With approval by the Washington University Human Research Protection Office (HRPO), we searched the Copath database of Barnes Jewish Hospital for temporal artery biopsy specimens from 1995 to 2008 and retrieved all specimens for which material was available. We identified approximately 60 cases, of which 43, (22 histologically positive and 21 histologically negative) had material available for review. There were five males and 17 females in the histologically positive GCA group with a mean age of 78.9 years. In the histologically negative group, there were four males and 17 females with a mean age of 67 years, and the control kidney vascular margin group had six males and nine females with a mean age of 38.2 years. There were no statistically significant difference in gender among the groups (p = 0.176). The clinical diagnosis of GCA was based on the criteria of the American College of Rheumatology[11]. However, for our study, we considered the gold standard to be histologic evidence of temporal arteritis. We also randomly selected 15 renal artery vascular resection margins from nephrectomy specimens in patients without any history of vasculitis for use as the negative controls.
Two methods were utilized to evaluate for HPV DNA. The first, INNO-LiPA HPV Genotyping Extra, was used to perform the testing in our research laboratory of the division of anatomic and molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis. All blocks were then submitted to an outside laboratory (CPA Laboratory, Louisville, KY) for independent preparation of genomic DNA and for testing employing the CervistaTM HPV HR (Hologic, Madison, WI, USA) assay.
In both laboratories to avoid potential contamination, a maximum physical separation between the pre- and post-amplification steps was used. Separate pipettes and other lab materials were used as a part of good laboratory practice. The FFPE blocks were cut and processed under strict conditions to prevent DNA from being carried over from one case to the next during microtomy. Also a new blade was used for each case and the area was cleaned. Ice cubes used to cool blocks were discarded between cases.
With regards to the technical limitations of this study, it is well known that formalin fixation randomly fragments DNA in a duration-dependent manner, resulting in a partial degradation. The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation. Due to this degradation, FFPE tissue is not suitable for amplification of large DNA segments. Nevertheless, PCR amplification of segments ranging up to 1300 base pair has been reported. On the other hand, incubation at an elevated temperature after proteinase K digestion partially removes formalin cross-linking of the DNA, improving yield as well as DNA performance in assays. Furthermore, in one of the techniques used in this study (INNO-LiPA HPV Genotyping), short-PCR-fragment (SPF 10) primers are employed, which amplify a 65 base pair segment of target DNA and this testing is considered to be one of the most sensitive PCR assays for the detection of HPV DNA.
Washington University testing
DNA extraction
DNA was extracted using PureGene Kit (Gentra,http://www.Gentra.com) as per the manufacturer's instructions from 10 μm sections cut from the paraffin blocks. The concentration of the prepped DNA was measured spectrophotometrically using Nanodrop. Detailed procedure information is available at their web site, but briefly, we placed five 10 micron sections of tissue into a 1.5 mL microcentrifuge tube and added 1.0 mL of xylene, vortexed, and incubated for five minutes with constant gentle mixing. Then we centrifuged it for five minutes at 13,000-16,000 x g. In the fume hood, discarded by pipetting the xylene supernatant and left behind the visible pellet (tissue). We repeated this xylene wash twice. We then added 1.0 mL 100% ethanol, vortexed, and incubated five minutes with constant gentle mixing at room temperature, centrifuged at 13,000-16,000 x g for five minutes to pellet the tissue, and discarded the ethanol. We repeated these ethanol washes twice. Subsequently we added 1.0 mL 70% ethanol, gently mixed, and centrifuged at 13,000-16,000 x g for five minutes at 4°C, removed all residual ethanol and allowed tissue pellet to dry by centrifugation under vacuum for five minutes. For cell lysis, we added 300 μl Cell Lysis Solution (Gentra Puregene™ kit) and gently vortexed for 30 seconds. Then we added three μl Puregene Proteinase K (20 mg/ml), and mixed by inverting 25 times and incubated the lysate at 55°C for three hours to overnight. We inverted the tube periodically during the incubation. Then was added three μl RNase A Solution to the cell lysate, and mixed by inverting the tube 25 times and incubated at 37°C for 15 min to one hour. For Protein Precipitation we cooled quickly the sample to room temperature by placing on ice and added 100 μl Protein Precipitation Solution (Gentra Puregene™ kit) to the cell lysate. The volume should be 1/3 of the Cell Lysis Solution in the tube. Subsequently, it is vortexed vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate. Then, we centrifuged it at 13,000-16,000 x g for five minutes. The precipitated proteins formed a tight pellet. If the protein pellet was not tight, we vortexed vigorously for 20 seconds at high speed, and then incubated on ice for 5 min. We then centrifuged at 13,000–16,000 x g for three minutes. Using a pipette, we removed the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a clean 1.5 mL microcentrifuge tube. We added two μl (for 400 μl supernatant) of a DNA carrier (glycogen; to final concentration of 50-150 mcg/μl) to aid recovery of small DNA quantities and then vortexed them. We then added 400 μl 100% of isopropanol. Subsequently, it was mixed by inverting gently ~50 times until the white threads of DNA formed a visible clump, and then it was centrifuged at 13,000-16,000 x g for 10 minutes and the supernatant was poured off. We added 500 μl of 70% ethanol and inverted the tube to wash the DNA pellet. We centrifuged at 13,000-16,000 x g for 5 minutes, and carefully poured off the ethanol and inverted and blotted the liquid from the tube on clean absorbent paper and allowed to air dry for 10-15 minutes. Finally, we added 50 μl DNA Hydration Solution and incubated at 65°C for one hour to dissolve the DNA. We incubated at room temperature overnight with gentle shaking. Samples could then be centrifuged briefly and transferred to a storage tube. The concentration of the DNA used for INNO-LiPA HPV Genotyping Extra testing in each case was 50 ng.
