14 December 2015 Accepted 7 February 2016 Available online 23 February 2016

Complete and concordant autosomal short tandem repeat (STR) DNA profiles were obtained from 2.0 mg human tissue samples of various types after they were preserved for 24 weeks in 100% ethanol and amplified with the GlobalFiler1 and the PowerPlex1 Fusion Amplification Kits. When 4.0 mg of the same tissues were preserved for 12 weeks in 10% Neutral Buffered Formalin (NBF) they yielded partial profiles when amplified with the same kits. However, these NBF preserved tissues yielded complete autosomal profiles when amplified with the AmpFlSTR1 MiniFilerTM Amplification Kit. Six tissue specimens from the male donor were also amplified with the PowerPlex1 Y-23 System. Y-STR profiles were successfully generated from 2.0 mg tissue specimens when preserved for 12 weeks in 100% Ethanol. Only partial profiles were obtained when the fixation time was increased to 24 weeks. Only partial Y-STR profiles were also obtained from 4.0 mg tissue specimen from the same donor when preserved in 10% NBF. In an attempt to optimize the method, the preserved samples that yielded partial profiles were homogenized using the BioMasher III disposable homogenizer and BioMasher III homogenizer and filter. These homogenized tissues did not yield significantly better or more complete profiles when using the GlobalFiler1, AmpFlSTR1 MiniFilerTM Amplification Kits, the PowerPlex1 Fusion System or the PowerPlex1 Y23 System. A total number of 240 tissue samples were analyzed in this project. The amplification of the tissues preserved in 10% NBF with kits such as AmpFlSTR1 MiniFilerTM and GlobalFiler1 Amplification Kits that contain mini STR primers can be beneficial in forensic testing. The results of the study indicate that in cases such as when a victim or a suspect is missing, the profiles obtained from minute amounts of chemically fixed tissues can be used as reference samples and compared to evidence found at the crime scene. ß 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic science Chemical fixation Quantifiler1 Trio DNA Quantification Kit GlobalFiler1 PowerPlex1 Fusion and PowerPlex1 Y23 Systems AmpFlSTR1 MiniFilerTM

1. Introduction Identification of individuals from body fluids found at crime scenes has been accomplished using molecular biology techniques such as STR analysis [1–5]. Additionally, such techniques are used in forensic pathology for identification and investigation of causes and manner of death [6]. However, postmortem specimen decomposition results in the breakdown of deoxyribonucleic Acid (DNA), ribonucleic Acid (RNA) and proteins, which may cause difficulties in using these specimens in routine forensic testing [7]. Additionally, mitochondrial and nuclear DNA in fixed tissues can undergo

* Corresponding author. Tel.: +1 814 867 2054; fax: +1 814 863 8372. E-mail addresses: [email protected] (M. Alqaydi), [email protected] (R. Roy). http://dx.doi.org/10.1016/j.forsciint.2016.02.011 0379-0738/ß 2016 Elsevier Ireland Ltd. All rights reserved.

degradation due to the chemical reactions caused by fixatives [8]. The main objective of using fixatives is to prevent autolysis events and to preserve the physical feature of tissues; therefore, allowing disease diagnosis and cause of death investigations [9]. The physical and chemical mechanisms of fixatives fall into wide range of categories, including crosslinkers, coagulants, additives, dehydrants, and combinations of these categories [10,11]. This study focuses on two types of chemical fixatives: 10% NBF which is a cross-linker and 100% ethanol (a coagulant). NBF is one of the most commonly used fixatives in pathology laboratories because it fixes and preserves broad range of tissue types and tissue components [12]. It is widely used due to its high reactivity, ease of preparation, and lower cost [13]. However, studies have shown that formaldehyde causes DNA degradation due to the gradual formation of formic acid from formaldehyde, resulting in small fragments of DNA that are not suitable for

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molecular biology and forensic testing [14,15]. Exposure of specimens to formaldehyde can cause protein-DNA crosslinks, protein modifications and interstrand DNA crosslinks [16]. It has been reported that there are at least four types of interactions of DNA with formaldehyde, which is the active component in formalin [12]. First, the addition of formaldehyde to nucleic acid bases forms hydroxymethyl (methylol) groups (–CH2 OH). This type of damage to nucleic acids is most common during fixation, and it can be reversed by applying a heating step in a buffered solution [17]. Second, nucleophilic attack of N-methylol, the primary form of formaldehyde in solution, on amino bases forming methylene bridge between the two adjacent amino groups. Third, apurinic and apyrimidinic sites can accumulate after formaldehyde exposure due to the hydrolysis of N-glycosylic bonds, which results in creating free pyrimidine and purine sites. Fourth, short chains of polydeoxyribose with pyrimidines attached to them can form via the slow hydrolysis of phosphodiester bonds induced by formaldehyde [12]. Ethanol or alcohol-based fixatives preserve tissues through coagulation rather than cross-linking [10]. When tissues are fixed in alcohol-based fixatives, the hydrophobic bonds are destabilized by the removal and replacement of free water in cells. Moreover, hydrophilic areas of the proteins are destabilized because of water removal. These chemical effects cause changes to the water solubility properties of proteins and their tertiary structure, which are mostly irreversible [10,18]. Previous studies reported that the alcohol-based fixatives preserve higher molecular weight DNA (>200 bp) and RNA, making these samples more suitable for Short Tandem Repeat (STR) typing in forensic applications than formalin-fixed tissues [19–21]. In contrast, buffered formalin preserves the histological structures of tissues better, making it more applicable for use in histological examinations [8]. Disrupting cells is an important step when extracting DNA and RNA. Research with the BioMasher disposable homogenizer showed that it is efficient in RNA extraction from various types of mouse and rat tissues. The BioMasher device is an easy to use device, available as sterilized tube, comes with a pestle and can eliminate cross-contamination between samples [22]. The objectives of this study were the following: the first was to compare the quantity and quality of DNA extracted from minute amounts of different types of tissues when preserved in 100% ethanol and 10% NBF over defined periods of time. A second objective was to amplify minute amounts of these preserved tissue samples using various recently available STR Amplification Kits. Two of these autosomal Amplification Kits and one Y-STR kit were evaluated for their efficacy in generating STR profiles from minute amounts of tissues which have been preserved in chemicals for several weeks. The goal of this research was to evaluate the STR profiles and compare the results with another kit introduced in the forensic community several years ago for use with degraded samples. The results from all amplification were compared for concordance within and between various types of tissue samples obtained from the same donor. The third objective included generating profiles from various types of tissues preserved in the two chemical fixatives to determine if particular specimens yield better quality STR profiles when compared to other types. Finally, the fourth objective was to determine the efficacy of the mechanical homogenization using the BioMasher III disposable homogenizer in increasing profile recovery from tissues preserved in the two fixatives mentioned above. 2. Materials and methods Blood and tissue samples were obtained from three-deceased individuals; two females and one male. A total of 16 tissue

