ORIGINAL ARTICLES
BIOPRESERVATION AND BIOBANKING Volume 11, Number 6, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2013.0030
Tissue Imprints: Assessing Their Potential for Routine Biobanking Collection Renata Greenspan,1 Amy O’Donnell,2 Jeff Meyer,2 Jennifer Kane,2 Kim Mamula,2 Sue Lubert,2 Brenda Deyarmin,2 Caroline Larson,2 Sean Rigby,2 Amber Greenawalt,2 Negin Vatanian,1 Richard Mural,2 Craig Shriver,1 and Stella Somiari2
Biomedical research depends on the availability of good quality biospecimens. Unfortunately, certain specimens are scarce due to disease rarity or size restrictions of surgical materials. To increase access to limited surgical specimens, Biobanks need to reassess and adjust their collection programs. We evaluated the feasibility of adapting ‘‘touch imprints’’ to gain access to limited surgical specimens as well as to maximize the use of ‘‘precious’’ specimens. We utilized 12 kidney samples for touch imprints on defined areas of microscope glass slides and FTA paper. DNA was isolated from glass slides on the day of preparation, Day 0, and from glass slide and FTA paper preparations after two weeks of storage at room temperature and - 80C. Yield and purity of DNA from reference kidney samples were compared to DNA from the touch imprints and the quality determined by realtime PCR using the amplification of Cyclophilin A (Cyc A) as an index. DNA quality for glass slides at Day 0 was not significantly different from DNA after two weeks at room temperature (glass at room temperature; p = 0.111 and 0.097, yield and purity, respectively) and after two weeks at - 80C (glass - 80C; p = 0.358 and 0.281, yield and purity, respectively). Glass slide DNA at room temperature and - 80C were not significantly different ( p = 0.795 and 0.146 for yield and purity, respectively). DNA from FTA paper at room temperature and from FTA paper at - 80C were significantly different from glass at room temperature and glass at - 80C ( p = 0.002, respectively). Threshold values for Cyc A were £ 28 for the reference DNA and £ 32 for DNA from glass and FTA paper. This study demonstrates that touch preparations on microscope glass slides and FTA paper can provide sufficient and good quality DNA suitable for PCR. Touch imprints could therefore be adopted by biobanks to collect and bank biological materials from limited surgical specimens. Introduction
T
he speed with which clinical research findings are translated from the bench to the bedside will determine how soon personalized medicine, the new paradigm for practicing medicine, can be completely embraced.1 The success of research translating to the clinic will partly depend on the availability of good quality, well-characterized biospecimens from consented donors. These specimens will allow scientists to study the development and progression of diseases at the molecular level. Dedicated biobanks established by academic institutions or private companies will start to play a greater role in the collection, annotation, storage, and distribution of biospecimens for research. Although the number of human tissue banks established by universities and private companies continues to increase, finding the required number, type, and quality of specimens, especially surgically-removed diseased tissue, for research
continues to be a challenge. A number of factors contribute to this scenario including: a) new advances in diagnostic procedures especially for cancer, which has resulted in earlier tumor detection; and b) minimally invasive surgical procedures resulting in little or no ‘‘leftover’’ tissue for research. It is expected that the demand for well-characterized human tissue will continue to increase as personalized medicine research increases. To ensure that research material continues to be available, even when specimen size is decreasing, it will be necessary for biobanks to reassess their tissue collection methods, and respond to the changes in the clinical setting. There will be a need to move away from the paradigm of collecting only tissue sections that are stored in the freezer or in paraffin after formalin fixation, to collecting and storing any material that contains molecular characteristics of the tissue. Any research-suitable, biological material collected without jeopardizing the routine clinical and
1
Walter Reed National Military Medical Center, Bethesda, Maryland. Windber Research Institute, Windber, Pennsylvania.
2
359
360 pathological diagnostic process will maximize the use of all surgical specimens for both clinical diagnosis and research. A method that could be used by biobanks to collect research materials from scarce surgical specimens is ‘‘touch preparation,’’ a technique that is widely used and well accepted in histology, cytology and DNA forensics. 2–6 Pressing a cut surface of a piece of tissue to a glass slide causes the cell clusters to adhere to the slide.2–4 Typically, the slide is air dried and the imprint fixed with ethanol prior to processing or storage for future use. Over the years, researchers have found that pressing cut surfaces of fresh or previously frozen and partially thawed human tissue against standard glass microscope slides or absorptive surfaces such as FTA (fast technology for analysis) paper would deposit multiple discrete clusters of cells suitable for molecular profiling.7 Also, manual exfoliation of tissue or scrapings commonly used for cytological diagnosis has been successfully used to collect cell populations for downstream molecular analysis.8,9 The fact that such imprints provide cytologists sufficient information for clinical diagnosis suggests that touch preparations could provide biobanks expanded access to specimens and lead to an increase in the number and type of research materials obtained from surgically removed tissue. The forensic community has been collecting DNA with paper for years, and DNA isolated from paper after 12 months of storage has been successfully used for short tandem repeat (STR) analysis.10 Our tissue bank collects and banks sections of surgically removed tissue, serum, plasma, OCT-embedded tissue, and formalin-fixed paraffin embedded tissue. Although we have over fifty-thousand specimens in our bank, we are still unable to provide all the types and numbers of specimens required by our scientists and collaborators. This is partly because we do not have access to ‘‘scarce’’ and ‘‘small’’ surgical specimens and our tissue banking standard operating procedure (SOP) preserves the last copy of ‘‘precious’’ tissue specimens in the bank. Unlike cell lines that can be replenished by culture, once a tissue section is consumed it can never be replaced, underscoring the need to conserve and maximize the use of every tissue section, even if it is abundant. To determine if touch preparations could be used to provide expanded access to tissue specimens, we hypothesized that imprints of frozen tissue from our bank will yield sufficient DNA for PCR. To test the hypothesis, we prepared touch preparations on glass slides and paper for analysis of isolated DNA by PCR after storage at room temperature and at - 80C for 2 weeks. All touch preparations were made from tissues stored in liquid nitrogen for up to 10 years. We were motivated because biobanking of tissue imprints would have several advantages, including expanded access to more tissue types, maximal use of all tissue types, and reduction in the overall cost of tissue collection/transport/storage. This study provides data that demonstrate that incorporating touch preparations as a standard tissue collection protocol could help biobanks expand access to scarce specimens, conserve rare specimens, maximize the use of wellcharacterized and annotated samples, and provide a cost effective method of distributing DNA at room temperature.
Materials and Methods Samples A total of 12 kidney specimens collected between 2002 and 2003 from donors classified as ‘‘normal’’ were used for the
GREENSPAN ET AL. Table 1. Age and Gender, Date of Collection, and DNA Yield and Purity from Donor Kidney Samples Donor No.
Age
Gender
Collection date
Yield (ng/mg tissue)
A260/280
01 02 03 04 05 06 07 08 09 10 11 12
60y 74y 69y 55y 45y 05m 77y 23y 41y 80y 35y 55y
M F M F F M F M F F F M
08/08/2002 08/10/2002 09/06/2002 09/07/2002 10/02/2002 08/07/2002 05/20/2003 06/22/2003 08/03/2003 12/19/2003 10/21/2003 10/29/2003
14.96 9.25 13.21 6.69 2.44 5.77 14.03 12.38 9.81 5.11 11.86 7.13
2.08 2.01 2.01 2.04 1.91 2.08 2.07 2.03 2.07 2.06 2.10 1.98
y, years; m, months; M, male; F, female.
study. Out of the 12, 5 were from male, and 7 were from female donors; the age of the donors ranged from 5 months to 80 years (Table 1). All specimens were obtained under an Institutional Review Board-approved protocol, and specimens were processed according to the Windber Research Institute Tissue Bank SOPs prior to storage.
