Aims: This study aims to investigate the potential influence of melanin content on the performance of the Idylla™ BRAF Mutation Test. Specifically, we assess whether melanin levels in samples impact the test’s reliability, thereby validating its clinical utility in accelerating melanoma diagnosis and potentially improving patient prognosis. Methods: We conducted a retrospective analysis of 98 confirmed melanoma samples collected between February 2020 and November 2020. Formalin-fixed paraffin-embedded (FFPE) slides were evaluated by two independent observers using light microscopy to categorise samples into three groups based on melanin content (no, low, or high) following a standardised system. The samples underwent the Idylla™ BRAF Mutation Test and were compared with results obtained from next-generation sequencing (NGS). Results: Quantification cycle (Cq) values were utilised to assess for interference from melanin levels on the Idylla™ BRAF Mutation Test results. Statistical analyses revealed no significant differences in Cq values based on melanin content categories. Furthermore, analysis of polymerase chain reaction PCR curves did not indicate any notable influence of melanin. Discordant results with NGS are discussed. Conclusions: The study demonstrates that melanin content in samples does not significantly affect the performance of the Idylla™ BRAF Mutation Test. These results provide robust evidence supporting the confident application of the test in clinical settings, even for samples with high melanin content. The ability to obtain rapid on-site results holds promising potential in guiding timely and appropriate treatment decisions, thereby contributing to improved patient prognosis. What is already known on this topic—Prior research conducted by Petty et al. (2020) including 23 melanoma samples suggested that melanin does not significantly interfere with the Idylla™ BRAF Mutation Test by stating they were concordant with reference laboratory testing. What this study adds—This current study builds upon prior research with a larger sample size of 98. In addition to examining concordance between the Idylla™ BRAF Mutation Test and next generation sequencing, this study examines PCR curves and effect on Cq values, providing more robust evidence that melanin content in FFPE samples does not have a significant impact on the accuracy of the Idylla™ BRAF Mutation Test. How this study might affect research, practice or policy—The additional evidence base provided by this study is valuable for researchers, clinicians, and policymakers, as it supports the integration of the Idylla™ BRAF Mutation Test as a rapid and accurate method for detecting these mutations in melanoma patients.

Background/Objectives: Celiac disease (CD) is a T-cell mediated autoimmune condition, triggered by gluten ingestion. Duodenal biopsy is the gold-standard diagnosis for CD, which is often limited by interobserver variability between pathologists. Immunohistochemistry (IHC) is a powerful technique for detecting biomarkers with potential diagnostic significance. This study aims to investigate five candidate biomarkers BTNL8, NKp46, TdT, THEMIS, and TCRδ that might improve the reproducibility of the diagnosis of CD. Methods: Formalin fixed paraffin-embedded material, surplus to diagnostic requirements was obtained from 46 subjects (untreated CD: n=21, CD treated with gluten-free diet: n=5; controls: n=20) and immunostained for BTNL8, NKp46, TdT, THEMIS and TCRδ. BTNL8 staining was scored on a 0-3 semi-quantitative scale. NKp46, TdT, THEMIS, and TCR delta-positive intra-epithelial lymphocytes (IELs) were quantified as mean counts per 100 epithelial cells (ECs). Results: TCRδ-positive IELs were markedly elevated in CD biopsies (median 9.4 IELs/100 ECs) compared to healthy controls (median 0.5 IELs/100 ECs; p2.1 TCRδ-positive IELs per 100 ECs yielding an AUC of 94% and interobserver agreement of 0.82. NKp46 expression was also increased in CD (median 13.8 IELs/100 ECs) versus controls (median 9.6; p12.8 NKp46-positive IELs per 100 ECs achieving an AUC of 86% and interobserver agreement of 0.82. Immunostaining for the other biomarkers demonstrated less clear differences between CD and healthy controls. Conclusions: Corroborating several recent publications, TCRδ immunostaining provides high diagnostic accuracy and good interobserver agreement in the diagnosis of CD on duodenal biopsy, even for patients on a gluten-free diet.