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specimens from these three donors were used in this study. Five tissue types, namely small bowel, kidney, colon, liver and muscle were obtained from each individual. In addition, one gall bladder specimen was also obtained from the male donor. A total number of 240 tissue samples were used in this research, which includes 16 pristine tissue specimen as well as tissues fixed in the two fixatives indicated in Table 1. These tissues were kept frozen until needed for analysis. Three blood samples were collected in Vacutainer1 tubes containing EDTA, a chelating agent that inhibits DNA degradation. As required by the policy of The Pennsylvania State University, all samples including the blood and tissues were obtained and processed following the guidelines approved by the Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC). Every sample used in this study was anonymized. 2.1. Sample preparation Each pristine blood and tissue sample was processed initially without adding any fixatives to it in order to establish concordance between and within samples obtained from each donor. DNA was extracted from 3 mL of blood from each donor and DNA profiles were generated (used as reference samples). 2.0 mg of each type of tissue was used for obtaining DNA profiles from the pristine tissue samples prior to adding any fixatives. Once these profiles were generated subsequent experiments with fixatives included 2.0 mg or 4.0 mg of each specimen. Initially, experiments were performed to compare the yield of DNA from tissues incubated in both fixatives for 1 to 13 days. Liver tissue from a female deceased individual was cut and 2.0 mg of each sample was immersed in each of the two fixatives: 100% ethanol (Electron Microscopy Sciences, Hatfield, PA) and 10% NBF (Globe Scientific INC, Paramus, NJ, USA). The total number of tissues used at this stage was 20. Specimens were preserved in test tubes at room temperature. The test tubes were corked and sealed with Parafilm1 to avoid evaporation. Each of these liver tissues was preserved in each fixative in increments of 1 day. Thus, each liver sample (total of three) was extracted and quantified the day before each was immersed in one of the fixatives (day 0) and then every day during the duration of preservation. This process required three liver samples, preserved from day 0 to day 13 in two chemical fixatives. Thus, a total of 84 samples were extracted and quantified at this stage of the study. After this stage of the study was completed and data was analyzed, each of the 16 tissue specimens from the three donors was cut, weighed and immersed in both of the two fixatives. Each 2.0 mg specimen was preserved in 100% ethanol for the length of time and incubation period described in Table 1. The duration of preservation in 100% ethanol ranged from 4 weeks to 24 weeks. Experiments using 10% NBF were performed with 4.0 mg of each tissue. Each of the 4.0 mg specimens was immersed in 10% NBF for a period of 4 weeks, 8 weeks, and 12 weeks. At the end of each of these periods, samples were taken out of each of the fixatives and DNA was extracted from them. A total of 16 tissue specimens from the three individuals were fixed in both chemical fixatives for each of the duration time mentioned in the table. 2.2. Tissue homogenization The BioMasher III disposable homogenizer (DiagnoCine LLC, Hackensack, NJ) is composed of a filter tube, a collection tube and a pestle [23]. The filter tube contains a filter paper with 80–145 mm pore size. When tissue is added to the BioMasher III along with lysis buffer, it is homogenized through hard textures on the pestle and the filter tube with grinding motions by hand. The accumulated liquid remains in the collection tube while the filter tube and the pestle are discarded.

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With BioMasherIII (16 samples) Without BioMasherIII (16 samples) With BioMasherIII (16 samples) Without BioMasherIII (16 samples) With BioMasherIII (16 samples) 4 mg 10% Neutral Buffered Formalin

Without BioMasherIII (16 samples)

With BioMasherIII +Filter (16 samples)

8 Weeks

(16 samples) (16 samples) 4 Weeks

Without BioMasher III 2 mg 100% Ethanol

Length of Preservation Weight of Each tissue Fixative

Table 1 List of the preserved tissues in the two fixatives for various durations of fixation.

Without BioMasher III

With BioMasherIII +Filter (16 samples)

(16 samples) 12 Weeks

Without BioMasher III

With BioMasherIII +Filter (16 samples)

Without BioMasherIII (16 samples)

24 Weeks

With BioMasherIII (16 samples)