Touch preparations Approximately 1 cm · 1 cm of a freshly cut piece of tissue was imprinted on a standard microscope glass slide or FTA paper. For glass slide preparations, tissue sections were blotted dry with a Kimwipe (Kimberly-Clark disposable wipes) and immediately pressed firmly multiple times on a clean 75 mm · 25 mm glass microscope slide. To allow for labeling, handling, and sample-to-sample comparison, DNA imprints were made within an area of 1000 mm2 (50 mm · 20 mm) on each glass slide. Nine glass slides were used to create touch preparations for each tissue. Imprinted slides were air dried for 5 min and immersed in 95% alcohol to fix. To ensure that the imprints were visible during recovery, the slides were stained with Hematoxylin and Eosin (H&E) using standard protocols. After the touch preparation, DNA was isolated immediately (without storage) from three glass slides (Day 0); three imprinted glass slides were stored at room temperature (Glass RT), and three imprinted glass slides were stored at - 80C (Glass - 80C) for 2 weeks before DNA isolation (Fig. 1). For paper preparations, each 1 cm · 1 cm tissue section was also imprinted on the surface of a 25 mm diameter Whatman FTA paper (Type WB120210, GE Healthcare). The area covered by the imprint was 490.63 mm2 (3.14 · 156.25 mm2). A total of six touch preparations were made for each tissue. No DNA was isolated from paper on Day 0 because for the best yield, it is recommended to store FTA paper for at least 2 weeks prior to DNA isolation (Gensolve DNA recovery protocol). After the preparation, three FTA papers each were placed in cryovials and stored at room temperature (Paper RT) and at - 80C (Paper - 80C) for 2 weeks before DNA isolation (Fig. 1). To enable determination of the potential effect of the imprinting process on DNA purity and quality, a Reference DNA sample was prepared from 25–30 mg of a mirror-image of the same piece of tissue used for imprinting on glass and filter paper as described below.
TISSUE IMPRINTS IN ROUTINE BIOBANKING
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FIG. 1. Work flow diagram illustrating the touch preparation experiment using kidney from 12 consented donors. For glass slides, 9 imprints were made per sample and the DNA isolated on the day of preparation (Day 0, n = 3) and after two weeks’ storage at room temperature (Glass RT, n = 3) and at - 80C (Paper - 80C, n = 3). All DNA samples were quantified using the Nanodrop 1000 spectrophotometer, and 25 ng of total DNA analyzed by PCR using Cyclophilin A (Cyc A) as a marker for DNA quality.
DNA isolation from glass slide and FTA paper DNA was isolated from the glass preparations using the QIAamp DNA Mini kit and protocol (Qiagen, Valencia, CA). Each touch preparation was scraped off the slide surface using a sterile scalpel, and the cells rinsed into a 2 mL tube with 180 mL of Buffer ATL (Qiagen). A new scalpel was used for each specimen. In order to isolate DNA from the Reference tissue sample, it was placed in a 2 mL sterile tube with 180 mL of Buffer ATL, and then homogenized with a pestle for mechanical disruption. Proteinase K was added to the buffer mixture of the Reference samples and samples from the slides prior to mixing and incubation at 56C overnight on a Thermomixer (Eppendorf, Hauppauge, NY). After overnight incubation, 200 mL of Buffer AL (Qiagen) was added to the mixture followed by vortexing for 15 sec and incubation for 10 min at 70C. Next, 200 mL of 100% ethanol was added, the mixture was vortexed for 15 sec before transfer into a spin column, and then it was centrifuged for 1 min at 6000 g. DNA was isolated from the FTA paper using the GenSolve Kit (GenVault, Carlsbad, CA) optimized for DNA isolation from FTA paper as previously described.10 The FTA paper with tissue imprint was cut into small pieces, Table 2.
placed into a 2 mL microtube, and the DNA isolated according to the recommended protocol. To avoid sample contamination, the scissors used to cut the paper was sterilized after each sample by washing with bleach and rinsing with 70% ethanol. The DNA isolated from glass slides and FTA papers was purified with the QIAamp DNA Mini kit (Qiagen) using the recommended protocol. DNA yield and purity were determined with the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). The purity of isolated DNA was assessed using the ratio of the absorbance at 260 nm and 280 nm (A260/280). The purified and quantified DNA was stored at - 80C until analyzed by PCR.
Real-time PCR To assess the quality of DNA, real-time PCR was performed using 25 ng of total DNA from Reference samples and touch imprint preparations. All DNA samples were normalized, and equal concentrations of DNA were run in duplicate. In this study, Cyclophilin A (Cyc A), a widely accepted housekeeping gene, was used as a marker for DNA quality. The primer sequence used for Cyc A amplification
Mean DNA Yield and Purity for Touch Preparations on Glass Slides and Filter Paper Glass slide Day 0
2 Weeks RT
Filter paper 2 Weeks - 80C
2 Weeks RT
2 Weeks - 80C
Donor No.
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
1 2 3 4 5 6 7 8 9 10 11 12
15.2 19.5 30.5 7.9 17.5 6.5 141.9 46.8 111.5 92.9 135.1 158.6
1.78 2.07 2.12 1.71 1.94 1.93 2.08 2.03 2.06 2.06 2.14 2.03
50.8 17.5 30.2 49.0 181.5 181.2 152.4 37.9 169.9 96.9 22.2 278.8
2.08 2.48 2.35 2.02 2.10 2.08 2.11 2.07 2.07 1.95 1.82 2.07
74.6 9.3 21.2 17.7 260.1 332.6 77.8 22.6 104.8 50.9 12.7 224.8
2.10 2.39 2.25 1.83 2.13 2.10 2.15 2.03 2.09 1.96 1.66 2.08
60.5 27.8 41.6 145.0 345.4 732.5 252.0 196.8 262.8 138.3 259.6 247.9
1.47 1.03 1.40 1.73 1.89 2.02 1.95 1.84 1.93 1.94 1.97 2.00
59.2 34.3 37.2 173.6 347.7 793.3 291.2 317.9 349.7 184.3 309.4 342.2
1.20 1.16 1.08 1.79 2.01 2.10 1.99 2.01 2.02 2.03 2.04 2.07
1.79 (6.41) 2.06 (0.21)
0.097
0.002
270.00 (208.92) 100.75 (115.89)
105.69 225.86 (94.61) (193.66) 0.022 2.10 1.76 (0.19) (0.33) 0.027 225.86 270.00 (193.66) (208.92) 0.003 1.76 1.79 (0.33) (0.41) 0.564 0.146 2.06 (0.19)
SD, standard deviation.
P value Purity (A260/280) (SD) P value
65.33 (62.50) Yield (ng) (SD)
105.69 (94.61) 0.111 2.00 2.10 (0.18) (0.19) 0.097
65.33 (62.50)
100.75 (115.89) 0.358 2.01 2.10 (0.18) (0.21) 0.281
105.69 (94.61)
0.795
2.06 (0.21)
100.75 (115.89)
Glass - 80C Paper RT Glass RT - 80C RT - 80C RT - 80C
Room Temp vs. - 80C Room Temp vs. - 80C Day 0 vs. - 80C
Day 0 RT Day 0 Compared parameters
DNA was isolated from glass slides on the day of preparation (Day 0) or after 2 weeks of storage at room temperature (Glass RT) or - 80C (Glass - 80C). As shown in Table 2, the yield of isolated DNA from imprints on glass ranged from 6.5 ng to 158.6 ng (Day 0), 17.5 ng to 278.8 ng (Glass RT), and 9.3 ng to 332.6 ng (Glass - 80C); the 260/280 ratio ranged from 1.71 to 2.14 (Day 0), from 1.82 to 2.48 (Glass RT), and from 1.66 to 2.39 (Glass - 80C). The mean DNA yield for Day 0 and Glass RT were 65.33 ng and 105.69 ng, respectively (Table 3). Statistical analysis showed no significant
Day 0 vs. Room Temp
Yield and purity of DNA isolated from glass slides
Filter paper
The yield and A260/280 ratio (purity) of DNA isolated from reference tissue is presented in Table 1, and the corresponding data for DNA isolated from glass slide and FTA paper are presented in Table 2. We compared the yield and purity within and between the different experimental groups to determine if there was any difference in DNA yield and quality. The yield reported for glass is the amount of DNA in ng isolated from an area of 1000 mm2, whereas the yield for FTA is the amount of DNA in ng isolated from an area of 490.63 mm2. The A260/280 ratio is a widely accepted indicator of nucleic acid purity. For pure DNA, the A260/280 is *1.8. Using the amplification of Cyc A by PCR as an index, we were able to determine the effect, if any, of the different collection and sample storage methods on DNA quality.