Background/Objectives: Celiac disease (CD) is a T-cell-mediated autoimmune condition, triggered by gluten ingestion. Duodenal biopsy is the gold-standard diagnosis for CD, which is often limited by interobserver variability between pathologists. Immunohistochemistry (IHC) is a powerful technique for detecting biomarkers with potential diagnostic significance. This study aims to investigate five candidate biomarkers, BTNL8, NKp46, TdT, THEMIS, and TCRδ, that might improve the reproducibility of the diagnosis of CD. Methods: Formalin-fixed paraffin-embedded material, surplus to diagnostic requirements, was obtained from 46 subjects (untreated CD: n = 21, CD treated with gluten-free diet: n = 5; controls: n = 20) and immunostained for BTNL8, NKp46, TdT, THEMIS and TCRδ. BTNL8 staining was scored on a 0–3 semi-quantitative scale. NKp46, TdT, THEMIS, and TCR delta-positive intra-epithelial lymphocytes (IELs) were quantified as mean counts per 100 epithelial cells (ECs). Results: TCRδ-positive IELs were markedly elevated in CD biopsies (median 9.4 IELs/100 ECs) compared to healthy controls (median 0.5 IELs/100 ECs; p < 0.001), with a threshold of >2.1 TCRδ-positive IELs per 100 ECs yielding an AUC of 0.94 and interobserver agreement of 0.82. NKp46 expression was also increased in CD (median 13.8 IELs/100 ECs) versus controls (median 9.6; p < 0.001), with >12.8 NKp46-positive IELs per 100 ECs achieving an AUC of 0.86 and interobserver agreement of 0.82. Immunostaining for the other biomarkers demonstrated less clear differences between CD and healthy controls. Conclusions: Corroborating several recent publications, TCRδ immunostaining provides high diagnostic accuracy and good interobserver agreement in the diagnosis of CD on duodenal biopsy, even for patients on a gluten-free diet.

Background/Objectives: T-cell lymphomas are relatively common in veterinary species, yet current diagnostic tools such as PCR-based clonality assays often lack sensitivity and specificity. In humans, we recently developed two related tissue-based diagnostic approaches based on the differential detection of the mutually exclusively expressed TCRbeta1 and 2 (TCRβ1 and 2) constant region proteins, or the corresponding TRBC1 and TRBC2 transcripts. Analogous to the detection of kappa/lambda light chains for the diagnosis of B-cell/plasma cell neoplasms in human clinical practice, our TCRβ1/2 diagnostic assay has the potential to transform veterinary diagnostic workflows. Methods: We identified and confirmed the sequences of the relevant TRBC1 and TRBC2 sequences in both cats and dogs, focusing on the 3′ untranslated region (UTR), where there is the least sequence homology between TRBC1 and TRBC2. To allow us to design appropriate probe sequences, we confirmed a lack of 3′UTR in either species, and we observed limited 3′ untranslated region UTR sequence polymorphism in the cat but not in the dog 3′UTR. We designed BaseScope™ RNA in situ hybridization probes targeting the 3′ UTR to distinguish between TRBC1 and TRBC2 transcripts in formalin-fixed paraffin-embedded tissues. Results: In normal tissues, we found the TRBC2:TRBC1 expression ratio to be similar to the 1.2:1 ratio in humans, between 1:1 and 3:1, skewing towards TRBC2, in both dogs and cats. These findings were corroborated using quantitative reverse transcription PCR. Applying our in situ hybridization probes to cases of T-cell lymphoma in dogs and cats, we demonstrated that an assay for differential expression of TRBC1 and TRBC2 in T-cell populations could identify clonal T-cell populations, as in human diagnostics. If further studies corroborate this proof-of-concept study, TRBC1/2 detection could obviate the need for slow, complex and expensive multiplexed PCR-based (PCR for antigen receptor rearrangements (PARR)) clonality assays. Conclusions: This study provides proof-of-concept data for a novel diagnostic approach that could simplify and substantially improve the accuracy of lymphoma diagnostics in veterinary medicine, by detecting TRBC1/2 transcripts.