20

At first, all tissues preserved in 100% ethanol for up to 24 weeks, and in 10% NBF for up to 12 weeks were processed without homogenization. As shown in Table 1, only tissues that did not yield complete profiles were subjected to homogenization using BioMasher III. This device was used to completely homogenize the fixed tissues preserved in 100% ethanol for 24 weeks. In addition, samples preserved in 10% NBF for 4 weeks, 8 weeks, and 12 weeks were also homogenized with BioMasher III. Thus a total of 16 tissues preserved at each preservation period were homogenized using the homogenization procedure. After these experiments were completed, a modified protocol was created in an attempt to increase the profile recovery from tissues that were homogenized with BioMasher III. Instead of discarding the filter paper in the filter tube, each filter paper was added to the collection tube. This set of samples was referred to as (BioMasherIII + Filter) in this study. The modified protocol was applied to the 16 tissues at the end of each preservation period with 10% NBF; namely the tissues preserved for 4, 8, and 12 weeks. 2.3. DNA extraction and Quantification Extraction and purification of all blood and tissue samples were performed using the Qiagen EZ1 DNA Investigator Kit (Qiagen, Hilden, Germany) [24]. Quantification of the three liver specimens that were preserved from 1 to 13 days in each of the two preservatives was performed using the Quantifiler1 Human DNA Quantification Kit (Life Technologies Corporation, Carlsbad, CA). The amount of DNA in all other tissue samples preserved in both fixatives was determined using Quantifiler1 Trio DNA Quantification Kit from Life Technologies [25]. 2.4. Amplification Kits Blood and tissue samples were amplified using GlobalFiler1 PCR Amplification Kit (Life Technologies Corporation, Carlsbad, CA) and PowerPlex1 Fusion System (Promega Corporation, Madison, WI) prior to subjecting them to fixatives. GlobalFiler1 PCR Amplification Kit is a 6-dye, 24-locus Amplification Kit whereas the PowerPlex1 Fusion System is a 5-dye, 24-locus Amplification Kit. The autosomal profiles generated from these amplifications kits were used as reference profiles for comparison with the results obtained from fixed tissues. Each tissue sample preserved in 100% ethanol was amplified with two recently available autosomal STR kits; GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System. All tissues preserved in 10% NBF were amplified with three autosomal Amplification Kits; GlobalFiler1 PCR Amplification Kit, PowerPlex1 Fusion System and AmpFlSTR1 MiniFilerTM PCR Amplification Kit (Life Technologies Corporation, Carlsbad, CA). In addition, the six tissue samples obtained from the male donor and preserved in each of the two fixatives were amplified using PowerPlex1 Y23 System (Promega Corporation, Madison, WI) for generation of Y-STR data. This kit is a 5-dye, 23-locus multiplex system for detection of male specific loci. 2.5. PCR amplification Except as described below, manufacturers’ recommendations were followed for amplification procedures [26–29]. An optimum amount of quantified DNA (1 ng) was used for amplification with all of the Amplification Kits mentioned above. All of the Amplification Kits used in this research recommend a reaction volume of 25.0 mL. Research conducted earlier with PowerPlex1 Fusion System indicated that reduced reaction volumes could be used for amplification [30]. At first experiments with the other kits using both the recommended volume and a reduced reaction

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volume were performed. Once it was established that the recommended volume and the reduced reaction volumes yielded concordant data, all amplification was carried out in a reduced reaction volume of 12.5 mL. This was applicable for all of the Amplification Kits. Autosomal and Y-STR Amplification was performed on a GeneAmp1 9700 Thermal Cycler (PE Applied Biosystems1, Foster City, CA). The manufacturer’s recommended thermal cycling conditions were followed for all of the Amplification Kits [26–29]. 2.6. Capillary electrophoresis Amplification products were analyzed using capillary electrophoresis injection on the Applied Biosystems 3130xl Genetic Analyzer. Analyses of the generated autosomal and Y-STR profiles were performed using GeneMarker1 HID Software Version 2.7 from SoftGenetics1 (State College, PA). Statistical analysis was conducted using the R Studio Software [30]. One-way ANOVA (Analysis of Variable) test was used to compare between mean values of different variables in this study with significance level of 0.05. 3. Results and discussion Pristine blood and the 16 tissue samples from the three deceased individuals were extracted and amplified with GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System. Complete and concordant autosomal STR profiles were generated from all of the blood and tissue samples which had not been subjected to any of the fixatives. The results were determined to be concordant between and within samples obtained from each of the three donors (data not shown). The six tissue samples from the male donor and his blood were also typed with PowerPlex1 Y23 System and results were concordant between all samples (data not shown). The autosomal and Y-STR profiles generated from the reference samples as well as the profiles generated from the tissues not subjected to chemical fixation were considered reference samples for analyzing the data from the fixed tissue samples. The physical and chemical mechanisms of how fixatives can be categorized fall into a wide range. The two fixatives chosen for this study were either alcohol-based (100% ethanol) which denatures proteins or aldehydes (10% NBF) which crosslinks proteins. 3.1. Quantity and quality of DNA from minute amounts of fixed tissues The first objective of this current research was to compare the quantity and the quality of DNA extracted from minute amounts of different types of tissues when preserved in 100% ethanol and 10% NBF. Use of liver tissues from each of the three donors preserved in the two fixatives in increments of one day, was necessary to establish when degradation actually started in each fixatives. This experiment was conducted to determine how much DNA could be recovered from each of the 2.0 mg of the three liver tissues obtained from three different donors, and submerged in the fixatives, over a period of 13 days. Thus, each sample of liver was tested on day 0 before each specimen was submerged in either ethanol or 10% NBF. Each fixed sample was then extracted and quantified every day during the duration of preservation. A total of 84 samples were used in this experiment and data was compared for the degradation pattern. Each sample was extracted and quantified each day after incubation using the Quantifiler1 Human DNA Quantification Kit. As shown in Fig. 1, the average concentration of DNA decreased in tissues preserved in both fixatives after the first day of preservation, and kept decreasing as the preservation time was increased. After 13 days of preservation, the average concentration

Fig. 1. Average concentration of DNA in tissues preserved in 100% ethanol and 10% NBF for 13 days.