Glass slide
Results
Table 3.
The yield and purity of DNA from the different experimental groups were compared. We also compared Ct values of the reference DNA sample with the Ct values obtained for the touch imprint preparations. Based on the experimental design, data were analyzed using a mixed model of random effects (interaction of donors and experimental methods) and fixed effects (experimental methods). Other fixed effects considered in the analysis included donor age, gender, and year of sample collection. Replicates were averaged for all experimental measurements and least squares means calculated for DNA yield, purity, and threshold cycle. The restricted maximum likelihood (REML) method was used to determine the variance due to the random effects and an F-test with a P value of 0.05 was used to determine significance of the fixed effects. JMP (v. 10) software was used for the statistical analysis.
Means for DNA Yield and Purity
Data analysis
RT vs. RT
Glass slide vs. Filter Paper
was 5¢-GGATGGCAAGCATGTGGTG-3¢ and 5¢-TGTCCA CAGTCAGCAATGG-3¢ for an expected fragment size of 123 base pairs. The reaction mixture consisted of 2 mL of DNA (25 ng total DNA), 1 mL each of 75 ng/mL primer mix, 7 mL nuclease free water and 10 mL of the SybrGreen Select Master Mix that contains all dNTPs (Life Technologies, Carlsbad, CA). To assess the efficiency of the PCR, two negative control reactions were included: a No Template Control (NTC) that did not contain any sample, and a No Amplification Control (NAC) that did not contain SyrGreen Select Master Mix. The real-time PCR assay was performed on the ViiA7 Real-Time PCR machine (Life Technologies, Carlsbad, CA). Cycling conditions were as follows; denaturation at 95C for 15 sec and annealing/extension at 60C for 1 min, repeated for 40 cycles.
Paper - 80C
GREENSPAN ET AL.
- 80C vs. - 80C
362
TISSUE IMPRINTS IN ROUTINE BIOBANKING difference in DNA yield and purity for Day 0 vs Glass RT ( p = 0.111 and p = 0.097 for yield and purity, respectively); for Day 0 vs Glass - 80C ( p = 0.358 and 0.281, respectively), and for Glass RT vs. Glass - 80C ( p = 0.795 and 0.146, respectively). Table 3 shows these comparisons together with the means (and standard deviation) for DNA yield and purity. Age, gender, and year of sample collection were not significant effects in any of the comparisons.
Yield and purity of DNA isolated from FTA paper The FTA paper preparations were stored for 2 weeks at room temperature (Paper RT) and - 80C (Paper - 80C) before DNA isolation. The yield of isolated DNA ranged from 27.8 ng to 732.5 ng for samples stored at room temperature and 34.3 ng to 793.3 ng for samples stored at - 80C (Table 2). The mean A260/280 ratio ranged from 1.03 to 2.02 for Paper RT, and from 1.08 to 2.10 for Paper - 80C samples. The mean DNA yield for Paper RT and Paper - 80C was 225.86 ng and 270.00 ng, respectively, and statistical analysis revealed a significant difference in DNA yield between the two storage methods ( p = 0.003; Table 3). The mean A260/280 ratio of the DNA sample was 1.76 and 1.79 for Paper RT and Paper - 80C, respectively, and there was no significant difference in purity between the storage methods ( p = 0.564; Table 3). Donor age, gender and year of sample collection were not significant effects in any of the comparisons.
Comparison of glass slides and FTA paper The yield and purity of DNA from Glass RT (Table 3) was significantly different from DNA from Paper RT ( p = 0.022 for yield and p = 0.027 for purity). While the DNA yield from Glass - 80C was significantly different from that of Paper - 80C ( p = 0.002), there was no statistical difference in the purity between the two comparisons ( p = 0.097).
Analysis of isolated DNA by real-time PCR Real-Time PCR was performed to assess the quality of DNA obtained from imprints. The mean threshold crossing (Ct) value for the reference DNA of individual donors ranged from 25.19 to 28.23, whereas the mean Ct range for glass slides was 26.06 to 31.29, and for filter paper 27.67 to 31.36 (Fig. 2). The Reference DNA therefore showed consistently
363 lower mean Ct values compared to that of DNA from Day 0, Glass RT, and Glass - 80C as shown in Figure 2. There was a significant difference in the Ct values between Reference DNA and that from Day 0 of the glass slides ( p = 0.002), Glass RT ( p = 0.005), and Glass - 80C ( p = 0.004) (Table 4). Threshold Ct values for the reference DNA were also significantly different (lower mean) from those for DNA from Paper RT (mean = 29.54) and Paper - 80C (mean = 30.08) ( p < 0.001 for both) (Table 4). Figure 2 shows the mean Ct values for reference DNA and DNA from the glass and paper imprints for each donor. Age, gender, and year of sample collection were not significant effects.
Discussion The overall goal of this study was to determine if methods used by cytologists to collect samples will be suitable for tissue banking operations. The specific aim was to determine if the quality of DNA isolated from tissue imprints stored at room temperature for 2 weeks will be suitable for PCR. We are constantly exploring ways to a) conserve scarce tissue; b) have full access to scarce and limited surgical biopsies; c) increase the type and variety of biospecimens; and d) maximize the use of all tissue types in the bank. Adapting methods that have been perfected for specimen collection by cytologists and forensic scientists has the potential to provide biobanks improved access to scarce clinical specimens and the ability to serve many more researchers. Additionally, cytological and forensic collection methods will be attractive since they are more cost effective due to the material and equipment required for collection, transport, and storage. The ability to store samples at room temperature provides an added benefit as frozen storage comes with a significant price as a result of electricity usage, equipment cost, and space requirements to store the freezers. The feasibility of collecting touch preparations as a standard tissue banking practice has not been systematically evaluated. We conducted a pilot study with eight different types of human tissues and organs, which demonstrated the practicality and feasibility of touch preparations using glass slides and FTA paper (results not shown). Based on the promising data from the pilot study, we designed a more streamlined but robust experiment that focused on quantifying and qualifying the DNA isolated from a well-defined
FIG. 2. Threshold values (Ct) of the reference DNA, DNA from the glass slide imprints at Day 0 (GS-0), room temperature (GS-RT), and - 80C (GS - 80C); and DNA from the filter paper imprints at room temperature (FP-RT), and - 80C (FP80C). Note that PCR was not performed for samples #4 and #6 of GS-0 because of low DNA yield.
364
GREENSPAN ET AL. Table 4.
