RT-QuIC detection of chronic wasting disease prions in third eyelids from white-tailed deer
New cases and strains of the contagious and fatal neurodegenerative chronic wasting disease (CWD) continue to emerge in cervid populations globally – . In the United States, it is currently found in free-ranging cervids in 36 out of 50 states as of January 2025. Chronic wasting disease has been shown to reach a prevalence of over 50% once established and have negative population level effects on cervids – . There is currently no vaccine or treatment available, and the infectious prion can persist in the environment for years, making the disease challenging to manage. Nevertheless, wildlife agencies are investing considerable effort and money , into CWD management as cervids are of great economic, cultural, and ecological value. Additionally, while there is no documented case of humans becoming infected after eating meat from infected cervids – there is still uncertainty about the zoonotic potential of CWD . Successful disease control in wildlife requires proper disease surveillance and monitoring which involves sample collection and diagnostic testing. There are currently two United States Department of Agriculture (USDA) approved tests for detecting CWD in captive cervids: enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) provided by American Association of Veterinary Laboratory Diagnostician (AAVLD) accredited diagnostic laboratories that are part of the National Animal Health Laboratory Network. These assays are performed on the medial retropharyngeal lymph nodes (referred to as lymph nodes hereafter) and/or brainstem (obex) . Sample collection requires head separation and extensive dissection as these tissues are found near the spinal column and adjacent to other structures that can resemble lymph nodes . Therefore, anatomical knowledge is required, and it is sometimes recommended that the tissues are collected by state-certified CWD technicians, veterinarians , , or collaborating meat processors and taxidermists. An assay for tissues that are more readily available and apparent to untrained individuals would increase the ability and success of hunters and field personnel to collect samples. A current method of increasing sample size for testing is by providing head collection bins or check stations that allow hunters to drop off deer heads and have them tested for CWD, which is without cost to hunters in some states. While the bins have increased the number of hunter-submitted samples they require wildlife agency personnel to collect the heads, extract the samples, and ship them to a laboratory for testing. In contrast, if each hunter could extract and ship samples individually, it would reduce the burden on agencies processing heads from the bins and likely decrease the time between collection and reporting of results. Additionally, more hunters may be willing to submit samples for testing if the tissue was easier to collect and still allow them to keep the head for mounting and taxidermy purposes. In identifying alternative tissue types for CWD testing, lymphoid tissues should remain a target as the infectious prions first accumulate there before spreading to the nervous system, providing a higher probability of detecting early-stage disease – . The third eyelid, or nictitating membrane, contains lymphoid follicles and is found just beneath the lower eyelid in several mammalian species, including deer (Fig. a and b ). Furthermore, the third eyelid is a USDA-approved tissue used to detect scrapie , a prion disease of domestic sheep , . More recently, real-time quaking-induced conversion assay (RT-QuIC) has been used to detect CWD prions in third eyelids from inoculated white-tailed deer ( Odocoileus virginianus ) and naturally infected elk ( Cervus canadensis nelson ) . RT-QuIC is an amplification assay which can be used to detect infectious CWD prions in an array of tissue types such as lymph nodes, brain, outer ear, third eyelids, skin, recto anal mucosal-associated lymphoid tissue also known as RAMALT, skeletal muscle, interdigital scent gland, fluids such as urine, saliva, feces, and cerebrospinal fluid – . RT-QuIC is highly sensitive and able to detect the presence of infectious prions at very low concentrations (such as in tissues from hosts that are in an asymptomatic phase of disease) compared to the current USDA-approved tests – . Despite the increasing application of RT-QuIC in CWD research, it is not currently considered a validated diagnostic test with nationally standardized methods. For RT-QuIC to become a validated diagnostic test for CWD, the assay needs to be standardized and specified in a way that laboratory and trained personnel at accredited veterinary diagnostic laboratories can perform it successfully and consistently. Currently, individual laboratories have been choosing RT-QuIC instrument settings and analytical methods based on internal optimizations and preferences. As a result, it is difficult to compare results between laboratories and we sought to find a potential consistent approach to instrument settings. We hypothesize that the third eyelid can be a good tissue source for CWD diagnostics in naturally infected white-tailed deer, as it works for inoculated white-tailed deer and scrapie in sheep . To advance the goal of standardizing RT-QuIC for CWD detection, we compare RT-QuIC testing (Fig. c and d ) and IHC (Fig. e and f ) of third eyelids from naturally infected white-tailed deer to their USDA-approved CWD diagnostic results which are ELISA and IHC performed on lymph node and obex in an AAVLD-accredited veterinary diagnostic laboratory. Finally, to maximize the number of samples that can be tested at a time, we determine the success of RT-QuIC to test pooled third eyelids as a useful tool to screen more individuals at the same time, particularly in a population that is expected to be free of CWD infection. 