of DNA in tissues preserved in 100% ethanol was above 60 ng/mL whereas the average concentration of DNA was close to 0.0 ng/mL when tissues were preserved in 10% NBF. Initial experiments with all 2.0 mg tissue samples preserved in 10% NBF yielded no STR profiles. Following this experiment, the amount of tissues preserved in 10% NBF was increased from 2.0 mg to 4.0 mg for all subsequent experiments. Tissues listed in Table 1 and fixed in these two chemical fixatives were extracted after defined periods of time (described as weeks). The extracted DNA was quantified using the Quantifiler1 Trio DNA Quantification Kit which provides quantitative assessment through targeting small autosomal targets (75 to 80 bp), large autosomal targets (>200 bp), and regions on the Y-chromosome (75 to 80 bp). The kit reports degradation indices (DI) for each sample. DI is the concentration of the small autosomal target divided by the concentration of the large autosomal target. All data obtained from Quantifiler Trio DNA Quantification Kit of tissues preserved in 100% ethanol and 10% NBF are summarized in Appendices A and B, respectively. The appendix includes quantities of large autosomal, small autosomal, and Y chromosome targets as well as IPC CT, and IPC CT flags. In addition, percentages of profile recoveries from all kits are listed. All values in Appendices A and B demonstrate average values obtained from all three donors except for data that was obtained from the male donor only. These values include data obtained from gall bladder tissue, quantities of Y target, and percentage of profile recovery from PowerPlex1 Y23 System. In the Quantifiler Trio DNA Quantification Kit, the IPC CT is set at 2 CT units. As a result, if the IPC CT value was more than 2 CT, the IPC CT flag will be displayed. IPC CT flags are indications of the amplification efficiency, which can include system failure or the presence of PCR inhibitors that interfere with the PCR amplification. In Appendices A and B, a value of 1 indicates that IPC CT flag was displayed and a zero value indicates that IPC CT was not flagged for that sample. All tissues preserved in 100% ethanol up to 24 weeks showed no IPC CT flags. Similarly, IPC CT flags were not shown in 10% NBF tissues expect for one kidney tissue of one female donor that was preserved for 4 weeks and was extracted without using the Biomasher III. The other two kidney tissues did not show any IPC CT flags, which indicates that the IPC CT flag was possibly triggered due to system failure. Table 2 demonstrates the average DIs of tissues fixed in 100% ethanol and 10% NBF and samples that were not subjected to homogenization. The average DIs of tissues preserved in 100% ethanol up to 24 weeks were close to the value of 1, indicating that DNA is not significantly degraded in these tissues. In contrast,

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Table 2 Average Degradation Indices (S.D.) of tissues preserved in 100% ethanol up to 24 weeks and specimens preserved in 10% NBF up to 12 weeks. Fixative

Duration of fixation 4 Weeks

8 Weeks

12 Weeks

24 Weeks

Average Standard deviation

100% Ethanol

1.8 (16 samples) 3.5

0.94 (16 samples) 0.5

0.99 (16 samples) 0.4

1.3 (16 samples) 1

Average Standard deviation

10% Neutral Buffered Formalin

10 (16 samples) 6

403 (16 samples) 582

408 (16 samples) 986

–

tissues preserved in 10% NBF had average DI values ranging from 10 after four weeks of preservation to 408 after 12 weeks of preservation. As indicated in the same table, these values are significantly higher in 10% NBF preserved specimen than DIs in specimen preserved in 100% ethanol (p = 0.003). Fig. 1 and Table 2 demonstrate that tissues fixed in 10% NBF yielded lower quantity of DNA and higher DIs than samples fixed in 100% ethanol. Since DIs of tissues preserved in 10% NBF for 8 and 12 weeks yielded similar values, tissues were not preserved in 10% NBF beyond the 12 week period. 3.2. Quality of autosomal and Y-STR DNA profiles obtained from preserved tissues The second objective of this research project included evaluating the quality of DNA profiles obtained from all of the tissues fixed in each of these fixatives and amplified with various autosomal and Y-STR Amplification Kits. Complete profiles were deemed present when all alleles were detected and were found to be consistent with the profiles generated as reference profiles. Percentage of alleles recovered and Peak Height Ratio (PHR) in heterozygous loci were used to compare the results of each kit. When complete profiles were not obtained, percentage of alleles recovered was used to compare quality of partial profiles. Tissues fixed in 100% Ethanol for up to 24 weeks were amplified with two kits; GlobalFiler1 PCR Amplification Kit, a six dye system and the PowerPlex1 Fusion System, a five dye system. GlobalFiler1 PCR Amplification Kit identifies 21 autosomal, one Y locus, amelogenin locus and a Y-InDel. PowerPlex1 Fusion System identifies 23 autosomal loci as well as the amelogenin sex determining locus. Fig. 2 compares the percent of alleles recovered from all of the 16 specimens preserved in 100% ethanol and amplified with both autosomal STR kits. As noted, the average profile recovery in the two kits and during the preservation periods is above 95%. Fig. 3 compares the peak height ratios of heterozygous loci common in both of the autosomal STR Amplification Kits mentioned above. The boxplot shows the range of PHRs of tissues preserved in 100% ethanol from 4 weeks to 24 weeks. As illustrated in this figure most PHRs using both kits are at or above 60%. In addition, both kits showed similar range of PHRs as well as similar outlier trends. Figs. 2 and 3 demonstrate that GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System performed similarly when tissues were fixed in 100% ethanol for up to 24 weeks. Concordance between and within tissues was determined by comparing STR profiles generated from them. Fig. 4a shows a complete autosomal profile generated from 2.0 mg kidney sample of a deceased female when preserved for 24 weeks in 100% ethanol and amplified with GlobalFiler1 PCR Amplification Kit. Fig. 4b shows STR profile from 2.0 mg colon tissue of another deceased female, fixed in 100% ethanol for 24 weeks and amplified with PowerPlex1 Fusion System. Tissues preserved in 10% NBF were amplified with three kits; GlobalFiler1 PCR Amplification Kit, PowerPlex1 Fusion System,