Means for Ct values Glass slide
Compared parameters Ct mean (SD) p value
Filter paper
Ref. DNA
Glass Day 0
Glass RT
Glass - 80C
Paper RT
Paper - 80C
27.29 (0.81)
28.48 (0.82) 0.002
28.18 (0.90) 0.005
28.17 (0.78) 0.004
29.54 (0.80) < 0.001
30.08 (0.72) < 0.001
Ref. DNA, reference DNA; SD, standard deviation.
area of a standard microscope glass slide and FTA paper, after 2 weeks of storage. Human kidney tissue obtained from 12 donors was used as the source of DNA. DNA from the slides and papers was isolated after storage, and the yield of isolated DNA measured. The purity of the isolated DNA was determined using the ratio of the absorbance at 260 nm and 280 nm, and the quality was determined by comparing the Ct values of Cyc A in the ‘‘reference’’ and ‘‘experimental’’ samples. Our study showed that there was no significant difference between the yield and purity of DNA isolated from glass slides on Day 0 and that isolated after storage for 2 weeks at room temperature ( p = 0.111 for yield and p = 0.097 for purity) or at - 80C ( p = 0.358 for yield and p = 0.281 for purity). Also, the DNA quality from the slides kept at room temperature was not significantly different from the DNA quality of the slides kept at - 80C ( p = 0.795 for yield, p = 0.146 for purity). Conversely, there was a significant difference in the yield of DNA isolated from filter paper after storage for 2 weeks at room temperature and at - 80C ( p = 0.003) but no significant difference in purity ( p = 0.564). Notably, the yield of DNA isolated from FTA at - 80C was generally higher than the yield after storage at room temperature. The yield of DNA isolated from glass was significantly different from that isolated from filter paper after storage for 2 weeks at room temperature and - 80C (Table 3). Interestingly, the yield of DNA recovered from filter paper was consistently higher than the yield of DNA recovered from glass slides following storage at room temperature (mean 225.86 ng for FTA paper vs. 105.69 ng for glass, p = 0.022) and - 80C (270.00 ng for FTA paper vs. 100.75 ng for glass, p = 0.002). The higher recovery from paper compared to glass must be due to the physical and chemical properties of paper, which apparently favor the absorption of more DNA than glass. The glass surface was not specially treated and the chemical agents applied to the FTA paper, which are supposed to protect nucleic acids from nucleases and oxidative/UV damage, may account for the difference in yield. This assumption is plausible because the area of imprint on glass was about twice the area of imprint on paper (glass 1000mm2 vs. paper 490.63 mm2). Filter paper, specifically FTA paper, may therefore be a better surface than glass if DNA yield is of most importance. Interestingly, DNA purity was not significantly different when Glass - 80C was compared with Paper - 80C ( p = 0.097; mean 1.79 for FTA paper vs. 2.06 for glass), but it was significantly different for storage at room temperature ( p = 0.027; mean 1.76 for FTA paper vs. 2.10 for glass). The Ct value, which represents the number of cycles required for the fluorescent signal to cross the background threshold, is inversely proportional to the amount of target nucleic acid in the sample (starting target DNA concentra-
tion). It is a relatively good measure of the concentration of the target in the PCR reaction. Typically, the higher the amount of undegraded target DNA, the earlier will be the threshold crossing, and hence lower Ct value. Although 25 ng of total DNA was used in all PCR reactions, Ct values for DNA from glass and paper were generally higher (Fig. 2) and significantly different than that of Reference DNA (Table 4). It is important to note that the Reference DNA was obtained from tissue that was not subjected to any experimental manipulation prior to DNA extraction and thus it is expected to have less undegraded target DNA and hence lower Ct values. Although Ct values were lower for DNA from Reference DNA compared to tissue imprints, Ct values for the imprints were < 32 which indicated that the DNA from imprints still had relatively abundant amounts of target DNA. The difference in Ct values observed between these DNA samples and the Reference DNA could be due to other factors beside the concentration of the target. The process of imprinting on slides and filter paper, the scraping from slides, and the DNA isolation process could all introduce artifacts. Any of these factors could result in templateindependent changes to the Ct value. The different levels of purity between the Reference DNA and experimental DNA samples could also be responsible for the difference in the Ct values obtained. The use of FTA paper for the collection of blood from which DNA is isolated is widely reported.10–14 Paper is an economical and efficient vehicle of sample collection that provides quality nucleic acids for forensics and genetic/epidemiological studies including but not limited to PCR and mutation analysis; paper has also been utilized for the storage of tumor cell suspensions for molecular testing.10–14 DNA specimens from FTA paper are claimed to be superior to DNA specimens from formalin-fixed paraffin-embedded (FFPE) tissue, the method is less cumbersome, and DNA can be obtained from relatively smaller specimens.15 In view of these advantages and the fact that FTA paper is already used widely by DNA forensic scientists and epidemiologists, biobanks could utilize FTA paper to collect DNA for storage and distribution. The results of this study clearly show that touch preparations on microscope slides and paper can produce sufficient, good-quality DNA even after 2 weeks storage at room temperature. Thus, tissue imprints on glass and paper could be used by biobanks to store and distribute DNA. The fact that we utilized tissue that had been stored at - 80C for 9– 10 years suggests that biobanks could adopt ‘‘touch preparation’’ as a way to provide access to DNA from ‘‘rare and precious’’ specimens. While we believe that banking of touch preparations will greatly expand the number and variety of DNA samples available, this study will need to be expanded to include other tissue types and an extended storage period at room temperature before the findings can be generalized.
TISSUE IMPRINTS IN ROUTINE BIOBANKING The DNA yield from filter paper was generally higher than that from glass slides, suggesting that when compared to glass, paper may be the most practical and preferred material for touch preparations, especially if the specimen can be held for 2 weeks before use. On the other hand, glass may be useful for collection and transport of urgently-needed DNA specimens (e.g., between 0 and 3 days after preparation). The introduction into routine biobanking of paper-based touch preparations in order to conserve scarce tissue specimens, provide access to limited surgical biopsy specimens, and importantly, to maximize the use of biobank resources, appears very feasible.
Acknowledgment We acknowledge Dr. Richard Somiari for his review and comments on the manuscript and IntegenX (Genvault) for providing DNA isolation reagents. This study was performed independent of any supplier.
Author Disclosure Statement No competing financial interests exist. This research was supported by a grant from the United States Department of Defense (Military Molecular Medicine Initiative MDA W81XWHH-05-2-0075, ROTOCOL01-20006). The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense.
References 1. Ginsburg GS, McCarthy JJ. Personalized medicine: Revolutionizing drug discovery and patient care. Trends Biotechnol 2001;19:491–496. 2. Komenaka IK, Torabi R, Nair G, et al. Intraoperative touch imprint and frozen seion analysis of sentinel lymph nodes after neoadjuvant chemotherapy for breast cancer. Ann Surg 2010;251: 319–322. 3. Khanna R, Bhadani S, Khanna S, et al. Touch imprint cytology evaluation of sentinel lymph node in breast cancer. World J Surg 2011;35:1254–1259.
