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Barrett's esophagus (BE) is a known precursor to esophageal adenocarcinoma (EAC). Guidelines recommend BE screening in populations with multiple risk factors, for which non-endoscopic esophageal cell collection with biomarker testing is considered as an acceptable alternative to esophagogastroduodenoscopy (EGD). The aim of this study was to evaluate analytical performance characteristics of EsoGuard® (EG), a DNA methylation biomarker assay, as a laboratory-developed test (LDT) in esophageal samples collected with the swallowable EsoCheck® (EC) device. EG is a next-generation sequencing (NGS) assay that evaluates methylated vimentin (VIM) and cyclin A1 (CCNA1), clinically validated biomarkers for the detection of BE and EAC. The studies were conducted according to standards of College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA), and New York (NY) state requirements for the analytical validation of molecular assays. Comparison to Sanger sequencing showed that EG was 100% accurate at all 31 CpG sites evaluated by the assay. The analytical sensitivity, specificity, and accuracy of the assay were 89%, 100%, and 96%, respectively. Intra- and inter-assay precision was 100%. The limit of detection (LOD) was 1 in 400 methylated cells, and the reference range was 84%. In summary, EsoGuard demonstrates high analytical accuracy, repeatability, and reproducibility in samples collected using the EsoCheck device.

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Perineural invasion (PNI) is defined as the dissemination of neoplastic cells within the perineural space. PNI can be a strong indicator of malignancy and is linked to poor prognosis and adverse outcomes in various malignant neoplasms; nevertheless, it can also be seen in benign pathologic conditions. In this review article, we discuss various signaling pathways and neurotrophic factors implicated in the development and progression of PNI. We also describe the methodology, benefits, and limitations of different in vitro, ex vivo, and in vivo models of PNI. The spectrum of presentation for PNI can range from diffuse spread within large nerves ("named" nerves) all the way through localized spread into unnamed microscopic nerves. Therefore, the clinical significance of PNI is related to its extent rather than its mere presence or absence. In this article, we discuss the guidelines for the identification and quantification of PNI in different malignant neoplasms based on the College of American Pathologists (CAP) and World Health Organization (WHO) recommendations. We also describe benign pathologic conditions and neoplasms demonstrating PNI and potential mimics of PNI. Finally, we explore avenues for the future development of targeted therapy options via modulation of signaling pathways involved in PNI.

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Tumor prognosis hinges on accurate cancer staging, a pivotal process influenced by the identification of lymphovascular invasion (LVI), i.e., blood vessel and lymphatic vessel invasion. Protocols by the College of American Pathologists (CAP) and the World Health Organization (WHO) have been established to assess LVI in various tumor types, including, but not limited to, breast cancer, colorectal cancer (CRC), pancreatic exocrine tumors, and thyroid carcinomas. The CAP refers to blood vessel invasion as "angioinvasion" (vascular invasion) to differentiate it from lymphatic vessel invasion (lymphatic invasion). For clarity, the latter terms will be used throughout this review. The presence of lymphatic and/or vascular invasion has emerged as a pivotal prognostic factor; therefore, its accurate identification is crucial not only for staging but also for providing the patient with an honest understanding of his/her prognosis. Given the prognostic importance of the correct identification of LVI, specific staining techniques are employed to distinguish lymphatic vessel invasion from angioinvasion and to differentiate true LVI from artifact. These encompass hematoxylin and eosin (H&E) staining, elastic staining, Factor VIII staining, Ulex europaeus I agglutinin staining, CD31, CD34, D2-40, ERG, and D2-40 (podoplanin) immunohistochemical (IHC) stains among others. Based on a review of numerous publications regarding the efficacy of various methods for LVI detection, elastin staining demonstrated superior accuracy and prognostic value, allowing for more targeted treatment strategies. The clinical significance of accurately detecting LVI cannot be overstated, as it is strongly linked to higher cancer-related mortality and an increased risk of tumor recurrence. This review aims to examine the existing literature on the use of elastin stains in the detection of vascular invasion among different types of tumors and its prognostic value.

