Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

Alzheimer\'s disease (AD), the most common form of dementia, remains challenging to understand and treat despite decades of research and clinical investigation. This might be partly due to a lack of widely available and cost-effective modalities for diagnosis and prognosis. Recently, the blood-based AD biomarker field has seen significant progress driven by technological advances, mainly improved analytical sensitivity and precision of the assays and measurement platforms. Several blood-based biomarkers have shown high potential for accurately detecting AD pathophysiology. As a result, there has been considerable interest in applying these biomarkers for diagnosis and prognosis, as surrogate metrics to investigate the impact of various covariates on AD pathophysiology and to accelerate AD therapeutic trials and monitor treatment effects. However, the lack of standardization of how blood samples and collected, processed, stored analyzed and reported can affect the reproducibility of these biomarker measurements, potentially hindering progress toward their widespread use in clinical and research settings. To help address these issues, we provide fundamental guidelines developed according to recent research findings on the impact of sample handling on blood biomarker measurements. These guidelines cover important considerations including study design, blood collection, blood processing, biobanking, biomarker measurement, and result reporting. Furthermore, the proposed guidelines include best practices for appropriate blood handling procedures for genetic and ribonucleic acid analyses. While we focus on the key blood-based AD biomarkers for the AT(N) criteria (e.g., amyloid-beta [Aβ]40, Aβ42, Aβ42/40 ratio, total-tau, phosphorylated-tau, neurofilament light chain, brain-derived tau and glial fibrillary acidic protein), we anticipate that these guidelines will generally be applicable to other types of blood biomarkers. We also anticipate that these guidelines will assist investigators in planning and executing biomarker research, enabling harmonization of sample handling to improve comparability across studies.

Prostate cancer (PCa) is the most common noncutaneous cancer in men in the Western world. In addition to accurate diagnosis, Gleason grading and tumor volume estimates are critical for patient management. Computer-aided detection (CADe) software can be used to facilitate the diagnosis and improve the diagnostic accuracy and reporting consistency. However, preanalytical factors such as fixation and staining of prostate biopsy specimens and whole slide images (WSI) on scanners can vary significantly between pathology laboratories and may, therefore, impact the quality of WSI and utility of CADe algorithms. We evaluated the performance of a CADe software in predicting PCa on WSIs of prostate biopsy specimens and focused on whether there were any significant differences in image quality between WSIs obtained on different scanners and specimens from different histopathology laboratories. Thirty prostate biopsy specimens from 2 histopathology laboratories in the United States were included in this study. The hematoxylin and eosin slides of the biopsy specimens were scanned on 3 scanners, generating 90 WSIs. These WSIs were then analyzed using a CADe software (INIFY Prostate, Algorithm), which identified and annotated all areas suspicious for PCa and calculated the tumor volume (percentage area of the biopsy core involved). Study pathologists then reviewed the Algorithm\'s annotations and tumor volume calculation to confirm the diagnosis and identify benign glands that were misclassified as cancer (false positive) and cancer glands that were misclassified as benign (false negative). The CADe software worked equally well on WSIs from all 3 scanners and from both laboratories, with similar sensitivity and specificity. The overall sensitivity was 99.4%, and specificity was 97%. The percentage of suspicious cancer areas calculated by the Algorithm was similar for all 3 scanners. For WSIs with small foci of cancer (<1 mm), the Algorithm identified all cancer glands (sensitivity, 100%). Preanalytical factors had no significant impact on whole slide imaging and subsequent application of a CADe software. Prediction accuracy could potentially be further improved by processing biopsy specimens in a centralized histology laboratory and training the Algorithm on WSIs from the same laboratory in order to minimize variations in preanalytical factors and optimize the diagnostic performance of the Algorithm.