and AmpFlSTR1 MiniFilerTM Amplification kit,. While the GlobalFiler1 and the PowerPlex1 Fusion System both detect 24 loci, the AmpFlSTR1 MiniFilerTM Kit is a five dye system that detects eight autosomal loci and the amelogenin gender locus. Fig. 5 compares the percent of alleles recovered from the three Amplification Kits when tissues were preserved in 10% NBF from 4 weeks to 12 weeks. The boxplot presents data from shared loci in all three kits. The AmpFlSTR1 MiniFilerTM Amplification Kit yielded the highest percent of alleles recovered which was at or above 80%. The profile recovery percentages from GlobalFiler1 PCR Amplification Kit were higher than PowerPlex1 Fusion System after 4 weeks of preservation in 10% NBF. However, tissues preserved in 10% NBF for 8 weeks and 12 weeks and amplified with PowerPlex1 Fusion System yielded higher values of profile recovery than samples amplified with GlobalFiler1 PCR Amplification Kit. The AmpFlSTR1 MiniFilerTM Amplification Kit yielded the highest percentage of alleles recovered from tissues preserved in 10% NBF. In order to determine if the small amplicon sizes or the formulation of the kit resulted in increased profile recovery, percentages of alleles recovered from loci that are at or below 283 bp in the GlobalFiler1 PCR Amplification Kit and the PowerPlex1 Fusion System were compared to the profiles recovery from the AmpFlSTR1 MiniFilerTM Amplification Kit (Fig. 6). A total of 16 loci fall completely below 283 bp in the GlobalFiler1 PCR Amplification Kit, which are D3S1358, vWA, D16S539, Y indel, Amelogenin, D8S1179, D21S11, D2S441, D19S433, TH01, D22S1045, D5S818, D13S317, D10S1248, D1S1656, and D12S391. The total number of loci that fall below 283 bp in the PowerPlex1 Fusion System is 13, which include Amelogenin, D3S1358, D1S1656, D2S441, D16S539, D18S51, TH01, vWA, D21S11, D8S1179, D12S391, D19S433, and DYS391. Fig. 6 compares the percent of alleles recovered from tissues preserved in 10% NBF for up to 12 weeks when only loci completely

Fig. 2. Percent of alleles recovered from specimens preserved in 100% ethanol for 24 weeks and amplified with GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System.

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Fig. 3. Boxplot comparing the peak height ratios of heterozygous loci common in GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System, when tissues were preserved in 100% ethanol from 4 weeks to 24 weeks.

below 283 bp were included. Primers in AmpFlSTR1 MiniFilerTM Amplification Kit yielded the highest range of profile recovery. This was followed by GlobalFiler1 PCR Amplification Kit and finally the PowerPlex1 Fusion System. As shown in Figs. 5 and 6, the range of profiles recovered increased by approximately two folds when only MiniSTR loci were analyzed in the GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System. Although both GlobalFiler1 and MiniFilerTM Amplification Kits contain

robust master mixes, GlobalFiler1 uses a six dye system and is a very complex kit with many more primers than the MiniFilerTM Amplification Kit. This level of complexity increases competition for other components in the Amplification Kit, especially when using chemical fixatives used in this research. It is postulated that this could result in the lower level of short amplicon recovery from GlobalFiler1 Amplification Kit and PowerPlex1 Fusion System.

Fig. 4. (a) Complete autosomal STR profile generated from 2.0 mg kidney sample of a deceased female when preserved for 24 weeks in 100% ethanol, and amplified with GlobalFiler1 PCR Amplification Kit. (b) Complete autosomal STR profile generated from 2.0 mg colon tissue of a deceased female, preserved in 100% ethanol for 24 weeks and amplified with PowerPlex1 Fusion System.

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Fig. 5. Boxplot comparing percent of alleles recovered from tissues preserved in 10% NBF from 4 weeks to 12 weeks, when amplified with GlobalFiler1 PCR Amplification Kit, AmpFlSTR1 MiniFilerTM Amplification Kit and PowerPlex1 Fusion System. The boxplot presents data from shared loci in all three kits.

Fig. 7 shows an example of a profile using the AmpFlSTR1 MiniFilerTM Kit when 4.0 mg colon tissue from another female was fixed in 10% NBF for 12 weeks. Although the larger loci did not amplify as well as the smaller ones, all of the alleles were detected and were found to be concordant with the reference profiles of the donors. Y-STR analysis was performed on the six tissue specimens obtained from the male donor using the PowerPlex1 Y23 System. Fig. 8 compares percent of alleles recovered from tissues fixed in 10% NBF and 100% ethanol for 12 weeks. The boxplot demonstrates that complete Y-STR profiles were obtained from all tissues preserved in 100% ethanol up to 12 weeks. As indicated, tissues fixed in 100% ethanol yielded complete profiles, thus the recovery with this method was 100%. In contrast, only partial Y-STR profiles were obtained from 10% NBF-fixed tissues during the entire duration of fixation. In addition, only partial Y-STR profiles were generated from tissues preserved in 100% ethanol for 24 weeks with an average of 63.5  38% of alleles recovered. The predictability of the DI on percentage of STR allele recovery was evaluated on both formalin and ethanol fixed tissues. The DI values do not significantly predict the percentage of profile recovery from tissues preserved in ethanol (p = 0.225). Similarly, the DI of formalin-fixed tissues was not significantly correlated with the percent of recovered alleles in all three Amplification Kits (p = 0.0219). Regardless of the DI values in formalin-fixed tissues, low percentage of profile recovery was mostly obtained.

Fig. 6. Boxplot comparing percent of alleles recovered from tissues preserved in 10% NBF from 4 weeks to 12 weeks, when amplified with GlobalFiler1 PCR Amplification Kit, AmpFlSTR1 MiniFilerTM Amplification Kit and PowerPlex1 Fusion System. The boxplot presents data of loci below 283 bp in all three kits.