365 4. Angelucci A, Pace G, Sanita P, et al. Tissue print of prostate biopsy: A novel tool in the diagnostic procedure of prostate cancer. Diagn Pathol 2011;6:34. 5. Goyal S, Mohan H, Saini V. Rinse fluid and imprint smear cytology of bronchial biopsies in diagnosis of lung tumors. Diagn Cytopathol 2010;40:98–103. 6. Faller E, Tunon de Lara C, Fournier M, et al. Accuracy of intraoperative imprint cytology of sentinel lymph nodes in cT1 infiltrating breast cancer. Bull Cancer 2011;98:1047–1057. 7. Mangia A, Chiriatti A, Chiarappa P, et al. Touch imprint cytology in tumor tissue banks for the confirmation of neoplastic cellularity and for DNA extraction. Arch Pathol Lab Med 2008;132:974–979. 8. Kolte SS, Satarkar RN. Role of scrape cytology in the intraoperative diagnosis of tumor. J Cytol 2010;27:86–90. 9. Mojica WD, Rapkiewicz AV, Liotta LA, et al. Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling. Cancer 2005;105:483–491. 10. Somiari RI, Adebiyi E, Ukachukwu L, et al. STR Analysis of human DNA samples after dry-state ambient temperature storage in GenPlates. Open Forensic Sci J 2011;4:30–35. 11. Harahap NI, Harahap IS, Kaszynski RH, et al. Spinal muscular atrophy patient detection and carrier screening using dried blood spots on filter paper. Genet Test Mol Biomarkers 2012;16:123–129. 12. Taylor BJ, Martin KA, Arango E, et al. Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples. Malar J 2011;10:244. 13. Jones S, Sutherland CJ, Hermsen C, et al. Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes. Malar J 2012;11:266. 14. Dobbs LJ, Madigan MN, Carter AB, et al. Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing. Arch Pathol Lab Med 2002;126:56–63. 15. Petras ML, Lefferts JA, Ward BP, et al. KRAS detection in colonic tumors by DNA extraction from FTA paper: The molecular touch-prep. Diagn Mol Pathol 2011;20:189–193.
Address correspondence to: Stella Somiari, PhD Windber Research Institute 620 Seventh Street Windber, PA 15963 E-mail: [email protected]
BIOPRESERVATION AND BIOBANKING Volume 11, Number 6, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2013.0030
Tissue Imprints: Assessing Their Potential for Routine Biobanking Collection Renata Greenspan,1 Amy O’Donnell,2 Jeff Meyer,2 Jennifer Kane,2 Kim Mamula,2 Sue Lubert,2 Brenda Deyarmin,2 Caroline Larson,2 Sean Rigby,2 Amber Greenawalt,2 Negin Vatanian,1 Richard Mural,2 Craig Shriver,1 and Stella Somiari2
Biomedical research depends on the availability of good quality biospecimens. Unfortunately, certain specimens are scarce due to disease rarity or size restrictions of surgical materials. To increase access to limited surgical specimens, Biobanks need to reassess and adjust their collection programs. We evaluated the feasibility of adapting ‘‘touch imprints’’ to gain access to limited surgical specimens as well as to maximize the use of ‘‘precious’’ specimens. We utilized 12 kidney samples for touch imprints on defined areas of microscope glass slides and FTA paper. DNA was isolated from glass slides on the day of preparation, Day 0, and from glass slide and FTA paper preparations after two weeks of storage at room temperature and - 80C. Yield and purity of DNA from reference kidney samples were compared to DNA from the touch imprints and the quality determined by realtime PCR using the amplification of Cyclophilin A (Cyc A) as an index. DNA quality for glass slides at Day 0 was not significantly different from DNA after two weeks at room temperature (glass at room temperature; p = 0.111 and 0.097, yield and purity, respectively) and after two weeks at - 80C (glass - 80C; p = 0.358 and 0.281, yield and purity, respectively). Glass slide DNA at room temperature and - 80C were not significantly different ( p = 0.795 and 0.146 for yield and purity, respectively). DNA from FTA paper at room temperature and from FTA paper at - 80C were significantly different from glass at room temperature and glass at - 80C ( p = 0.002, respectively). Threshold values for Cyc A were £ 28 for the reference DNA and £ 32 for DNA from glass and FTA paper. This study demonstrates that touch preparations on microscope glass slides and FTA paper can provide sufficient and good quality DNA suitable for PCR. Touch imprints could therefore be adopted by biobanks to collect and bank biological materials from limited surgical specimens. Introduction
T
he speed with which clinical research findings are translated from the bench to the bedside will determine how soon personalized medicine, the new paradigm for practicing medicine, can be completely embraced.1 The success of research translating to the clinic will partly depend on the availability of good quality, well-characterized biospecimens from consented donors. These specimens will allow scientists to study the development and progression of diseases at the molecular level. Dedicated biobanks established by academic institutions or private companies will start to play a greater role in the collection, annotation, storage, and distribution of biospecimens for research. Although the number of human tissue banks established by universities and private companies continues to increase, finding the required number, type, and quality of specimens, especially surgically-removed diseased tissue, for research
continues to be a challenge. A number of factors contribute to this scenario including: a) new advances in diagnostic procedures especially for cancer, which has resulted in earlier tumor detection; and b) minimally invasive surgical procedures resulting in little or no ‘‘leftover’’ tissue for research. It is expected that the demand for well-characterized human tissue will continue to increase as personalized medicine research increases. To ensure that research material continues to be available, even when specimen size is decreasing, it will be necessary for biobanks to reassess their tissue collection methods, and respond to the changes in the clinical setting. There will be a need to move away from the paradigm of collecting only tissue sections that are stored in the freezer or in paraffin after formalin fixation, to collecting and storing any material that contains molecular characteristics of the tissue. Any research-suitable, biological material collected without jeopardizing the routine clinical and
1
Walter Reed National Military Medical Center, Bethesda, Maryland. Windber Research Institute, Windber, Pennsylvania.
2
359
360 pathological diagnostic process will maximize the use of all surgical specimens for both clinical diagnosis and research. A method that could be used by biobanks to collect research materials from scarce surgical specimens is ‘‘touch preparation,’’ a technique that is widely used and well accepted in histology, cytology and DNA forensics. 2–6 Pressing a cut surface of a piece of tissue to a glass slide causes the cell clusters to adhere to the slide.2–4 Typically, the slide is air dried and the imprint fixed with ethanol prior to processing or storage for future use. Over the years, researchers have found that pressing cut surfaces of fresh or previously frozen and partially thawed human tissue against standard glass microscope slides or absorptive surfaces such as FTA (fast technology for analysis) paper would deposit multiple discrete clusters of cells suitable for molecular profiling.7 Also, manual exfoliation of tissue or scrapings commonly used for cytological diagnosis has been successfully used to collect cell populations for downstream molecular analysis.8,9 The fact that such imprints provide cytologists sufficient information for clinical diagnosis suggests that touch preparations could provide biobanks expanded access to specimens and lead to an increase in the number and type of research materials obtained from surgically removed tissue. The forensic community has been collecting DNA with paper for years, and DNA isolated from paper after 12 months of storage has been successfully used for short tandem repeat (STR) analysis.10 Our tissue bank collects and banks sections of surgically removed tissue, serum, plasma, OCT-embedded tissue, and formalin-fixed paraffin embedded tissue. Although we have over fifty-thousand specimens in our bank, we are still unable to provide all the types and numbers of specimens required by our scientists and collaborators. This is partly because we do not have access to ‘‘scarce’’ and ‘‘small’’ surgical specimens and our tissue banking standard operating procedure (SOP) preserves the last copy of ‘‘precious’’ tissue specimens in the bank. Unlike cell lines that can be replenished by culture, once a tissue section is consumed it can never be replaced, underscoring the need to conserve and maximize the use of every tissue section, even if it is abundant. To determine if touch preparations could be used to provide expanded access to tissue specimens, we hypothesized that imprints of frozen tissue from our bank will yield sufficient DNA for PCR. To test the hypothesis, we prepared touch preparations on glass slides and paper for analysis of isolated DNA by PCR after storage at room temperature and at - 80C for 2 weeks. All touch preparations were made from tissues stored in liquid nitrogen for up to 10 years. We were motivated because biobanking of tissue imprints would have several advantages, including expanded access to more tissue types, maximal use of all tissue types, and reduction in the overall cost of tissue collection/transport/storage. This study provides data that demonstrate that incorporating touch preparations as a standard tissue collection protocol could help biobanks expand access to scarce specimens, conserve rare specimens, maximize the use of wellcharacterized and annotated samples, and provide a cost effective method of distributing DNA at room temperature.