BACKGROUND: Sufficient tumor collection has become of utmost importance in therapeutic experimental protocols. Rapid on-site evaluation (ROSE) ensures adequate sampling for quantification of biomarkers, molecular analyses, and other ancillary studies. The objectives of this study were to evaluate the role of ROSE in trial-associated fine-needle aspiration (FNA) and to analyze predictors of adequacy and cumulative survival from in-house FNA cases used in clinical trials.
METHODS: Clinical trial FNA biopsies performed at a large academic institution were analyzed over 10 months using a comprehensive chart review of the electronic medical records. SPSS version 28 was used for statistical analysis.
RESULTS: Three hundred twenty-five FNAs were collected for 57 clinical trials. In total, 225 individual patients had an average of 1.4 FNA procedures each as a result of a multidepartmental collaborative effort. ROSE was performed for all patients, and adequacy was evaluated by cytotechnologists. Seventy-eight percent of samples were considered adequate, 14% were considered less than optimal, and 8% were considered inadequate, with the latter two categories designated together as less than adequate. The imaging modalities were mainly ultrasound-guided (n = 267; 82%) and computed tomography-guided (n = 58; 18%). There was a statistically significant association between adequate sampling and ultrasound-guided biopsies (83%) compared with computed tomography-guided biopsies (59%; p < .01). The effect of body mass index (BMI) on mortality was also a significant finding. The authors observed a survival benefit in patients who had elevated BMIs (range, 25.0-34.9 kg/m2 ) compared with those who were underweight (BMI, <18.5 kg/m2 ) or class III obese (BMI, >35.0 kg/m2 ; p < .01). Therefore, the best predictors of adequacy and mortality were imaging modality and BMI, respectively.
CONCLUSIONS: Ultrasound-guided modalities are recommended for obtaining adequate FNA sampling for clinical trials. In addition, patients with cancer who had slightly elevated BMIs (25.0-34.0 kg/m2 ) had increased overall survival in this cohort.

BACKGROUND: Phosphatidylethanol (PEth) is a marker of alcohol consumption used in clinical and forensic settings. PEth positivity in individuals expected to abstain from alcohol can have serious consequences. PEth is located on erythrocytes, thus packed red blood cell (pRBC) transfusion is a potential cause of false-positive results. This report is the first to demonstrate this phenomenon in an authentic patient who was negative for PEth immediately prior to transfusion.
METHODS: Residual blood samples collected for clinical testing before and after pRBC transfusion and citrated pRBC segments were tested for PEth homologues 16:0/18:1 (POPEth) and 16:0/18:2 (PLPEth) by liquid chromatography - tandem mass spectrometry with limit of quantitation 10 ng/mL (0.01 µmol/L).
METHODS: A 56-year-old male with new-onset leukemia required transfusion of 4 pRBC units on hospital days 1-2. Blood collected at admission (day 0) showed POPEth and PLPEth < 10 ng/mL (<0.01 µmol/L). Blood collected after completion of the fourth pRBC transfusion demonstrated POPEth = 57 ng/mL (0.08 µmol/L), PLPEth = 38 ng/mL (0.05 µmol/L). One citrated segment demonstrated extremely elevated PEth, supporting pRBC transfusion as the source.
CONCLUSIONS: This case demonstrates pRBC transfusion elevating PEth to concentrations associated with moderate alcohol consumption. Studies suggest that healthy individuals (potential donors) could have PEth concentrations sufficient to cause significant elevation of PEth from a single pRBC unit. This is concerning for populations such as liver transplant candidates who are required to abstain from alcohol, but whose disease sequelae may require pRBC transfusion.
CONCLUSIONS: pRBC transfusion can artificially elevate PEth into clinically and forensically relevant ranges. Individuals interpreting toxicology testing should consider recent pRBC transfusion when evaluating PEth concentrations.

Non-cryopreservation temperature exposure (NCE) is a vital preanalytical factor for assessing plasma quality. NCE can introduce undesirable errors in clinical diagnosis or when developing biomarkers of diseases. Biomarkers that can effectively indicate the changes in sample quality caused by long-term NCE (0-several days) are limited. Low-molecular-weight (LMW) peptides in the plasma are modulated by endogenous proteases. These protease activities are significantly correlated with NCE temperatures and duration, indicating a potential link of these protease reactions with the preanalytical quality of plasma samples. In this study, two groups of plasma samples were aged at room temperature (RT, 57 samples) and 4 °C (69 samples) for different durations (0, 1, 2, 5, and 10 days), and LMW peptidomics were analyzed through nanopore-assisted matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The analysis revealed 10 peptides that consistently exhibited time-dependent changes, which were used to develop multiple-variable models for predicting the changes in sample quality resulting from extended NCE. These biomarker models exhibited outstanding performance in distinguishing poor-quality samples aged at both RT and 4 °C. To validate the findings, tests on samples from validation sets were conducted by analysts who were blinded to the detailed conditions, which revealed a high specificity (94.3-96.9%) and sensitivity (90.5-99.3%). These results indicate the potential of these peptides as novel biomarkers of quality control.