Similar to the correlation between DI and percentage of profile recovery in STR, these two variables were not significantly correlated in Y-STRs profile from tissues preserved in ethanol and formalin (p = 0.1842 and p = 0.6145, respectively). The results discussed above include the effect of increasing duration of fixation on the quality of the STR profiles generated from these chemically fixed tissue specimens. As demonstrated in Figs. 2 and 3, increasing the duration of fixation did not affect the quality of autosomal DNA profiles generated from tissues preserved in 100% ethanol for up to 24 weeks. The recovery of Y-STR profiles was not affected when the preservation duration was increased to 12 weeks but it decreased when preserved for 24 weeks in 100% ethanol. Increasing duration of preservation decreased the percent of autosomal and Y-STR profile recovery when tissues were fixed in 10% NBF (Figs. 5–8). 3.3. Comparison between tissue types and the quality of DNA profiles To determine the result of the third objective in this research which included generating profiles from minute amounts of various types of tissues from three deceased donors, the generated profiles from all samples preserved over a period of time in both fixatives were compared to see if one type of fixed tissue yielded better quality profiles than others. All tissues were subjected to the same conditions as described in Table 1. Fig. 9 compares PHRs in heterozygous loci from different types of tissues preserved in 100% ethanol, amplified with GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System. The range of PHRs is similar among different tissue types, except for the gallbladder when preserved for 4 weeks. This phenomenon was not observed in any other tissue type and was not consistent with results generated from the other tissues of the same male donor. PHRs were at or above 60% in most tissue types when amplified with both of the autosomal Amplification Kits. Similarly, tissue types did not have any effect on the percent of profile recovery from specimens preserved in 10% NBF during the entire duration of the fixation in this research. This was true when each fixed specimen was amplified with GlobalFiler1 PCR Amplification Kit, PowerPlex1 Fusion System, and with AmpFlSTR1 MiniFilerTM Amplification Kits (data not shown). In addition, the type of specimens did not affect the quality of Y-STR profiles generated from the six types of specimens obtained from the male donor, preserved in 100% ethanol and 10% NBF during the entire preservation period when amplified with PowerPlex1 Y23 System (data not shown).

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Fig. 7. Complete autosomal STR profile generated from 4.0 mg colon tissue of a deceased female, fixed in 10% NBF for 12 weeks and amplified with AmpFlSTR1 MiniFilerTM Amplification Kit.

3.4. Efficacy of the homogenization process The last objective of this study was undertaken to test the efficacy of homogenization using the BioMasher III device in increasing profile recovery from the fixed samples that yielded partial profiles. When the recommended protocol was followed, tissue residues were observed on the filter paper in the BioMasher III device. Therefore, a modification of the BioMasher III protocol was created through depositing the filter paper in the collection tube instead of discarding it. Therefore, a modification of the recommended protocol included the filter paper in the homogenization process in an effort to increase profile recovery.

As noted earlier, homogenization procedure was used only for tissues that at first did not yield complete profiles when preserved with both fixatives. Fig. 10 compares the percent of alleles recovered from tissues preserved in 10% NBF for up to 12 weeks and amplified with three autosomal Amplification Kits; GlobalFiler1 and AmpFlSTR1 MiniFilerTM Amplification Kits and the PowerPlex1 Fusion System. The boxplot shows results from tissues that were not homogenized (Without Homogenization), tissues homogenized with BioMasher III (BioMasherIII), and tissues homogenized with BioMasher III with modification which included addition of filter (BioMasherIII + Filter). Within specimens amplified with AmpFlSTR1 MiniFilerTM Kit, the (BioMasherIII + Filter) samples yielded the

Fig. 8. Boxplot comparing percent of alleles recovered from Y-STR profiles generated from six male tissue samples fixed in 10% NBF and 100% ethanol for 12 weeks, and amplified with PowerPlex1 Y23 System.

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M. Alqaydi, R. Roy / Forensic Science International 262 (2016) 18–29

Fig. 9. Boxplot comparing PHRs in heterozygous loci from different types of tissues preserved in 100% ethanol, and amplified with GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System.

Fig. 10. Boxplot comparing percent of alleles recovered from tissues preserved in 10% NBF for up to 12 weeks and amplified with GlobalFiler1 PCR Amplification Kit, AmpFlSTR1 MiniFilerTM Amplification Kit and PowerPlex1 Fusion System.

highest profile recovery and the smallest range of values during the entire duration of preservation. Fig. 10 also demonstrates that consistent trends were not observed when homogenizing the tissues with BioMasher III and BioMasherIII + Filter. Overall, using homogenization procedures with the BioMasher III and BioMasherIII + Filter and amplifying the extracted DNA with the all three autosomal Amplification Kits did not significantly increase profile recovery from tissues fixed in 10% NBF up to 12 weeks (p = 0.273). Similarly, tissues fixed in 10% NBF for up to 12 weeks and homogenized with BioMasher III and BioMasherIII + Filter did not significantly increase profile recovery when amplified with PowerPlex1 Y23 System (p = 0.44). Partial Y-STR profiles were generated from specimens preserved in 100% ethanol for 24 weeks. Homogenizing these tissues with BioMasher III did not significantly increase the percentage of

alleles recovered when compared to profile recovery from tissues that were not homogenized (p = 0.49). 4. Conclusion Preserving minute amounts of tissues in 10% NBF significantly increased DNA degradation, as indicated by the DI values when compared with specimens in 100% ethanol. Autosomal STR profiles were successfully generated from samples fixed in 100% ethanol for at least 24 weeks. GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System yielded concordant results and the quality of DNA profiles from minute amounts of tissues preserved in 100% ethanol were similar. The six tissue samples obtained from the male donor and preserved in 100% ethanol and amplified with PowerPlex1 Y23