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Introduction: Therapeutic drug monitoring (TDM) of immunosuppressants is essential for optimal care of transplant patients. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) are the most commonly used methods for TDM. However, immunoassays can suffer from interference from heterophile antibodies and structurally similar drugs and metabolites. Additionally, nominal-mass LC-MS assays can be difficult to optimize and are limited in the number of detectable compounds. Objectives: The aim of this study was to implement a mass spectrometry-based test for immunosuppressant TDM using online solid-phase extraction (SPE) and accurate-mass full scan-single ion monitoring (FS-SIM) data acquisition mode. Methods: LC-MS analysis was performed on a TLX-2 multi-channel HPLC with a Q-Exactive Plus mass spectrometer. TurboFlow online SPE was used for sample clean up. The accurate-mass MS was set to positive electrospray ionization mode with FS-SIM for quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A. MS2 fragmentation pattern was used for compound confirmation. Results: The method was validated in terms of precision, analytical bias, limit of quantitation, linearity, carryover, sample stability, and interference. Quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A correlated well with results from an independent reference laboratory (r = 0.926-0.984). Conclusions: Accurate-mass FS-SIM can be successfully utilized for immunosuppressant TDM with good correlation with results generated by standard methods. TurboFlow online SPE allows for a simple "protein crash and shoot" sample preparation protocol. Compared to traditional MRM, analyte quantitation by FS-SIM facilitates a streamlined assay optimization process.

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Background: Telepathology utilizing high-throughput static whole slide image scanners is proposed to address the challenge of limited pathology services in resource-restricted settings. However, the prohibitive equipment costs and sophisticated technologies coupled with large amounts of space to set up the devices make it impractical for use in resource-limited settings. Herein, we aimed to address this challenge by validating a portable whole slide imaging (WSI) device against glass slide microscopy (GSM) using lymph node biopsies from suspected lymphoma cases from Sub-Saharan Africa. Material and methods: This was part of a multicenter prospective case-control head-to-head comparison study of liquid biopsy against conventional pathology. For the portable WSI scanner validation, the study pathologists evaluated 105 surgical lymph node specimens initially confirmed by gold-standard pathology between February and December 2021. The tissues were processed according to standard protocols for Hematoxylin and Eosin (H&E) and Immunohistochemistry (IHC) staining by well-trained histotechnicians, then digitalized the H& E and IHC slides at each center. The digital images were anonymized and uploaded to a HIPAA-compliant server by the histotechnicians. Three study pathologists independently accessed and reviewed the images after a 6-week washout. The agreement between diagnoses established on GSM and WSI across the pathologists was described and measured using Cohens' kappa coefficient (κ). Results: On GSM, 65.5% (n=84) of specimens were lymphoma; 25% were classified as benign, while 9.5% were metastatic. Morphological quality assessment on GSM and WSI established that 79.8% and 53.6% of cases were of high quality, respectively. When diagnoses by GSM were compared to WSI, the overall concordance for various diagnostic categories was 93%, 100%, and 86% for lymphoma, metastases, and benign conditions respectively. The sensitivity and specificity of WSI for the detection of lymphoma were 95.2% and 85.7%, respectively, with an overall inter-observer agreement (κ) of 0.86; 95% CI (0.70-0.95). Conclusions: We demonstrate that mobile whole slide imaging (WSI) is non-inferior to conventional glass slide microscopy (GSM) for the primary diagnosis of malignant infiltration of lymph node specimens. Our results further provide proof of concept that mobile WSI can be adapted to resource-restricted settings for primary surgical pathology and would significantly improve patient outcomes.