Samples from white-tailed deer with known CWD-status All samples were collected from white-tailed deer in Pennsylvania, USA. Lymph nodes and/or obex and third eyelids were opportunistically collected from each animal postmortem by CWD-certified personnel as part of statewide CWD surveillance either from road-killed deer, hunter-harvested deer, free-ranging clinical suspects, or culled captive deer. The majority of the individuals with lymph node and/or obex that tested ELISA/IHC positive were from the CWD-endemic area in southcentral Pennsylvania which includes the counties Bedford, Fulton, Blair and Huntington (12% prevalence (143/1193) in 2024/2025) , and the remaining individuals were both from within and outside the CWD-endemic area. The third eyelid was collected by grasping the visible portion located in the inner corner of the eye (Fig. a ) with forceps, pulling the tissue forward, and excising as much of the third eyelid tissue as possible with a scalpel (Fig. b ). Forceps and scalpels were disinfected between sampling of each individual by thoroughly removing any organic matter on the instruments with Dawn dish detergent, followed by soaking in a 10:1 water and household bleach solution for at least 10 min and rinsed by water. Obex and lymph nodes from captive individuals were processed and fixed in formalin at the Pennsylvania Animal Diagnostic Laboratory (PADLS) in Harrisburg, Pennsylvania and sent to the National Veterinary Services Laboratory in Ames, Iowa for IHC testing. All samples from free-ranging deer were sent to the PADLS laboratory at the University of Pennsylvania’s New Bolton Center, which is an AAVLD-accredited laboratory, where the lymph nodes and/or obices were tested by ELISA and/or IHC following USDA-approved protocols . Because both lymph node and obex were collected for some but not all the study animals, we refer to ELISA and IHC of “lymph node and/or obex” in this paper. The associated third eyelids for each animal were stored immediately upon receipt in a −80˚C freezer until they were tested by RT-QuIC at the University of Pennsylvania’s Wildlife Futures Program RT-QuIC laboratory and CWD Evolution laboratory in Colorado. Third eyelids were processed for IHC at the University of Pennsylvania’s PADLS New Bolton Center. Included in this study are the third eyelids from 250 white-tailed deer (captive n = 35 and wild n = 215) collected between 2021 and 2024 in Pennsylvania, with known CWD-status based on ELISA and/or IHC results from lymph node and/or obex. Of these, 121 individuals were confirmed CWD-positive, and 129 individuals were considered non-detect/negative for CWD. 

All samples were collected from white-tailed deer in Pennsylvania, USA. Lymph nodes and/or obex and third eyelids were opportunistically collected from each animal postmortem by CWD-certified personnel as part of statewide CWD surveillance either from road-killed deer, hunter-harvested deer, free-ranging clinical suspects, or culled captive deer. The majority of the individuals with lymph node and/or obex that tested ELISA/IHC positive were from the CWD-endemic area in southcentral Pennsylvania which includes the counties Bedford, Fulton, Blair and Huntington (12% prevalence (143/1193) in 2024/2025) , and the remaining individuals were both from within and outside the CWD-endemic area. The third eyelid was collected by grasping the visible portion located in the inner corner of the eye (Fig. a ) with forceps, pulling the tissue forward, and excising as much of the third eyelid tissue as possible with a scalpel (Fig. b ). Forceps and scalpels were disinfected between sampling of each individual by thoroughly removing any organic matter on the instruments with Dawn dish detergent, followed by soaking in a 10:1 water and household bleach solution for at least 10 min and rinsed by water. Obex and lymph nodes from captive individuals were processed and fixed in formalin at the Pennsylvania Animal Diagnostic Laboratory (PADLS) in Harrisburg, Pennsylvania and sent to the National Veterinary Services Laboratory in Ames, Iowa for IHC testing. All samples from free-ranging deer were sent to the PADLS laboratory at the University of Pennsylvania’s New Bolton Center, which is an AAVLD-accredited laboratory, where the lymph nodes and/or obices were tested by ELISA and/or IHC following USDA-approved protocols . Because both lymph node and obex were collected for some but not all the study animals, we refer to ELISA and IHC of “lymph node and/or obex” in this paper. The associated third eyelids for each animal were stored immediately upon receipt in a −80˚C freezer until they were tested by RT-QuIC at the University of Pennsylvania’s Wildlife Futures Program RT-QuIC laboratory and CWD Evolution laboratory in Colorado. Third eyelids were processed for IHC at the University of Pennsylvania’s PADLS New Bolton Center. Included in this study are the third eyelids from 250 white-tailed deer (captive n = 35 and wild n = 215) collected between 2021 and 2024 in Pennsylvania, with known CWD-status based on ELISA and/or IHC results from lymph node and/or obex. Of these, 121 individuals were confirmed CWD-positive, and 129 individuals were considered non-detect/negative for CWD. 

Third eyelids were thawed and trimmed to weights of 50 ± 5 mg, transferred to 2mL lysing tubes (Precellys hard tissue homogenizing CK28, Bertin Technologies, Montigny-le-Bretonneux, France) before adding 1X phosphate buffered saline pH 7.4 to achieve a 10% weight/volume concentration. Disposable forceps, scalpels and plastic plates were used for each sample to avoid cross-contamination. The samples were then homogenized using a Precellys Evolution homogenizer (Bertin Technologies) at 7500RPMs, 12 cycles of 30 s duration with 30 s breaks between each cycle. Homogenized samples were diluted to a concentration of 10 −2 using a diluted, commercially available dilution buffer (CWD 10X Sample Dilution Buffer, VMRD, Pullman, Washington, USA), resulting in a buffer containing 0.1% sodium dodecyl sulfate and 1X phosphate buffered saline. A concentration of 10 −2 is in this study achieved by diluting 5 µl of the 10% weight/volume homogenate in 45 µl of the commercially available buffer resulting in a 10 −1 dilution, and then diluting 5 µl of the 10 −1 dilution in 45 µl of the commercially available buffer to reach a final dilution of 10 −2 . 