M. Alqaydi, R. Roy / Forensic Science International 262 (2016) 18–29

System yielded complete and concordant Y-STR profiles for tissue preserved for up to 12 weeks. Partial Y-STR profiles were generated from tissues preserved in the same fixative for 24 weeks. Partial autosomal STR profiles were generated from samples preserved in 10% NBF for four to 12 weeks. Tissues immersed in 10% NBF and amplified with AmpFlSTR1 MiniFilerTM PCR Amplification Kit generated the highest percentage of profile recovery autosomal STR profiles. However, it should be noted that AmpFlSTR1 MiniFilerTM PCR Amplification Kit detects only nine loci compared to GlobalFiler1 PCR Amplification Kit and PowerPlex1 Fusion System which amplifies 23 loci. For forensic case work and analysis of evidence samples, each laboratory should consider whether from a biostatistical point of view a partial megaplex profile would yield more discrimination power than a complete profile obtained with MinifilerTM Amplification Kit. YSTR analysis of these tissues showed partial Y-STR profiles through the entire duration of the study. Duration of fixation did not impact profile recovery and PHRs of specimens preserved in 100% ethanol. However, increasing the fixation time in 10% NBF significantly increased the percentage of allelic dropout in these specimens. In addition, autosomal and YSTR profiles obtained from different types of tissues fixed in the two chemicals were similar in quality when amplified with the three autosomal and one Y-STR Amplification Kits. Homogenizing preserved tissues with BioMasher III disposable homogenizer following the recommended protocol and with modification to the protocol (BioMasherIII + Filter) did not significantly impact the percentage of alleles recovered. Experiments conducted in this

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research indicate that minute amounts of tissues preserved in 100% ethanol for at least 24 weeks are more suitable for forensic STR DNA analysis compared to those preserved in 10% NBF. Funding This study was supported by the Forensic Science Program at The Pennsylvania State University, University Park, PA, USA. Ethical approval The authors declare that they have no conflict of interest or financial gain from this research. All procedures performed in studies involving human participants were in accordance with the collection procedure approved by the Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC). All samples were coded and anonymized. Acknowledgements The authors are grateful for receiving helpful suggestions and comments from Bill Gartside, DNA Technical Leader, San Bernardino Sheriff’s Department, Scientific Investigation Division and Dr. Teresa Snyder-Leiby, Product Manager at SoftGenetics LLC. The authors are also grateful to Mark Guilliano and Mary Jones Dukes (Qiagen), and Dawn Waltman (Themo Fisher Scientific, Inc.) for their many comments and assistance with this research project.

Appendix A. Summary of data obtained from Quantifiler Trio DNA Quantification Kit and percentages of profile recoveries of tissues preserved in 100% Ethanol. Fixation Period (weeks)

Use of BioMasher

Tissue Type

LA*

SA**

Y***

DI

IPC Ct

IPC Ct Flag?

Percent of profile recovery GlobalFiler

PowerPlex Fusion

PowerPlex Y-23

4

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

33.6 14.7 39.2 16.8 10.6 9.9

29.1 15.6 28.1 19.5 13.5 17.8

10.5 10.3 16.8 6.7 6.9 21.7

0.9 1.0 0.7 1.3 0.6 1.8

27.8 27.2 28.1 27.4 26.6 26.8

0.0 0.0 0.0 0.0 0.0 0.0

99.2 99.6 99.2 98.8 99.6 100.0

100.0 100.0 100.0 99.6 100.0 86.7

100.0 100.0 100.0 100.0 100.0 100.0

8

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

36.3 17.4 41.5 15.0 17.4 11.7

25.6 18.2 26.4 17.9 9.5 25.2

12.8 9.3 20.0 12.7 4.2 27.4

0.7 1.2 0.6 1.3 0.5 2.2

27.8 27.7 28.0 27.5 27.6 26.7

0.0 0.0 0.0 0.0 0.0 0.0

88.6 99.1 100.0 98.9 100.0 100.0

94.8 96.9 100.0 83.3 100.0 95.6

100.0 100.0 100.0 100.0 100.0 100.0

12

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

27.4 17.8 40.0 16.3 17.4 9.1

20.0 18.5 31.6 20.1 10.5 17.9

9.2 21.3 51.4 11.4 7.8 20.3

0.7 1.2 0.8 1.3 0.6 2.0

27.4 27.7 28.2 26.7 27.9 26.5

0.0 0.0 0.0 0.0 0.0 0.0

100.0 100.0 100.0 100.0 100.0 100.0

100.0 100.0 100.0 100.0 100.0 97.8

100.0 100.0 100.0 100.0 100.0 100.0

24

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

13.4 15.9 30.4 9.5 18.1 9.7

16.2 18.0 23.6 13.9 11.5 20.1

5.7 31.5 33.9 27.2 17.3 21.4

1.7 1.1 0.8 2.3 0.6 2.1

27.7 27.3 28.0 27.4 28.0 27.0

0.0 0.0 0.0 0.0 0.0 0.0

99.2 100.0 100.0 99.2 100.0 100.0

100.0 100.0 100.0 100.0 100.0 100.0

43.0 100.0 17.0 30.0 100.0 91.0

24

With BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

7.8 10.0 28.5 5.3 10.2 5.7

11.3 13.0 23.8 7.1 6.2 18.6

5.4 8.9 11.9 9.5 7.3 19.3

4.7 1.3 0.8 3.4 0.6 3.3

27.5 27.8 28.2 27.1 27.1 26.3

0.0 0.0 0.0 0.0 0.0 0.0

98.7 100.0 100.0 99.6 100.0 100.0

97.8 100.0 100.0 97.6 100.0 100.0

39.0 100.0 100.0 87.0 100.0 39.0

* **

Average quantity of large autosomal target (ng/mL). Average quantity of small autosomal target (ng/mL). Average quantity of Y chromosome target (ng/mL).

***

M. Alqaydi, R. Roy / Forensic Science International 262 (2016) 18–29

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Appendix B. Summary of data obtained from Quantifiler Trio DNA Quantification Kit and percentages of profile recoveries of tissues preserved in 10% NBF (S) Quantity was undetermined or no profile was obtained.