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The Histocompatibility and Identity Testing Committee offers an overview of the College of American Pathologists' (CAP) Proficiency Testing (PT) program, commemorating its significant 75th anniversary in 2024. The CAP PT program has undergone significant growth and evolution over the years, ultimately achieving Centers for Medicare and Medicaid Services approval. In 1979, CAP's partnership with the American Association for Clinical Histocompatibility Testing marked a pivotal moment, leading to the creation of the first proficiency testing survey in 1980. This laid the foundation for various PT programs managed by the CAP Histocompatibility and Identity Testing Committee, including HLA antibody testing, HLA molecular typing, engraftment monitoring, parentage/relationship testing, HLA disease associations and drug risk, and HLA-B27 typing. Each program's distinctive considerations, grading methodologies, and future prospects are detailed here, highlighting the continual evolution of histocompatibility and identity testing PT to support emerging technologies and evolving laboratory practices in the field.

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Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean. Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

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One nucleotide substitution in codon 217 of HLA-DRB1*09:31:01 results in a novel allele HLA-DRB1*09:31:02.

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BACKGROUND: Pathological margin assessment is an essential component of surgical management of oral cavity squamous cell carcinoma (OCSCC), however, in many studies, variable definitions of involved margins have been used. The purpose of the present study was to compare the prognostic ability of involved margins according to Royal College of Pathologists (RCPath) and College of American Pathologists (CAP) guidance. METHODS: Retrospective study of 300 patients with previously untreated OCSCC undergoing definitive surgical management. Main specimen margin status was defined according to RCPath guidance and CAP guidance. "Final margin status", incorporated the results of frozen sections and extra tumour bed resections. The prognostic impact of each margin definition was studied using univariate analysis, and in multivariate models including T-stage (AJCC 8th edition), nodal status (pN+), extranodal extension (ENE), and use of adjuvant radiotherapy. RESULTS: Both RCPath and CAP positive margins were associated with local recurrence (LR), disease-specific survival (DSS), and overall survival (OS) on univariate analysis, while final margin status was associated with LR and DSS, but not OS. On multivariate analysis, only CAP positive main specimen margin status was independently associated with LR (odds ratio 2.44, 95% CI 1.37, 4.34), DSS (odds ratio 2.28, 95% CI 1.31, 3.82), and OS (odds ratio 1.59, 95% CI 1.04, 2.42). CONCLUSIONS: Involved main specimen margin as defined by CAP guidance has the advantage of being an independent prognosticator of LR and survival in our cohort.

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Clinical laboratories have many regulations to follow, requiring complete adherence to specific standards and regulations in order to be granted accreditation. As part of the accreditation process, a laboratory must be inspected. Whether it is an initial or biennial inspection, there are some standard tasks and duties a laboratory can do to prepare in advance to reduce stress, improve the inspection process, and reduce the risk of getting a deficiency. Good Clinical Laboratory Practice (GCLP) is an important part of preparing a clinical laboratory for Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) inspections. GCLP standards have been developed by CLIA and were developed with the goal of providing a sole source of requirements that clinical laboratories using human patient samples need to follow to ensure reproducible and reliable clinical laboratory results. The Laboratory Accreditation Program (LAP) of the College of American Pathologists (CAP) also has ongoing activities and guidelines for clinical laboratories to follow. Although this is a voluntary program, it is driven by peer review, education, and compliance to established performance standards. CAP is focused on laboratory improvement and views its inspections as collaborations between inspector and laboratory. The CAP checklists, based on their standards for good lab practice, are used by inspectors to ensure that each inspection is consistent and thorough and to enable CAP to determine if the laboratory meets the standards for accreditation. © 2021 Wiley Periodicals LLC.