To assess the performance of screening multiple third eyelids simultaneously using RT-QuIC, we combined 5 µl of the 10% weight/volume homogenized third eyelids with known CWD-status based on ELISA/IHC results of lymph node and/or obex in one tube. We tested the assay on pools of 10 samples and 5 samples to determine whether the number of pooled samples would increase or lower sensitivity and specificity. Each pool with 10 samples included either 10, 8, 6, 4, 2, 1, or 0 positive third eyelid homogenates and the remaining samples negative (Fig. , bottom row). Each pool with 5 samples included either 4, 2, 1, or 0 positive third eyelid homogenates and the remaining samples were negative (Fig. , top row). Each pooled sample was diluted to a concentration of 10 −2 using the same dilution buffer as described for the individual third eyelid homogenates above. We tested 32 pools of 5 samples (21 positive and 11 negative pools) and 46 pools of 10 samples (39 positive and 7 negative pools). Additionally, for the pools containing 5 samples we conducted a blinded test with 14 pools where 11 of these included third eyelids from negative deer only, and the remaining 3 pools contained third eyelids from 1 positive deer and 4 negative deer. The blinded test involved having one person combine different samples to create pools and have a second person conduct the assay and analyze the results without knowing the CWD-status of the samples in each pool. 

The RT-QuIC reaction solution was prepared using commercially available AA-90–231Syrian Hamster substrate (0.1 mg/mL, CWD Amplification Reagent, VMRD, expressed and purified as described previously , , ) and reaction solution (CWD 5X Reaction Buffer, VMRD) containing 5XPBS pH 7.4, 0.85 M NaCl, 5mM EDTA, and 50 µl Thioflavin T as previously described (however, N-2 cell culture media supplement was not added in our experiments). A total volume of 98 µl reaction solution and 2 µl 10 −2 diluted sample was added to each well of a 96-well optical-bottom plate (Item no: 655096, Greiner Bio-One, Monroe, North Carolina, USA). Homogenized lymph nodes from white-tailed deer in Pennsylvania with known CWD-status were used as positive and negative controls at 10 −3 dilutions using the same dilution buffer. A diluted lymph node homogenate with known high fluorescence value was selected as the positive control since RT-QuIC results on lymph nodes have already been proven to correlate 100% with ELISA/IHC on lymph nodes in free-ranging cervid species including white-tailed deer in Pennsylvania , . The plates were assayed on a BMG FLUOstar plate reader (BMG Labtech, Offenburg, Germany) for 62 h using the following settings: 42˚C, 250 cycles (700RPM with 60s shake/60s rest cycles), fluorescent scans every 15 min, at a gain of 1350. 

We tested the same protocol as described above but adjusted the gain setting to the positive control. An often-varied instrument setting is the gain, which determines the fluorescence measurement range on the microplate reader. The gain can be adjusted according to the intensity of fluorescence of the sample wells, with a higher gain being better able to intensify dim signals from sample wells and a lower gain setting preventing oversaturation from sample wells with higher signals . The plate reader manufacturer recommends that the gain is adjusted “on the sample with the expected highest signal output” . Differences in gain settings can change the time it takes for a sample to cross the set threshold and followingly be considered positive or negative. Therefore, we compared the results between a predetermined gain value (1350) and a gain that was adjusted based on the known positive control (i.e. the sample with the expected highest signal output) included on each plate as described under assay conditions above. We selected 30 third eyelids from deer that had tested positive ( n = 15) or negative ( n = 15) by ELISA/IHC on lymph nodes/obex, and deliberately selected 5 third eyelids that had false negative results by RT-QuIC at the initial gain of 1350 to see whether a gain adjustment could increase the sensitivity of the assay on one 96-well plate. Additionally, we compared 30 of the pooled samples which contained samples from a total of 10 animals in each pool using the adjusted gain setting with results from the initial gain of 1350. RT-QuIC data analysis – Wildlife Futures Program RT-QuIC laboratory The criteria to identify a sample as positive or negative was determined before the assays were run and was similar to methods previously described for CWD detection by RT-QuIC . A threshold value was calculated by averaging the fluorescence values of all wells, which contained either a sample or control, from the first three cycle scans and adding 10X the standard deviation. All samples were tested in replicates of 3, where a sample was considered positive when the fluorescence value of at least two out of three (2/3) wells crossed the threshold within 48 h. If 2/3 wells crossed the threshold after 48 h or not at all (time to threshold = 0), the sample was considered negative. The 48-hour cutoff was selected based on the positive samples’ times to threshold and because it yielded the highest sensitivity and specificity (Fig. ). Means, standard deviations (SD), and graphs were created in R version 4.3.1 (R Core Team, 2023), using RStudio version 2023.06.0. 

The criteria to identify a sample as positive or negative was determined before the assays were run and was similar to methods previously described for CWD detection by RT-QuIC . A threshold value was calculated by averaging the fluorescence values of all wells, which contained either a sample or control, from the first three cycle scans and adding 10X the standard deviation. All samples were tested in replicates of 3, where a sample was considered positive when the fluorescence value of at least two out of three (2/3) wells crossed the threshold within 48 h. If 2/3 wells crossed the threshold after 48 h or not at all (time to threshold = 0), the sample was considered negative. The 48-hour cutoff was selected based on the positive samples’ times to threshold and because it yielded the highest sensitivity and specificity (Fig. ). Means, standard deviations (SD), and graphs were created in R version 4.3.1 (R Core Team, 2023), using RStudio version 2023.06.0. 