Fixation Period (weeks)

Use of BioMasher

Tissue Type

LA

SA

Y

DI

IPC Ct

IPC Ct Flag?

Percent of profile recovery (%) GlobalFiler

PowerPlex Fusion

MiniFiler

PowerPlex Y23

4

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.02 0.13 0.08 0.08 0.00 0.03

0.31 1.30 0.67 0.68 0.01 0.57

0.86 2.01 0.88 0.27 0.00 0.76

17.99 10.38 7.27 8.64 5.06 19.53

26.60 25.50 26.66 26.44 26.46 26.78

0.00 0.33 0.00 0.00 0.00 0.00

61.79 72.97 64.39 81.64 43.54 68.18

28.89 43.70 41.48 41.48 11.07 37.78

35.67 31.33 36.67 36.67 26.67 38.00

4.00 22.00 35.00 30.00 – 17.00

8

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.00 0.00 0.00 0.00 0.00 0.00

0.23 0.42 0.03 0.47 0.00 0.58

0.11 0.64 0.04 0.35 0.00 0.57

482.32 314.23 351.28 671.70 64.02 459.55

26.87 25.73 26.65 26.32 26.56 26.61

0.00 0.00 0.00 0.00 0.00 0.00

41.67 46.61 35.24 46.53 10.98 52.27

22.22 23.70 20.00 22.96 2.11 24.44

36.30 36.30 35.56 36.30 19.26 37.78

17.00 13.00 13.00 4.00 – 9.00

12

Without BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.00 0.00 0.00 0.00 0.00 0.00

0.27 0.20 0.15 0.24 0.01 0.51

0.12 0.11 0.39 0.30 0.02 0.45

287.56 1353.03 307.36 206.21 173.17 728.83

26.17 26.10 25.78 25.66 25.58 26.03

0.00 0.00 0.00 0.00 0.00 0.00

39.87 46.26 39.58 45.38 24.43 43.18

24.44 17.78 19.26 21.48 8.15 22.22

34.81 34.81 36.30 33.33 31.11 37.78

4.00 4.00 9.00 13.00 – 9.00

4

With BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.14 0.14 0.06 0.06 0.00 0.03

1.34 1.22 0.25 0.54 0.01 1.28

0.69 1.90 0.00 0.75 0.01 1.12

9.32 7.95 6.54 9.22 4.30 41.84

26.59 26.09 26.58 26.42 26.44 26.31

0.00 0.00 0.00 0.00 0.00 0.00

82.05 76.13 60.55 85.58 45.00 47.73

42.22 40.00 25.56 43.70 12.59 28.89

36.30 36.30 31.11 36.67 35.56 35.56

17.00 30.00 4.00 22.00 – 13.00

8

With BioMasher

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.00 0.00 0.01 0.00 – 0.00

0.20 0.25 0.27 0.14 0.00 0.60

0.23 0.15 0.30 0.31 0.00 0.51

142.70 582.66 1439.96 321.00 – 31383.5

26.81 26.76 25.61 26.44 26.83 25.57

0.00 0.00 0.00 0.00 0.00 0.00

46.00 46.21 36.81 50.00 11.70 45.45

26.67 23.70 20.70 25.19 – 26.67

36.30 36.30 34.81 36.30 17.04 37.78

17.00 9.00 – 13.00 9.00 13.00

12

With BioMasher

Small Bowel Kidney Colon Liver Muscle

0.00 0.00 0.00 0.00 0.00

0.17 0.25 0.06 0.22 0.15

0.17 0.13 0.01 0.29 0.25

178.76 214.11 140.51 188.05 538.65

25.84 25.96 25.75 25.81 26.09

0.00 0.33 0.00 0.00 0.00

43.18 45.30 32.37 44.11 30.98

16.30 22.96 14.81 20.00 8.89

34.81 34.81 28.15 35.56 29.63

9.00 9.00 – 9.00 No Profile

4

With BioMasher + Filter

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.19 0.15 0.13 0.08 0.00 0.02

1.37 1.28 1.22 0.66 0.01 0.65

1.00 1.79 1.63 0.76 0.01 0.86

8.20 7.59 9.75 9.18 5.50 31.51

26.14 26.14 26.02 25.77 25.80 26.69

0.00 0.00 0.00 0.00 0.00 0.00

86.78 90.17 88.02 81.87 52.52 68.18

86.78 90.17 88.02 81.87 52.52 68.18

36.30 36.37 36.30 36.30 32.59 37.78

17.00 22.00 17.00 No Profile – 13.00

8

With BioMasher + Filter

Small Bowel Kidney Colon Liver Muscle Gall Bladder

0.00 0.01 0.00 0.01 0.00 0.00

0.43 0.49 0.14 0.30 0.00 0.41

0.09 0.35 0.02 0.10 0.00 0.46

904.95 164.46 115.22 70.56 12.87 289.53

25.83 26.21 26.31 25.26 26.07 23.48

0.00 0.00 0.00 0.00 0.00 0.00

42.24 42.92 40.47 44.46 17.40 45.45

35.57 42.92 40.47 44.46 12.25 45.45

36.30 36.30 36.30 36.30 33.33 37.78

– 9.00 – 13.00 – 4.00

12

With BioMasher + Filter

Small Bowel Kidney Colon Liver Muscle

0.01 0.01 0.01 0.00 0.00

0.21 0.54 0.60 0.22 0.01

0.13 0.13 0.17 0.06 0.00

64.29 94.35 128.43 249.58 25.22

26.18 25.63 25.00 26.41 26.45

0.00 0.00 0.00 0.00 0.00

45.16 40.11 42.70 46.63 18.62

45.16 40.11 42.70 46.63 18.62

34.81 35.56 35.56 36.44 29.63

13.00 13.00 9.00 – –

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