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Malaria rapid diagnostic tests (RDTs) have had an enormous global impact which contributed to the World Health Organization paradigm shift from empiric treatment to obtaining a parasitological diagnosis prior to treatment. Microscopy, the classic standard, requires significant expertise, equipment, electricity, and reagents. Alternatively, RDT's lower complexity allows utilization in austere environments while achieving similar sensitivities and specificities. Worldwide, there are over 200 different RDT brands that utilize three antigens: Plasmodium histidine-rich protein 2 (PfHRP-2), Plasmodium lactate dehydrogenase (pLDH), and Plasmodium aldolase (pALDO). pfHRP-2 is produced exclusively by Plasmodium falciparum and is very Pf sensitive, but an alternative antigen or antigen combination is required for regions like Asia with significant Plasmodium vivax prevalence. RDT sensitivity also decreases with low parasitemia (<100 parasites/uL), genetic variability, and prozone effect. Thus, proper RDT selection and understanding of test limitations are essential. The Center for Disease Control recommends confirming RDT results by microscopy, but this is challenging, due to the utilization of clinical laboratory standards, like the College of American Pathologists (CAP) and the Clinical Lab Improvement Act (CLIA), and limited recourses. Our focus is to provide quality assurance and quality control strategies for resource-constrained environments and provide education on RDT limitations.

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Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

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BACKGROUND: The evaluation of 24 h urinary oxalate excretion is the gold standard for diagnosing hyperoxaluria in patients with recurrent urolithiasis. However, 24 h urine sample collection is cumbersome. Therefore we aim to see if oxalate to creatinine ratio in random urine sample can be used as an alternative. MATERIALS AND METHODS: A cross-sectional study was conducted at Section of Chemical Pathology, Department of Pathology and Laboratory Medicine Aga Khan University Karachi from 1st February to December 31, 2019. A total of 62 adult patients, 18-60 years of age with history of kidney stones presenting to the clinical laboratory for 24 h urine oxalate estimation were invited to participate in the study after informed consent. Clinical details were recorded on a structured questionnaire and patients were guided to submit 24 h urine and a random spot urine sample. Urinary oxalate was measured on Micro lab 300 using a kit based on oxalate oxidase principle by Trinity Biotech plc, Wicklow, Ireland following standard operating procedures. Urinary creatinine was measured on ADVIA 1800 by Siemens, US using kinetic Jaffe reaction according to the manufacturer's instructions. The data was analyzed on SPSS. RESULTS: In a period of ten months, a total of 62 subjects were recruited; mean age was 32.4 ± 2.6 years. Males were 49 (79.0%) and females were 13 (20.9%). Correlation was found to be (r = 0.289) by Spearman correlation (p value < 0.005). Taking 24 h urinary oxalate as gold standard the sensitivity, specificity, positive predictive value and negative predictive value of spot oxalate to creatinine ratio was 83.3%, 17.8%, 9.8% and 90.9% respectively. CONCLUSION: The random spot urine test cannot replace the 24 h urinary oxalate estimation in patients with urolithiasis.

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OBJECTIVES: The level of glycated hemoglobin A (HbA1C) in blood is the preferred marker for diabetes monitoring and treatment. Here, we evaluate the analytical performance of the Roche Diagnostics Cobas c 513, a stand-alone HbA1C immunoassay analyzer. DESIGN AND METHODS: Performance was assessed with regards to imprecision, accuracy, linearity, method comparison against the Roche Cobas Integra 800 CTS, specimen stability, interference from common hemoglobin variants and hemoglobin F, and throughput. RESULTS: Within-run and between-run precisions were 0.5-0.7 and 0.8-1.3%CV, respectively. An average bias of -1.6% to proficiency survey samples was observed. The c 513 correlated well with the Integra (slope = 0.94, y-intercept = 0.50, and correlation coefficient = 0.998). The effect of hemoglobin variants on this assay was negligible while specimens containing ≥10% HbF demonstrated a negative bias. The c 513 instrument can process up to 340 samples per hour. CONCLUSIONS: The c 513 is a precise, accurate, automated high throughput analyzer for measuring HbA1C in large laboratories.