Materials and Methods Samples A total of 12 kidney specimens collected between 2002 and 2003 from donors classified as ‘‘normal’’ were used for the
GREENSPAN ET AL. Table 1. Age and Gender, Date of Collection, and DNA Yield and Purity from Donor Kidney Samples Donor No.
Age
Gender
Collection date
Yield (ng/mg tissue)
A260/280
01 02 03 04 05 06 07 08 09 10 11 12
60y 74y 69y 55y 45y 05m 77y 23y 41y 80y 35y 55y
M F M F F M F M F F F M
08/08/2002 08/10/2002 09/06/2002 09/07/2002 10/02/2002 08/07/2002 05/20/2003 06/22/2003 08/03/2003 12/19/2003 10/21/2003 10/29/2003
14.96 9.25 13.21 6.69 2.44 5.77 14.03 12.38 9.81 5.11 11.86 7.13
2.08 2.01 2.01 2.04 1.91 2.08 2.07 2.03 2.07 2.06 2.10 1.98
y, years; m, months; M, male; F, female.
study. Out of the 12, 5 were from male, and 7 were from female donors; the age of the donors ranged from 5 months to 80 years (Table 1). All specimens were obtained under an Institutional Review Board-approved protocol, and specimens were processed according to the Windber Research Institute Tissue Bank SOPs prior to storage.
Touch preparations Approximately 1 cm · 1 cm of a freshly cut piece of tissue was imprinted on a standard microscope glass slide or FTA paper. For glass slide preparations, tissue sections were blotted dry with a Kimwipe (Kimberly-Clark disposable wipes) and immediately pressed firmly multiple times on a clean 75 mm · 25 mm glass microscope slide. To allow for labeling, handling, and sample-to-sample comparison, DNA imprints were made within an area of 1000 mm2 (50 mm · 20 mm) on each glass slide. Nine glass slides were used to create touch preparations for each tissue. Imprinted slides were air dried for 5 min and immersed in 95% alcohol to fix. To ensure that the imprints were visible during recovery, the slides were stained with Hematoxylin and Eosin (H&E) using standard protocols. After the touch preparation, DNA was isolated immediately (without storage) from three glass slides (Day 0); three imprinted glass slides were stored at room temperature (Glass RT), and three imprinted glass slides were stored at - 80C (Glass - 80C) for 2 weeks before DNA isolation (Fig. 1). For paper preparations, each 1 cm · 1 cm tissue section was also imprinted on the surface of a 25 mm diameter Whatman FTA paper (Type WB120210, GE Healthcare). The area covered by the imprint was 490.63 mm2 (3.14 · 156.25 mm2). A total of six touch preparations were made for each tissue. No DNA was isolated from paper on Day 0 because for the best yield, it is recommended to store FTA paper for at least 2 weeks prior to DNA isolation (Gensolve DNA recovery protocol). After the preparation, three FTA papers each were placed in cryovials and stored at room temperature (Paper RT) and at - 80C (Paper - 80C) for 2 weeks before DNA isolation (Fig. 1). To enable determination of the potential effect of the imprinting process on DNA purity and quality, a Reference DNA sample was prepared from 25–30 mg of a mirror-image of the same piece of tissue used for imprinting on glass and filter paper as described below.
TISSUE IMPRINTS IN ROUTINE BIOBANKING
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FIG. 1. Work flow diagram illustrating the touch preparation experiment using kidney from 12 consented donors. For glass slides, 9 imprints were made per sample and the DNA isolated on the day of preparation (Day 0, n = 3) and after two weeks’ storage at room temperature (Glass RT, n = 3) and at - 80C (Paper - 80C, n = 3). All DNA samples were quantified using the Nanodrop 1000 spectrophotometer, and 25 ng of total DNA analyzed by PCR using Cyclophilin A (Cyc A) as a marker for DNA quality.
DNA isolation from glass slide and FTA paper DNA was isolated from the glass preparations using the QIAamp DNA Mini kit and protocol (Qiagen, Valencia, CA). Each touch preparation was scraped off the slide surface using a sterile scalpel, and the cells rinsed into a 2 mL tube with 180 mL of Buffer ATL (Qiagen). A new scalpel was used for each specimen. In order to isolate DNA from the Reference tissue sample, it was placed in a 2 mL sterile tube with 180 mL of Buffer ATL, and then homogenized with a pestle for mechanical disruption. Proteinase K was added to the buffer mixture of the Reference samples and samples from the slides prior to mixing and incubation at 56C overnight on a Thermomixer (Eppendorf, Hauppauge, NY). After overnight incubation, 200 mL of Buffer AL (Qiagen) was added to the mixture followed by vortexing for 15 sec and incubation for 10 min at 70C. Next, 200 mL of 100% ethanol was added, the mixture was vortexed for 15 sec before transfer into a spin column, and then it was centrifuged for 1 min at 6000 g. DNA was isolated from the FTA paper using the GenSolve Kit (GenVault, Carlsbad, CA) optimized for DNA isolation from FTA paper as previously described.10 The FTA paper with tissue imprint was cut into small pieces, Table 2.
placed into a 2 mL microtube, and the DNA isolated according to the recommended protocol. To avoid sample contamination, the scissors used to cut the paper was sterilized after each sample by washing with bleach and rinsing with 70% ethanol. The DNA isolated from glass slides and FTA papers was purified with the QIAamp DNA Mini kit (Qiagen) using the recommended protocol. DNA yield and purity were determined with the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). The purity of isolated DNA was assessed using the ratio of the absorbance at 260 nm and 280 nm (A260/280). The purified and quantified DNA was stored at - 80C until analyzed by PCR.
Real-time PCR To assess the quality of DNA, real-time PCR was performed using 25 ng of total DNA from Reference samples and touch imprint preparations. All DNA samples were normalized, and equal concentrations of DNA were run in duplicate. In this study, Cyclophilin A (Cyc A), a widely accepted housekeeping gene, was used as a marker for DNA quality. The primer sequence used for Cyc A amplification
Mean DNA Yield and Purity for Touch Preparations on Glass Slides and Filter Paper Glass slide Day 0
2 Weeks RT
Filter paper 2 Weeks - 80C
2 Weeks RT
2 Weeks - 80C
Donor No.
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
Yield (ng)
Abs 260/280
1 2 3 4 5 6 7 8 9 10 11 12
15.2 19.5 30.5 7.9 17.5 6.5 141.9 46.8 111.5 92.9 135.1 158.6
1.78 2.07 2.12 1.71 1.94 1.93 2.08 2.03 2.06 2.06 2.14 2.03
50.8 17.5 30.2 49.0 181.5 181.2 152.4 37.9 169.9 96.9 22.2 278.8
2.08 2.48 2.35 2.02 2.10 2.08 2.11 2.07 2.07 1.95 1.82 2.07
74.6 9.3 21.2 17.7 260.1 332.6 77.8 22.6 104.8 50.9 12.7 224.8
2.10 2.39 2.25 1.83 2.13 2.10 2.15 2.03 2.09 1.96 1.66 2.08
60.5 27.8 41.6 145.0 345.4 732.5 252.0 196.8 262.8 138.3 259.6 247.9
1.47 1.03 1.40 1.73 1.89 2.02 1.95 1.84 1.93 1.94 1.97 2.00
59.2 34.3 37.2 173.6 347.7 793.3 291.2 317.9 349.7 184.3 309.4 342.2
1.20 1.16 1.08 1.79 2.01 2.10 1.99 2.01 2.02 2.03 2.04 2.07
1.79 (6.41) 2.06 (0.21)
0.097
0.002
270.00 (208.92) 100.75 (115.89)
105.69 225.86 (94.61) (193.66) 0.022 2.10 1.76 (0.19) (0.33) 0.027 225.86 270.00 (193.66) (208.92) 0.003 1.76 1.79 (0.33) (0.41) 0.564 0.146 2.06 (0.19)
SD, standard deviation.