Of the total 250 third eyelids, 60 of them (30 CWD-positive and 30 CWD-negative) were shipped to CWD Evolution, Fort Collins, Colorado, USA, for testing by RT-QuIC to validate the method across two separate locations and technicians using the exact same substrate, buffers, protocol, and RT-QuIC assay conditions. Samples were run in triplicate, for a total runtime of 48 h at a gain of 1350 and analyzed using the same threshold calculations as the Wildlife Futures RT-QuIC laboratory, which is described above. If there were any discrepant results between the two laboratories, a reevaluation of the lymph node and/or obex IHC would be completed to confirm the individual’s CWD status. 

A total of 60 third eyelids were prepared for formalin fixation and staining. All but one of these third eyelids were from the same group that was shipped for the inter-laboratory cross validation. This was because the contralateral third eyelid had insufficient remaining tissue and was therefore replaced by a contralateral third eyelid from another individual in the study. Each third eyelid was fixed by placing the eyelid flat, bulbar side (closest to or against the eye itself rather than palpebral or exposed side) down on a piece of thick paper followed by placing the paper and third eyelid in a 90mL specimen jar with 10% neutral buffered formalin, similar to a technique proposed for scrapie-diagnosis in sheep (Method 1 in citation, the difference in our method is we faced the bulbar side down to further prevent the tissue from curling upward during formalin fixation and O’Rourke et al. faced the bulbar side upward). After formalin fixation of at least 72 h, we transferred the third eyelids from the specimen jars to tissue cassettes. The peripheral edges of each third eyelid were trimmed to fit the cassette and cut transversely in up to three pieces, to ease paraffin embedding and cutting by microtome. Tissues were stained using a commercially available Anti-Prion (99) Research Kit (Roche Diagnostics, Indianapolis, Indiana, USA) at the PADLS laboratory at the University of Pennsylvania’s New Bolton Center. IHC slides were blindly interpreted (unknown RT-QuIC status) by a board-certified veterinary pathologist (author KN). Positive and negative interpretation was made based on experience of interpreting immunological staining in lymph nodes and obex. The third eyelid IHC results were primarily compared to the RT-QuIC results of the contralateral third eyelid for a tissue-specific comparison, but we also include a comparison of the ELISA/IHC results of the lymph nodes/obex in the results. Genetic analysis - Pennsylvania Veterinary Laboratory We conducted genetic analysis of any deer that had discrepant results, meaning deer which had tested CWD-positive by ELISA/IHC of their lymph nodes/obex but did not test positive by RT-QuIC on their third eyelids. DNA was extracted using the MagMAX CORE Nucleic Acid Purification kit (Applied Biosystems, Waltham, Massachusetts, USA) on tongue specimens collected using disposable forceps and scalpels between individuals to avoid cross-contamination. The prion protein gene ( PRNP ) was amplified using Platinum 2X Universal Mix (Invitrogen, Waltham, Massachusetts, USA) with 0.4 µM of each of primers CWD-13 (5′-TTTTGCAGATAAGTCATGGTGAAA3′) and CWD-LA (5′AGAAGATAATGAAAACAGGAAGGTTGC-3′). PCR conditions were as follows: 95 °C for 5 min, 10 cycles of denaturation at 95 °C for 45 s (s), annealing at 58 °C for 45 s, and extension at 72 °C for 90 s, followed by 35 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 90 s with a final extension at 72 °C for 5 min, as previously described . PCR products were purified (Qiagen, Hilden, Germany) and sequenced using amplification primers with Sanger sequencing, as previously described (Eurofins Scientific, Luxembourg) . All sequences were individually analyzed for conflicts and secondary peaks and aligned to the O. virginianus reference sequence, AF 156,185 (GenBank, Bethesda, Maryland, USA), considering only functional genes but not the pseudogene . The nucleotides and corresponding amino acid polymorphisms at codons 95 (Glutamine Q or Histidine H), 96 (Glycine G or Serine S), 116 (Alanine A or Glycine G), and 226 (Glutamine Q or Lysine K) were evaluated. The results were examined to determine if variants in the PRNP gene may correlate with delayed progression of CWD prions to the lymphoid tissue in the third eyelids. 

We conducted genetic analysis of any deer that had discrepant results, meaning deer which had tested CWD-positive by ELISA/IHC of their lymph nodes/obex but did not test positive by RT-QuIC on their third eyelids. DNA was extracted using the MagMAX CORE Nucleic Acid Purification kit (Applied Biosystems, Waltham, Massachusetts, USA) on tongue specimens collected using disposable forceps and scalpels between individuals to avoid cross-contamination. The prion protein gene ( PRNP ) was amplified using Platinum 2X Universal Mix (Invitrogen, Waltham, Massachusetts, USA) with 0.4 µM of each of primers CWD-13 (5′-TTTTGCAGATAAGTCATGGTGAAA3′) and CWD-LA (5′AGAAGATAATGAAAACAGGAAGGTTGC-3′). PCR conditions were as follows: 95 °C for 5 min, 10 cycles of denaturation at 95 °C for 45 s (s), annealing at 58 °C for 45 s, and extension at 72 °C for 90 s, followed by 35 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 90 s with a final extension at 72 °C for 5 min, as previously described . PCR products were purified (Qiagen, Hilden, Germany) and sequenced using amplification primers with Sanger sequencing, as previously described (Eurofins Scientific, Luxembourg) . All sequences were individually analyzed for conflicts and secondary peaks and aligned to the O. virginianus reference sequence, AF 156,185 (GenBank, Bethesda, Maryland, USA), considering only functional genes but not the pseudogene . The nucleotides and corresponding amino acid polymorphisms at codons 95 (Glutamine Q or Histidine H), 96 (Glycine G or Serine S), 116 (Alanine A or Glycine G), and 226 (Glutamine Q or Lysine K) were evaluated. The results were examined to determine if variants in the PRNP gene may correlate with delayed progression of CWD prions to the lymphoid tissue in the third eyelids. 