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BACKGROUND: Training in patient safety, quality, and management is a key component of Graduate Medical Education (GME) training in all specialties. However, residency programs, especially Pathology programs, often find it challenging to create strong learning opportunities in these areas. OBJECTIVES: Focused quality assurance (QA) projects are one approach to teach and engage trainees in these key areas. Residents have been historically involved in different QA projects in our department but mainly in small secondary roles. Leading a large QA project that can enhance residents' management skills and improve clinical operations in our laboratory was the main objective of our project. DESCRIPTION: A new process for laboratory self-inspection led by residents was implemented that simulates the exact process of a formal outside College of American Pathologists (CAP) inspection. We aim to prove that resident-led QA activities not only have profound educational benefit but can also result in significant performance and operational improvement. RESULTS: For this paper, we focus on the Histology laboratory since the ramifications from the self-inspection process during a three year period were profound leading to change in management, workflow changes, and notable improvement in staff morale. CONCLUSION: The self-inspection process exposed the residents to operational issues and corrective actions that provided them the opportunity to take a more active role in laboratory management and helped prepare them for post-graduation challenges. It also helped the department identify and rectify many operational issues, confirmed by the enumeration of CAP deficiencies and significant improvement of staff morale.

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The advent of fiberoptic endoscopy with biopsy has revolutionized procurement of specimens from deep sites. This has translated into more cytologic specimens whereby the material is limited and best handled by cytology laboratory staff. While the diagnosis of the pathologic process is of utmost importance, there is increasing expectation that the diagnosis be specific and accurate as not to require additional biopsy for initiation of treatment. This expectation has driven demand in immunohistochemical (IHC) and molecular studies conducted specifically on material processed as cytology specimens. The Clinical Laboratory Improvement Amendments of 1988 requires laboratories in the United States of America to verify the performance of patient tests. Due to varying laboratory practices with respect to validation of IHC assays, the College of American Pathologists introduced guidelines for analytic validation of IHC tests. These guidelines address how to perform validation by recommending the number of cases in the validation set, comparator concordance, and when to revalidate. The main thrust of the guidelines is based on formalin-fixed paraffin-embedded tissue with only one expert consensus opinion referring to validation of IHC tests on cytology specimens which delegates to the medical director, the determination of number of positive and negative cases to be tested. This article will outline how an academic center approaches validation of IHC studies performed on cytology cell block specimens using the College of American Pathologists guidelines. A stepwise approach from selection of antibodies to validate followed by building the validation panel and evaluating the stain results for concordance against the gold standard of histology tissue specimen will be described. A rationale for dealing with discordant results and future innovations will conclude the report.

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BACKGROUND: The prognostic value of positive circumferential resection margins (CRM) in resected esophageal squamous cell carcinoma (ESCC) is unclear. The Royal College of Pathologists criteria and the College of American Pathologists criteria are the two commonly used definitions of CRM involvement. The aim of this report was to compare the prognostic performance of the two criteria and to propose a modified stratification in patients who underwent radical esophagectomy for ESCC. METHODS: We retrospectively reviewed 112 patients with pathologically confirmed T3N0M0 ESCC and without neoadjuvant therapy from June 2009 and July 2011. The optimal cutoff point was obtained by the X-tile. The prognostic performance of different classifications of CRM was assessed in terms of homogeneity, discriminatory ability, and monotonicity. RESULTS: According to the Royal College of Pathologists criteria, a positive CRM was detected in 87 patients (77.7%); and 24 patients (21.4%) were found with positive CRM according to the College of American Pathologists criteria. Non-significant associations between overall survival and CRM were observed according to either of the two criteria. The analysis of reclassifying the CRM criteria demonstrated that the optimal cutoff CRM value for best prognostic power was 600 µm. Patients with CRM more than 600 µm showed better overall survival (P<0.05) than the cases with CRM less than 600 µm. Furthermore, the improved homogeneity, discriminatory ability, and monotonicity gradients were also found in this modified criteria, as compared with the two existing criteria. CONCLUSIONS: Our study highlighted that CRM was an independent prognostic factor for survival in esophageal cancer patients, and the modified CRM criteria had better prognostic power than the traditional criteria in patients with ESCC.