Ethanol is a volatile substance, and specimens need to be tightly capped prior to analysis to prevent evaporative loss. However, add-on requests in previously decapped tubes are commonly received, yet ethanol stability in this setting is unclear. We compared the stability of ethanol in capped vs decapped tubes in the context of routine laboratory automation, storage time, and specimen volumes. Serum specimens were pooled and spiked with ethanol followed by simulating an add-on scenario. Additionally, to evaluate ethanol stability at room temperature for extended times, ethanol concentrations were measured in capped or decapped tubes containing 0.5 mL or 0.1 mL samples over a 4 h time course. Finally, the risk of misclassification of ethanol results in decapped tubes was evaluated near the critical value threshold (∼54 mmol/L). The add-on tubes had a mean recovery of 101.5 % (95 % CI: 97.7-105.4 %) relative to the direct tubes. The time-course experiment showed an average recovery of 87.4 % (95 % CI: 81.8-94.0 %) at the 4 h time point in decapped 0.5 mL specimens. An average recovery of 85.4 % (95 % CI: 84.2-86.1 %) was observed for specimens spiked near the critical value threshold. Importantly, all measurements with 0.5 mL specimen volume were within 25 %, which is the total allowable error (TAE) of the assay.However, with a 0.1 mL volume, specimens cross the TAE threshold just after 1 h, and the percent recovery at 4 h dropped to 52.9 % (95 % CI: 50.2-55.7 %). In conclusion, ethanol testing in decapped tubes remains within the TAE for up to 4 h in specimens with a 0.5 mL volume. Therefore, add-on ethanol testing using routine laboratory automation and storage conditions can be successfully performed.

This study investigated the effect of sample storage duration on the quantification of oxidative stress markers in the gastrocnemius, heart, and brain of mice submitted to a maximum swimming exercise. Thiobarbituric acid reactive substances (TBARSs), protein carbonyl derivatives, total antioxidant capacity (TAC), and the activity of superoxide dismutase (SOD) and catalase (CAT) were quantified in fresh tissues and in samples stored at -80°C for 1, 3, or 6 months, from exercised (n = 13) and nonexercised mice (n = 13). Except for protein carbonyl derivatives in the heart, the exercise resulted in the modification of all markers in all fresh-evaluated samples (p < 0.001). The storage duration did not modify the effect of exercise on protein carbonyl derivatives and TAC. TBARS was stable for 3 months in the gastrocnemius and for 1 month in frozen heart and brain. Accordingly, the exercise effect on TBARS levels observed in fresh samples was absent in the gastrocnemius frozen for 6 months (p = 0.98) and in the heart and brain frozen for 3 months (p = 0.07 and 0.28, respectively) or more (p = 0.21 for heart and p > 0.99 for brain). In addition, CAT and SOD activities were reduced by storage duration in all tissues evaluated (p < 0.05). Our findings show that sample storage duration alters the quantification of oxidative stress markers in mice submitted to maximum exercise, and its effect is tissue and marker dependent. Some recommendations to achieve more accurate and reproducible data in the exercise physiology and oxidative stress markers field are presented.

Background: The role of cerebrospinal fluid (CSF) alpha-synuclein as a potential biomarker has been challenged mainly due to variable preanalytical measures between laboratories. To evaluate the impact of the preanalytical factors contributing to such variability, the different subforms of alpha-synuclein need to be studied individually. Method: We investigated the effect of exposing CSF samples to several preanalytical sources of variability: (1) different polypropylene (PP) storage tubes; (2) use of non-ionic detergents; (3) multiple tube transfers; (4) multiple freeze-thaw cycles; and (5) delayed storage. CSF oligomeric- and total-alpha-synuclein levels were estimated using our in-house sandwich-based enzyme-linked immunosorbent assays. Results: Siliconized tubes provided the optimal preservation of CSF alpha-synuclein proteins among other tested polypropylene tubes. The use of tween-20 detergent significantly improved the recovery of oligomeric-alpha-synuclein, while multiple freeze-thaw cycles significantly lowered oligomeric-alpha-synuclein in CSF. Interestingly, oligomeric-alpha-synuclein levels remained relatively stable over multiple tube transfers and upon delayed storage. Conclusion: Our study showed for the first-time distinct impact of preanalytical factors on the different forms of CSF alpha-synuclein. These findings highlight the need for special considerations for the different forms of alpha-synuclein during CSF samples\' collection and processing.

Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute\'s Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12-24 hr to avoid confounding effects of these preanalytical factors on IHC.