RT-QuIC testing– Wildlife Futures Program RT-QuIC laboratory Out of 121 white-tailed deer that were CWD-positive by ELISA and/or IHC of lymph node/ obex, 114 tested positive by RT-QuIC (94% sensitivity, Table ) on third eyelid tissue. All 129 white-tailed deer previously confirmed to be CWD-negative by ELISA and/or IHC-negative of lymph node and/or obex tested negative by RT-QuIC (100% specificity, Table ) on third eyelid tissue. Additionally, for pools with 10 samples we found that 37 of 39 positive pools which included third eyelid tissue from at least one confirmed CWD-positive deer (based on ELISA/IHC of lymph node and/or obex) also tested positive with RT-QuIC (95% sensitivity, Table ). All 7 negative pools with 10 samples which only contained third eyelids from confirmed negative deer tested negative (100% specificity, Table ). When pooling 10 samples together we found that pools which contained third eyelid from only one positive deer had a sensitivity of 75% (6 out of 8 pools tested positive, Table ). All 21 positive pools with 5 samples which included at least one third eyelid from a positive deer tested positive by RT-QuIC (100% sensitivity, Table ). Furthermore, the blinded test on 11 negative pools with third eyelids from 5 negative deer all tested negative by RT-QuIC (100% specificity, Table ), and the remaining 3 positive pools with third eyelid from one positive deer in the blinded test all tested positive (100% sensitivity). As the assays with 5 samples per pool were conducted after the gain optimization results below, we used the optimized gain adjustment for these 96-well plates (gain was 1546 and 1628). RT-QuIC plate reader gain optimization– Wildlife Futures Program RT-QuIC laboratory The gain was set to 1423 when adjusted to the positive control on the plate with individual samples. The 5 third eyelids that were from CWD-positive deer (lymh nodes and/or obex positive by ELISA/IHC) but had not tested positive by RT-QuIC initially did not test positive the second time despite the different gain setting. Overall, the optimized gain adjustment did not change the sensitivity or specificity of the assay but significantly reduced the time to threshold for the 10 positive samples that crossed the threshold from an average of 28.1 (SD = 4.2) to 20.3 (SD = 2.6) hours (paired T-test, t = 7.6, p = 0.00003, df = 9, Fig. a ). The pooled samples were tested on two plates where the gain was set to 1628 and 1409 when adjusted to the positive control. By adjusting the gain for the pooled samples, we were able to increase the sensitivity of the assay from 90% (27/30) to 93% (28/30, Fig. b ). Additionally, the gain adjustment significantly reduced the time to threshold from an average of 37.4 (SD = 8.6) to 28.3 (SD = 9.7) for the 30 pools that contained at least one third eyelid from a deer diagnosed with CWD (paired Wilcoxon signed-rank test, V = 398, p = 0.0003). The difference between the times to threshold before gain adjustment and after for the individual samples was normally distributed (Shapiro-Wilk test, p = 0.6905), and non-normally distributed for the pooled samples (Shapiro-Wilk test, p = 0.0273). Inter-laboratory cross validation Both laboratories diagnosed 29 third eyelids as CWD-positive by RT-QuIC out of a total of 30 third eyelids that were from CWD-positive deer (97% sensitivity, with the same third eyelid sample testing falsely negative, Table ). The third eyelids from negative deer ( n = 30) had one discrepant result between the two labs. The one discrepant sample (ID = 97) had 2/3 replicate wells cross the threshold at 46 and 49 h in the Wildlife Futures laboratory but was still considered negative since less than two wells crossed the threshold within the 48-hour cutoff (Table ). However, this sample crossed the threshold at 10, 20, and 40 h in the CWD Evolution laboratory, meaning it would be considered a CWD-positive result by RT-QuIC (Table ). Due to the discrepancy, we reevaluated the lymph node IHC results to confirm the individual’s CWD status and discovered that there was weak staining in multiple follicles in the IHC-stained lymph node, consistent with being considered CWD-positive. Third eyelid tissue sampling and preparation for IHC – PADLS New Bolton Center The third eyelids of 60 deer were tested by IHC and included 32 that were RT-QuIC negative and 28 that were RT-QuIC positive of the contralateral third eyelid (Table ). Seven of these (11.7%, 2 RT-QuIC positive, 5 RT-QuIC negative) were considered non-diagnostic as they lacked visible lymphoid tissue in the sections and were excluded from the following results. The lack of lymphoid tissue visibility in this subset was due to a combination of factors including artifactual tearing of the tissue near the cartilage, the undulations during fixation preventing enough tissue surface area, and the cartilage hindering consistent sticking of the tissue to the glass slide. Of the 32 third eyelids that were RT-QuIC negative, 19 were considered IHC negative (Fig. c ). The 8 that were RT-QuIC negative but interpreted as IHC positive had faint but consistent intra-follicular staining. One of these 8 was from a deer that had tested positive by IHC/ELISA on the lymph node, meaning that RT-QuIC was unable to diagnose this third eyelid as positive, but IHC was. Of the 28 third eyelids that were RT-QuIC positive, 21 were considered IHC positive (81%, Fig. a and b ) based on a spectrum of weak to strongly positive staining exclusive to the lymphoid follicle. Genetic analysis - Pennsylvania Veterinary Laboratory Genetic analysis was performed on a total of 8 discrepant samples; the 7 deer which were CWD-positive by testing lymph nodes with ELISA/IHC but where RT-QuIC was negative when testing third eyelids and one deer that had discrepant results in the inter-laboratory cross validation (ID = 97, initially ELISA/IHC of lymph node was negative and RT-QuIC of third eyelid was negative in the Wildlife Futures RT-QuIC laboratory but ended up being positive in the CWD Evolution laboratory). Codons 95, 96, 116 and 226 were all reviewed for their genotypic variation. All 7 individuals with discrepancy between ELISA/IHC on lymph node and RT-QuIC on third eyelid were homozygous at codons 116 and 226 expressing AA and QQ respectively. One individual was heterozygous at codon 95, expressing QH, and the other 6 were QQ. Two other individuals were heterozygous at 96 expressing GS, with all other samples homozygous expressing GG. The one deer with discrepant results in the inter-laboratory cross validation was also homozygous for all the studied codons (95QQ, 96GG, 116AA, 226QQ). These results are similar to previously published studies which showed little genetic variability in white-tailed deer from Pennsylvania , . For this reason, and due to the limited number of false negative results in this study, additional genotyping was not done as there is not a statistically significant difference from the previously published studies. 