P value Purity (A260/280) (SD) P value
65.33 (62.50) Yield (ng) (SD)
105.69 (94.61) 0.111 2.00 2.10 (0.18) (0.19) 0.097
65.33 (62.50)
100.75 (115.89) 0.358 2.01 2.10 (0.18) (0.21) 0.281
105.69 (94.61)
0.795
2.06 (0.21)
100.75 (115.89)
Glass - 80C Paper RT Glass RT - 80C RT - 80C RT - 80C
Room Temp vs. - 80C Room Temp vs. - 80C Day 0 vs. - 80C
Day 0 RT Day 0 Compared parameters
DNA was isolated from glass slides on the day of preparation (Day 0) or after 2 weeks of storage at room temperature (Glass RT) or - 80C (Glass - 80C). As shown in Table 2, the yield of isolated DNA from imprints on glass ranged from 6.5 ng to 158.6 ng (Day 0), 17.5 ng to 278.8 ng (Glass RT), and 9.3 ng to 332.6 ng (Glass - 80C); the 260/280 ratio ranged from 1.71 to 2.14 (Day 0), from 1.82 to 2.48 (Glass RT), and from 1.66 to 2.39 (Glass - 80C). The mean DNA yield for Day 0 and Glass RT were 65.33 ng and 105.69 ng, respectively (Table 3). Statistical analysis showed no significant
Day 0 vs. Room Temp
Yield and purity of DNA isolated from glass slides
Filter paper
The yield and A260/280 ratio (purity) of DNA isolated from reference tissue is presented in Table 1, and the corresponding data for DNA isolated from glass slide and FTA paper are presented in Table 2. We compared the yield and purity within and between the different experimental groups to determine if there was any difference in DNA yield and quality. The yield reported for glass is the amount of DNA in ng isolated from an area of 1000 mm2, whereas the yield for FTA is the amount of DNA in ng isolated from an area of 490.63 mm2. The A260/280 ratio is a widely accepted indicator of nucleic acid purity. For pure DNA, the A260/280 is *1.8. Using the amplification of Cyc A by PCR as an index, we were able to determine the effect, if any, of the different collection and sample storage methods on DNA quality.
Glass slide
Results
Table 3.
The yield and purity of DNA from the different experimental groups were compared. We also compared Ct values of the reference DNA sample with the Ct values obtained for the touch imprint preparations. Based on the experimental design, data were analyzed using a mixed model of random effects (interaction of donors and experimental methods) and fixed effects (experimental methods). Other fixed effects considered in the analysis included donor age, gender, and year of sample collection. Replicates were averaged for all experimental measurements and least squares means calculated for DNA yield, purity, and threshold cycle. The restricted maximum likelihood (REML) method was used to determine the variance due to the random effects and an F-test with a P value of 0.05 was used to determine significance of the fixed effects. JMP (v. 10) software was used for the statistical analysis.
Means for DNA Yield and Purity
Data analysis
RT vs. RT
Glass slide vs. Filter Paper
was 5¢-GGATGGCAAGCATGTGGTG-3¢ and 5¢-TGTCCA CAGTCAGCAATGG-3¢ for an expected fragment size of 123 base pairs. The reaction mixture consisted of 2 mL of DNA (25 ng total DNA), 1 mL each of 75 ng/mL primer mix, 7 mL nuclease free water and 10 mL of the SybrGreen Select Master Mix that contains all dNTPs (Life Technologies, Carlsbad, CA). To assess the efficiency of the PCR, two negative control reactions were included: a No Template Control (NTC) that did not contain any sample, and a No Amplification Control (NAC) that did not contain SyrGreen Select Master Mix. The real-time PCR assay was performed on the ViiA7 Real-Time PCR machine (Life Technologies, Carlsbad, CA). Cycling conditions were as follows; denaturation at 95C for 15 sec and annealing/extension at 60C for 1 min, repeated for 40 cycles.
Paper - 80C
GREENSPAN ET AL.
- 80C vs. - 80C
362
TISSUE IMPRINTS IN ROUTINE BIOBANKING difference in DNA yield and purity for Day 0 vs Glass RT ( p = 0.111 and p = 0.097 for yield and purity, respectively); for Day 0 vs Glass - 80C ( p = 0.358 and 0.281, respectively), and for Glass RT vs. Glass - 80C ( p = 0.795 and 0.146, respectively). Table 3 shows these comparisons together with the means (and standard deviation) for DNA yield and purity. Age, gender, and year of sample collection were not significant effects in any of the comparisons.
Yield and purity of DNA isolated from FTA paper The FTA paper preparations were stored for 2 weeks at room temperature (Paper RT) and - 80C (Paper - 80C) before DNA isolation. The yield of isolated DNA ranged from 27.8 ng to 732.5 ng for samples stored at room temperature and 34.3 ng to 793.3 ng for samples stored at - 80C (Table 2). The mean A260/280 ratio ranged from 1.03 to 2.02 for Paper RT, and from 1.08 to 2.10 for Paper - 80C samples. The mean DNA yield for Paper RT and Paper - 80C was 225.86 ng and 270.00 ng, respectively, and statistical analysis revealed a significant difference in DNA yield between the two storage methods ( p = 0.003; Table 3). The mean A260/280 ratio of the DNA sample was 1.76 and 1.79 for Paper RT and Paper - 80C, respectively, and there was no significant difference in purity between the storage methods ( p = 0.564; Table 3). Donor age, gender and year of sample collection were not significant effects in any of the comparisons.
Comparison of glass slides and FTA paper The yield and purity of DNA from Glass RT (Table 3) was significantly different from DNA from Paper RT ( p = 0.022 for yield and p = 0.027 for purity). While the DNA yield from Glass - 80C was significantly different from that of Paper - 80C ( p = 0.002), there was no statistical difference in the purity between the two comparisons ( p = 0.097).
Analysis of isolated DNA by real-time PCR Real-Time PCR was performed to assess the quality of DNA obtained from imprints. The mean threshold crossing (Ct) value for the reference DNA of individual donors ranged from 25.19 to 28.23, whereas the mean Ct range for glass slides was 26.06 to 31.29, and for filter paper 27.67 to 31.36 (Fig. 2). The Reference DNA therefore showed consistently
363 lower mean Ct values compared to that of DNA from Day 0, Glass RT, and Glass - 80C as shown in Figure 2. There was a significant difference in the Ct values between Reference DNA and that from Day 0 of the glass slides ( p = 0.002), Glass RT ( p = 0.005), and Glass - 80C ( p = 0.004) (Table 4). Threshold Ct values for the reference DNA were also significantly different (lower mean) from those for DNA from Paper RT (mean = 29.54) and Paper - 80C (mean = 30.08) ( p < 0.001 for both) (Table 4). Figure 2 shows the mean Ct values for reference DNA and DNA from the glass and paper imprints for each donor. Age, gender, and year of sample collection were not significant effects.