INNO-LiPA HPV Genotyping testing
The INNO-LiPA HPV Genotyping Extra is based on the principle of reverse hybridization. Part of the L1 region of the human papillomavirus (HPV) genome is amplified using short-PCR-fragment assay (SPF10 primers), and the resulting biotinylated amplicons are then denatured and hybridized with specific oligonucleotide probes.
An additional primer pair for the amplification of the human HLA-DPB1 gene is added to monitor sample quality and extraction. The length of the HLA-DPB1 fragment is 280 base pairs. All probes are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added, which binds to any biotinylated hybrid previously formed.
Incubation with BCIP (5-Bromo-4-Chloro-3’-Indolyphosphate p-Toluidine Salt)/NBT (Nitro-Blue Tetrazolium Chloride) chromogen yields a purple precipitate, and the results are visually interpreted using the reference guide provided. An amplification kit (INNO-LiPA HPV Genotyping Extra Amp) is used for standardized preparation of biotinylated amplified material. This amplification kit is based on the polymerase chain reaction (PCR) using SPF10 primers.
Amplification products are subsequently hybridized using a single typing strip on which 28 sequence-specific DNA probe lines and 4 control lines are fixed, which permits specific detection of 28 HPV genotypes, including all 18 high-risk genotypes, and 10 low-risk genotypes (HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74, and 82) as described by the manufacturer (Figure 3).
CPA Laboratory testing
DNA extraction
Slides were cut from the original blocks, and The QIAamp DNA FFPE Tissue kit (QIAGEN,http://www.qiagen.com) was used for purification of genomic DNA from formalin-fixed, paraffin-embedded tissues according to the manufacturer’s instructions with exception of incubation time. Overnight incubation in proteinase K for digestion of proteins/contaminants is not recommended by Qiagen but is something that CPA Laboratory has found useful to increase the nucleic acid elution.
Briefly, the QIAamp DNA FFPE Tissue procedure consisted of several steps including removal of paraffin from slides using xylene, subsequent specimen lysis under denaturing conditions with proteinase K, incubation at 90°C to reverse all formalin cross-linking, and DNA binding to the membrane for removal of contaminants. Residual contaminants were washed away and pure, concentrated DNA was eluted from the membrane.
CervistaTM HPV HR testing
CervistaTM HPV HR[12] is a qualitative, diagnostic test for the detection of DNA from 14 high-risk HPV types (i.e., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The CervistaTM HPV HR test uses the Invader® chemistry which is a signal amplification technique for detection of specific nucleic acid sequences. In this method two types of isothermal reactions are used: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal. During the primary reaction, two types of sequence specific oligonucleotides (i.e. a probe oligonucleotide and an Invader® oligonucleotide) bind to the target DNA recognition site. When those sequence specific oligonucleotides overlap by at least one base pair on the target sequence, an invasive structure forms that acts as a substrate for the Cleavase® enzyme. The enzyme cleaves the 5’ portion (flap) of the probe at the position of the overlap.
The probes are present in large molar excess and cycle rapidly on and off the target sequence generating many cleaved 5’ flaps per target sequence. The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide creating another invasive structure that the Cleavase® enzyme recognizes as a substrate. The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces a fluorescence signal as the cleaved flaps cycle on and off. For each copy of target, the combined primary and secondary reactions result in 106– 107 fold signal amplification per hour. The reagents for this test are provided as three oligonucleotide master mixtures, which identify the 14 types of HPV arranged according to phylogenetic relatedness. Master mixture 1 identifies the positivity of genotypes 51, 56 and 66(MM1: HPV 51, 56 and 66). Master mixture 2 (MM2) shows the positivity of genotypes HPV 18, 39, 45, 59 and 68 and master mixture 3 (MM3) reveals the positivity of HPV genotypes 16, 31, 33, 35, 52 and 58. By design, the released 5'-flaps bind only to their respective FRET oligonucleotides to generate target-specific signal. A positive result indicates that at least one of the 14 high-risk types is present in the DNA sample. For each case 150 to 200 ng of DNA prepped from the FFPE tissues at CPA Labs were utilized for the Cervista HPV testing. The sensitivity of this test is set at 5000 copies of HPV DNA. The Cervista assay has an internal control to verify if the assay worked. If the internal control does not pass, it suggests either the sample is degraded or extraction failed or the PCR did not work.
Statistics
Statistical analyses were performed using the Chi-Square, T-test and Fisher's exact tests to evaluate the correlation between the presence of HPV DNA and other variables. P values < 0.05 were considered significant. All statistical analyses were performed using the SAS v.9.1 system software (SAS Institute Inc., Cary, NC).