Out of 121 white-tailed deer that were CWD-positive by ELISA and/or IHC of lymph node/ obex, 114 tested positive by RT-QuIC (94% sensitivity, Table ) on third eyelid tissue. All 129 white-tailed deer previously confirmed to be CWD-negative by ELISA and/or IHC-negative of lymph node and/or obex tested negative by RT-QuIC (100% specificity, Table ) on third eyelid tissue. Additionally, for pools with 10 samples we found that 37 of 39 positive pools which included third eyelid tissue from at least one confirmed CWD-positive deer (based on ELISA/IHC of lymph node and/or obex) also tested positive with RT-QuIC (95% sensitivity, Table ). All 7 negative pools with 10 samples which only contained third eyelids from confirmed negative deer tested negative (100% specificity, Table ). When pooling 10 samples together we found that pools which contained third eyelid from only one positive deer had a sensitivity of 75% (6 out of 8 pools tested positive, Table ). All 21 positive pools with 5 samples which included at least one third eyelid from a positive deer tested positive by RT-QuIC (100% sensitivity, Table ). Furthermore, the blinded test on 11 negative pools with third eyelids from 5 negative deer all tested negative by RT-QuIC (100% specificity, Table ), and the remaining 3 positive pools with third eyelid from one positive deer in the blinded test all tested positive (100% sensitivity). As the assays with 5 samples per pool were conducted after the gain optimization results below, we used the optimized gain adjustment for these 96-well plates (gain was 1546 and 1628). 

The gain was set to 1423 when adjusted to the positive control on the plate with individual samples. The 5 third eyelids that were from CWD-positive deer (lymh nodes and/or obex positive by ELISA/IHC) but had not tested positive by RT-QuIC initially did not test positive the second time despite the different gain setting. Overall, the optimized gain adjustment did not change the sensitivity or specificity of the assay but significantly reduced the time to threshold for the 10 positive samples that crossed the threshold from an average of 28.1 (SD = 4.2) to 20.3 (SD = 2.6) hours (paired T-test, t = 7.6, p = 0.00003, df = 9, Fig. a ). The pooled samples were tested on two plates where the gain was set to 1628 and 1409 when adjusted to the positive control. By adjusting the gain for the pooled samples, we were able to increase the sensitivity of the assay from 90% (27/30) to 93% (28/30, Fig. b ). Additionally, the gain adjustment significantly reduced the time to threshold from an average of 37.4 (SD = 8.6) to 28.3 (SD = 9.7) for the 30 pools that contained at least one third eyelid from a deer diagnosed with CWD (paired Wilcoxon signed-rank test, V = 398, p = 0.0003). The difference between the times to threshold before gain adjustment and after for the individual samples was normally distributed (Shapiro-Wilk test, p = 0.6905), and non-normally distributed for the pooled samples (Shapiro-Wilk test, p = 0.0273). 

Both laboratories diagnosed 29 third eyelids as CWD-positive by RT-QuIC out of a total of 30 third eyelids that were from CWD-positive deer (97% sensitivity, with the same third eyelid sample testing falsely negative, Table ). The third eyelids from negative deer ( n = 30) had one discrepant result between the two labs. The one discrepant sample (ID = 97) had 2/3 replicate wells cross the threshold at 46 and 49 h in the Wildlife Futures laboratory but was still considered negative since less than two wells crossed the threshold within the 48-hour cutoff (Table ). However, this sample crossed the threshold at 10, 20, and 40 h in the CWD Evolution laboratory, meaning it would be considered a CWD-positive result by RT-QuIC (Table ). Due to the discrepancy, we reevaluated the lymph node IHC results to confirm the individual’s CWD status and discovered that there was weak staining in multiple follicles in the IHC-stained lymph node, consistent with being considered CWD-positive. 