Discussion The overall goal of this study was to determine if methods used by cytologists to collect samples will be suitable for tissue banking operations. The specific aim was to determine if the quality of DNA isolated from tissue imprints stored at room temperature for 2 weeks will be suitable for PCR. We are constantly exploring ways to a) conserve scarce tissue; b) have full access to scarce and limited surgical biopsies; c) increase the type and variety of biospecimens; and d) maximize the use of all tissue types in the bank. Adapting methods that have been perfected for specimen collection by cytologists and forensic scientists has the potential to provide biobanks improved access to scarce clinical specimens and the ability to serve many more researchers. Additionally, cytological and forensic collection methods will be attractive since they are more cost effective due to the material and equipment required for collection, transport, and storage. The ability to store samples at room temperature provides an added benefit as frozen storage comes with a significant price as a result of electricity usage, equipment cost, and space requirements to store the freezers. The feasibility of collecting touch preparations as a standard tissue banking practice has not been systematically evaluated. We conducted a pilot study with eight different types of human tissues and organs, which demonstrated the practicality and feasibility of touch preparations using glass slides and FTA paper (results not shown). Based on the promising data from the pilot study, we designed a more streamlined but robust experiment that focused on quantifying and qualifying the DNA isolated from a well-defined
FIG. 2. Threshold values (Ct) of the reference DNA, DNA from the glass slide imprints at Day 0 (GS-0), room temperature (GS-RT), and - 80C (GS - 80C); and DNA from the filter paper imprints at room temperature (FP-RT), and - 80C (FP80C). Note that PCR was not performed for samples #4 and #6 of GS-0 because of low DNA yield.
364
GREENSPAN ET AL. Table 4.
Means for Ct values Glass slide
Compared parameters Ct mean (SD) p value
Filter paper
Ref. DNA
Glass Day 0
Glass RT
Glass - 80C
Paper RT
Paper - 80C
27.29 (0.81)
28.48 (0.82) 0.002
28.18 (0.90) 0.005
28.17 (0.78) 0.004
29.54 (0.80) < 0.001
30.08 (0.72) < 0.001
Ref. DNA, reference DNA; SD, standard deviation.
area of a standard microscope glass slide and FTA paper, after 2 weeks of storage. Human kidney tissue obtained from 12 donors was used as the source of DNA. DNA from the slides and papers was isolated after storage, and the yield of isolated DNA measured. The purity of the isolated DNA was determined using the ratio of the absorbance at 260 nm and 280 nm, and the quality was determined by comparing the Ct values of Cyc A in the ‘‘reference’’ and ‘‘experimental’’ samples. Our study showed that there was no significant difference between the yield and purity of DNA isolated from glass slides on Day 0 and that isolated after storage for 2 weeks at room temperature ( p = 0.111 for yield and p = 0.097 for purity) or at - 80C ( p = 0.358 for yield and p = 0.281 for purity). Also, the DNA quality from the slides kept at room temperature was not significantly different from the DNA quality of the slides kept at - 80C ( p = 0.795 for yield, p = 0.146 for purity). Conversely, there was a significant difference in the yield of DNA isolated from filter paper after storage for 2 weeks at room temperature and at - 80C ( p = 0.003) but no significant difference in purity ( p = 0.564). Notably, the yield of DNA isolated from FTA at - 80C was generally higher than the yield after storage at room temperature. The yield of DNA isolated from glass was significantly different from that isolated from filter paper after storage for 2 weeks at room temperature and - 80C (Table 3). Interestingly, the yield of DNA recovered from filter paper was consistently higher than the yield of DNA recovered from glass slides following storage at room temperature (mean 225.86 ng for FTA paper vs. 105.69 ng for glass, p = 0.022) and - 80C (270.00 ng for FTA paper vs. 100.75 ng for glass, p = 0.002). The higher recovery from paper compared to glass must be due to the physical and chemical properties of paper, which apparently favor the absorption of more DNA than glass. The glass surface was not specially treated and the chemical agents applied to the FTA paper, which are supposed to protect nucleic acids from nucleases and oxidative/UV damage, may account for the difference in yield. This assumption is plausible because the area of imprint on glass was about twice the area of imprint on paper (glass 1000mm2 vs. paper 490.63 mm2). Filter paper, specifically FTA paper, may therefore be a better surface than glass if DNA yield is of most importance. Interestingly, DNA purity was not significantly different when Glass - 80C was compared with Paper - 80C ( p = 0.097; mean 1.79 for FTA paper vs. 2.06 for glass), but it was significantly different for storage at room temperature ( p = 0.027; mean 1.76 for FTA paper vs. 2.10 for glass). The Ct value, which represents the number of cycles required for the fluorescent signal to cross the background threshold, is inversely proportional to the amount of target nucleic acid in the sample (starting target DNA concentra-
tion). It is a relatively good measure of the concentration of the target in the PCR reaction. Typically, the higher the amount of undegraded target DNA, the earlier will be the threshold crossing, and hence lower Ct value. Although 25 ng of total DNA was used in all PCR reactions, Ct values for DNA from glass and paper were generally higher (Fig. 2) and significantly different than that of Reference DNA (Table 4). It is important to note that the Reference DNA was obtained from tissue that was not subjected to any experimental manipulation prior to DNA extraction and thus it is expected to have less undegraded target DNA and hence lower Ct values. Although Ct values were lower for DNA from Reference DNA compared to tissue imprints, Ct values for the imprints were < 32 which indicated that the DNA from imprints still had relatively abundant amounts of target DNA. The difference in Ct values observed between these DNA samples and the Reference DNA could be due to other factors beside the concentration of the target. The process of imprinting on slides and filter paper, the scraping from slides, and the DNA isolation process could all introduce artifacts. Any of these factors could result in templateindependent changes to the Ct value. The different levels of purity between the Reference DNA and experimental DNA samples could also be responsible for the difference in the Ct values obtained. The use of FTA paper for the collection of blood from which DNA is isolated is widely reported.10–14 Paper is an economical and efficient vehicle of sample collection that provides quality nucleic acids for forensics and genetic/epidemiological studies including but not limited to PCR and mutation analysis; paper has also been utilized for the storage of tumor cell suspensions for molecular testing.10–14 DNA specimens from FTA paper are claimed to be superior to DNA specimens from formalin-fixed paraffin-embedded (FFPE) tissue, the method is less cumbersome, and DNA can be obtained from relatively smaller specimens.15 In view of these advantages and the fact that FTA paper is already used widely by DNA forensic scientists and epidemiologists, biobanks could utilize FTA paper to collect DNA for storage and distribution. The results of this study clearly show that touch preparations on microscope slides and paper can produce sufficient, good-quality DNA even after 2 weeks storage at room temperature. Thus, tissue imprints on glass and paper could be used by biobanks to store and distribute DNA. The fact that we utilized tissue that had been stored at - 80C for 9– 10 years suggests that biobanks could adopt ‘‘touch preparation’’ as a way to provide access to DNA from ‘‘rare and precious’’ specimens. While we believe that banking of touch preparations will greatly expand the number and variety of DNA samples available, this study will need to be expanded to include other tissue types and an extended storage period at room temperature before the findings can be generalized.
TISSUE IMPRINTS IN ROUTINE BIOBANKING The DNA yield from filter paper was generally higher than that from glass slides, suggesting that when compared to glass, paper may be the most practical and preferred material for touch preparations, especially if the specimen can be held for 2 weeks before use. On the other hand, glass may be useful for collection and transport of urgently-needed DNA specimens (e.g., between 0 and 3 days after preparation). The introduction into routine biobanking of paper-based touch preparations in order to conserve scarce tissue specimens, provide access to limited surgical biopsy specimens, and importantly, to maximize the use of biobank resources, appears very feasible.
Acknowledgment We acknowledge Dr. Richard Somiari for his review and comments on the manuscript and IntegenX (Genvault) for providing DNA isolation reagents. This study was performed independent of any supplier.
Author Disclosure Statement No competing financial interests exist. This research was supported by a grant from the United States Department of Defense (Military Molecular Medicine Initiative MDA W81XWHH-05-2-0075, ROTOCOL01-20006). The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense.
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Address correspondence to: Stella Somiari, PhD Windber Research Institute 620 Seventh Street Windber, PA 15963 E-mail: [email protected]