The third eyelids of 60 deer were tested by IHC and included 32 that were RT-QuIC negative and 28 that were RT-QuIC positive of the contralateral third eyelid (Table ). Seven of these (11.7%, 2 RT-QuIC positive, 5 RT-QuIC negative) were considered non-diagnostic as they lacked visible lymphoid tissue in the sections and were excluded from the following results. The lack of lymphoid tissue visibility in this subset was due to a combination of factors including artifactual tearing of the tissue near the cartilage, the undulations during fixation preventing enough tissue surface area, and the cartilage hindering consistent sticking of the tissue to the glass slide. Of the 32 third eyelids that were RT-QuIC negative, 19 were considered IHC negative (Fig. c ). The 8 that were RT-QuIC negative but interpreted as IHC positive had faint but consistent intra-follicular staining. One of these 8 was from a deer that had tested positive by IHC/ELISA on the lymph node, meaning that RT-QuIC was unable to diagnose this third eyelid as positive, but IHC was. Of the 28 third eyelids that were RT-QuIC positive, 21 were considered IHC positive (81%, Fig. a and b ) based on a spectrum of weak to strongly positive staining exclusive to the lymphoid follicle. 

Genetic analysis was performed on a total of 8 discrepant samples; the 7 deer which were CWD-positive by testing lymph nodes with ELISA/IHC but where RT-QuIC was negative when testing third eyelids and one deer that had discrepant results in the inter-laboratory cross validation (ID = 97, initially ELISA/IHC of lymph node was negative and RT-QuIC of third eyelid was negative in the Wildlife Futures RT-QuIC laboratory but ended up being positive in the CWD Evolution laboratory). Codons 95, 96, 116 and 226 were all reviewed for their genotypic variation. All 7 individuals with discrepancy between ELISA/IHC on lymph node and RT-QuIC on third eyelid were homozygous at codons 116 and 226 expressing AA and QQ respectively. One individual was heterozygous at codon 95, expressing QH, and the other 6 were QQ. Two other individuals were heterozygous at 96 expressing GS, with all other samples homozygous expressing GG. The one deer with discrepant results in the inter-laboratory cross validation was also homozygous for all the studied codons (95QQ, 96GG, 116AA, 226QQ). These results are similar to previously published studies which showed little genetic variability in white-tailed deer from Pennsylvania , . For this reason, and due to the limited number of false negative results in this study, additional genotyping was not done as there is not a statistically significant difference from the previously published studies. 

Our findings support that RT-QuIC of individual and pooled third eyelids from white-tailed deer can be used to detect the CWD prion in white-tailed deer in a diagnostic setting. This demonstrates that RT-QuIC is reliable to detect CWD from naturally infected captive and wild white-tailed deer across laboratories building upon previous results from inoculated white-tailed deer in a research facility . The RT-QuIC assay did not identify any false positives (100% specificity) in the Wildlife Futures laboratory but was unable to detect 7 third eyelids from deer that had lymph node and/or obex previously diagnosed as CWD-positive by ELISA/IHC (sensitivity 94%). Genetic analysis was performed to understand if there was a genetic bias among these discrepant samples, and results showed that 3/7 carried either QH or GS heterozygosity (considered more resistant) but the remainder were homozygous (considered less resistant). Overall, the number of discrepancies between ELISA/IHC of lymph node and/or obex and RT-QuIC on third eyelids were rather low in our study (8/250 or 3%, including the discrepancy for sample 97). As 3/7 deer did harbor heterozygous genetic variations in the PRNP gene that are believed to delay the disease progression of CWD (one 95 H/Q and two 96G/S) compared to homozygous variations that are more susceptible (QQ/GG/AA/QQ) 53 , it is possible that the genotype could have contributed to these false negative results. However, in previous studies it was found that the more susceptible homozygous genotypes for PRNP were most prevalent in Pennsylvania white-tailed deer and that the few individuals that were heterozygous (HQ or GS) and thus carrying a more resistant PRNP genotype still tested positive indicating that these genotypes likely do not have a significant impact on RT-QuIC test performance , . However, we consider this sample size to be too small to make any conclusions on whether QH or GS would be more likely to test negative for CWD by RT-QuIC on third eyelids. An expanded study in wider geographic regions can further tease out discrepancies encountered with testing and use of RT-QuIC. The discrepancy between the positive ELISA/IHC results of the lymph node and/or obex and the negative RT-QuIC results of third eyelids could be because the infectious prion protein had not yet aggregated in the third eyelid of these individuals, or that the 50 ± 5 mg piece of third eyelid we tested did not contain lymphoid tissue or detectable concentration of infectious prions, similar to what has been previously reported for IHC of lymph nodes . This could also explain the discrepancy which was observed for the CWD-positive deer where one third eyelid was considered negative by RT-QuIC but the contralateral eyelid was considered positive by IHC. Additional studies could further determine differences in timing of when different lymphatic tissues test positive during the course of a CWD infection. Nevertheless, the high specificity of this assay yields a confident positive result, which is particularly important when evaluating results from samples in an area close to and/or outside an established CWD endemic zone. The inter-laboratory cross validation supports that results are reproducible in separate laboratories and by different technicians, with minor differences. The discrepancy between the two laboratories’ results was that one third eyelid from a deer that previously tested negative by ELISA/IHC on the lymph node tested positive by RT-QuIC in the CWD Evolution laboratory, while it was considered negative by RT-QuIC in the Wildlife Futures RT-QuIC laboratory. Reevaluation of the IHC staining for that individual’s lymph node was consistent with a weak CWD-positive result. Thus, it is likely that this individual was in the early phase of the disease with a low number of stained follicles in the lymph node that were missed in the initial screening of the histology slide. This finding emphasizes the high sensitivity of RT-QuIC as individual 97’s third eyelid had multiple wells cross the threshold within 10–49 h in both laboratories but was not considered positive at initial IHC screening of the lymph node associated with that deer. Despite this discrepancy between laboratory results, the sensitivity and specificity were very similar (Table ) which illustrates how RT-QuIC of third eyelids can produce reliable results across laboratories and technicians. By following the plate reader manufacturer’s recommendations of adjusting the gain by the wells with the highest expected fluorescence value (known positive control), we were able to significantly reduce the time it took for a sample to cross the set threshold to be considered positive without compromising the sensitivity and specificity. For individual samples, we were able to reduce the time to threshold by 8 h and 9 h for pooled samples which is beneficial as it can facilitate faster result reporting and the possibility to overall reduce the assay length. The lower sensitivity that was observed in the pools of 10 samples occurred in pools that included only 1 or 2 third eyelids from deer with lymph node and/or obex that had previously tested CWD-positive. For the initial testing where the gain was manually set to 1350, 3/5 pools that included 1 third eyelid from a CWD-positive deer and 4/5 pools that included 2 third eyelids from CWD-positive deer were considered positive. However, by adjusting the gain to a known positive control, we were able to increase the sensitivity for the pools of 10 samples which contained 2 third eyelids from CWD-positive deer from 4/5 to 5/5 positive results, which increased the overall sensitivity of pools of 10 samples from 90 to 93%. Combining 10 samples in a pool was less ideal due to a sensitivity of 75% (6/8) for pools which only contained one third eyelid from a CWD-positive deer, whereas combining 5 samples per pool offered 100% sensitivity (21/21) and 100% specificity (11/11), including all pools which only contained one third eyelid from a CWD-positive deer (7/7 positive). Pooling 10 samples increases the possibility of false negatives in situations where only 1/10 samples is from a CWD-positive deer, possibly due to dilution or inhibitory factor of pooling this many samples together. In contrast, the pooling of 5 samples was promising and would allow laboratories to test 150 individuals in 30 pooled samples, which is 5 times the number of individuals that can be tested individually by RT-QuIC using 3 replicates per sample on a 96-well plate. This demonstrates the ability of RT-QuIC testing on third eyelids to increase the total number of individuals that are screened for CWD without reducing sensitivity and specificity. As RT-QuIC is currently not a validated test for diagnosing CWD, immunohistochemical staining of third eyelids showed to be a promising follow-up or confirmatory test following a positive RT-QuIC result. However, interpretation of the third eyelids by IHC was challenging for several reasons, including the overall paucity of visible lymphoid follicles compared to lymph nodes, the cartilage hindering smooth and consistent cutting of the microtome resulting in tearing and shattering of the section placed on the slide, and a wide variety of immune staining intensity including unexpectedly common false positive or non-specific staining. Considering third eyelids are unique compared to lymph node and obex with respect to tissue size, shape, and texture, further optimization of this tissue is required during all stages of testing. This includes the collection phase to minimize crushing of the tissue upon extraction of the deer, fixation stage to ensure the tissue is flat to maximize the surface area to view on the slide, slide processing stage to reduce the cartilage hindering the ability to smoothly and consistently cut tissue sections with the microtome, and staining stage to ensure appropriate duration of antigen-antibody binding to minimize non-specific or false positive staining. Additionally, determining a minimum number of lymphoid follicles that need to be present and examined to ensure a non-detection similar to lymph nodes would be a valuable step in validation and should be established prior to widespread use. As lymphoid tissue in the third eyelid can be less developed in juveniles , age of the animal was considered as one reason for the paucity of interpretable lymphoid tissue. However, all three fawns included in this analysis were correctly identified as positive ( n = 1) or negative ( n = 2) suggesting factors other than age are more likely to affect the required amount of lymphoid tissue available to interpret, including the aforementioned factors related to fixation, trimming, and slide preparation. Nonetheless, IHC was considered useful as a confirmatory test and often (case from Fig. ), but not always, showed strong, intense immunoreactivity that was easily interpretable by diagnosticians experienced in interpreting IHC slides. Further optimization of IHC-staining third eyelids is necessary before the authors would like to recommend it as part of a diagnostic tool for CWD. Our results support that third eyelids could be used as an alternative or in addition to the current accepted tests for detecting CWD, with three major management implications. First, as the tissue is easier to collect than lymph nodes, and does not require deep dissection, hunters could more easily collect this sample, which would greatly reduce the financial cost and workload of wildlife agencies. Further investigations that include third eyelid and lymph nodes and/or obex submitted by hunters should be conducted to evaluate the assay’s sensitivity and specificity. Secondly, since third eyelids can be tested in pools, it enables laboratories to screen a higher number of samples at once, decreasing the costs of running each sample individually, and increasing the turnaround time for reporting results. Third, easier sample collection and the ability to screen a higher number of samples at a time enable wildlife agencies to expand their surveillance capabilities. While IHC is considered the gold standard by the USDA to diagnose CWD, it needs to be appreciated that samples from wild cervids are not always in an ideal condition for this assay, where delays in submission, decomposition, and freezing can make disease detection, particularly in early stages, challenging to assess by a pathologist. In addition, many states have areas that are under-surveyed for CWD (such as areas thought to have no CWD prevalence) where providing a highly sensitive (94%) assay with the above considerations can improve a state’s disease surveillance plan. We demonstrated that third eyelids from white-tailed deer can be tested by RT-QuIC individually, and in pooled samples with high sensitivity and specificity. IHC of third eyelids can be used as a follow-up test to confirm a sample that tested positive by RT-QuIC but should not yet be used in isolation. Together, these results provide support for further investigation of third eyelids as a potential target sample for CWD diagnostics in an effort to reduce the cost of testing while expanding surveillance capacity. 

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Supplementary Material 4 Supplementary Material 5 Supplementary Material 6 Supplementary Material 7 

