Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens.

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell-cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.

A comprehensive assessment of exome capture methods for RNA sequencing of formalin-fixed and paraffin-embedded samples.

RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research and drug development. However, the processing and storage of FFPE samples are known to cause extensive degradation of RNAs, which limits the discovery of gene expression or gene fusion-based biomarkers using RNA sequencing, particularly methods reliant on Poly(A) enrichment. Recently, researchers have developed an exome targeted RNA-Seq methodology that utilizes biotinylated oligonucleotide probes to enrich RNA transcripts of interest, which could overcome these limitations. Nevertheless, the standardization of this experimental framework, including probe designs, sample multiplexing, sequencing read length, and bioinformatic pipelines, remains an essential requirement. In this study, we conducted a comprehensive comparison of three main commercially available exome capture kits and evaluated key experimental parameters, to provide the overview of the advantages and limitations associated with the selection of library preparation protocols and sequencing platforms. The results provide valuable insights into the best practices for obtaining high-quality data from FFPE samples.

High-Resolution Multiparameter DNA Flow Cytometry for Accurate Ploidy Assessment and the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin-Embedded Tissues.

This article contains detailed protocols for the simultaneous flow cytometric identification of tumor cells and stromal cells and measurement of DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for accurate DNA content assessments of FFPE carcinoma tissues. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 (near-haploidy) and of keratin-positive tumor cells with a DNA index close to 1.0 in overall DNA aneuploid samples, thus improving DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiparameter DNA content analysis of FFPE carcinomas Alternate Protocol 1: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue and red excitation Alternate Protocol 2: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue excitation Support Protocol: Sorting cell population from FFPE carcinomas.

Instant Pot for antigen retrieval: a simple, safe and economical method for use in immunohistochemistry.

Here, the authors report a simple method to perform antigen retrieval using a commonly available commercial Instant Pot® for immunohistochemistry. It provides a validated alternative to previous antigen retrieval methods that employ water baths, microwave ovens or scientific-grade pressure cookers. The Instant Pot can be set to obtain a variety of desired temperatures and is straightforward to use, making it extremely amenable to optimization. The Instant Pot method is an easy, safe and inexpensive alternative means to perform immunohistochemistry on formalin-fixed paraffin-embedded sections. It has been validated using several different monoclonal antibodies including ones directed against cell surface or intracellular antigens. As a result, it should be useful for a variety of research labs as well as undergraduate laboratory courses.

A Low-Cost Modular Imaging System for Rapid, Multiplexed Immunofluorescence Detection in Clinical Tissues.

To image 4-plex immunofluorescence-stained tissue samples at a low cost with cellular level resolution and sensitivity and dynamic range required to detect lowly and highly abundant targets, here we describe a robust, inexpensive (<$9000), 3D printable portable imaging device (Tissue Imager). The Tissue Imager can immediately be deployed on benchtops for in situ protein detection in tissue samples. Applications for this device are broad, ranging from answering basic biological questions to clinical pathology, where immunofluorescence can detect a larger number of markers than the standard H&E or chromogenic immunohistochemistry (CIH) staining, while the low cost also allows usage in classrooms. After characterizing our platform's specificity and sensitivity, we demonstrate imaging of a 4-plex immunology panel in human cutaneous T-cell lymphoma (CTCL) formalin-fixed paraffin-embedded (FFPE) tissue samples. From those images, positive cells were detected using CellProfiler, a popular open-source software package, for tumor marker profiling. We achieved a performance on par with commercial epifluorescence microscopes that are >10 times more expensive than our Tissue Imager. This device enables rapid immunofluorescence detection in tissue sections at a low cost for scientists and clinicians and can provide students with a hands-on experience to understand engineering and instrumentation. We note that for using the Tissue Imager as a medical device in clinical settings, a comprehensive review and approval processes would be required.

Assessment of stained direct cytology smears of breast cancer for whole transcriptome and targeted messenger RNA sequencing.

BACKGROUND: Rather than surgical resection, cytologic specimens are often used as first-line clinical diagnostic procedures due to higher safety, speed, and cost-effectiveness. Archival diagnostic cytology slides containing cancer can be equivalent to tissue biopsies for DNA mutation testing, but the accuracy of transcriptomic profiling by RNA sequencing (RNA-seq) is less understood. METHODS: This study compares the results from whole transcriptome RNA-seq and a targeted RNA-seq assay of stained cytology smears (CS) versus matched tumor tissue samples preserved fresh-frozen (FF) and processed as formalin-fixed paraffin-embedded (FFPE) sections. Cellular cytology scrapes from all 11 breast cancers were fixed and stained using three common protocols: Carnoy's (CS_C) or 95% ethanol (CS_E) fixation and then Papanicolaou stain or air-dried then methanol fixation and DiffQuik stain (CS_DQ). Agreement between samples was assessed using Lin's concordance correlation coefficient. RESULTS: Library yield for CS_DQ was too low, therefore it was not sequenced. The distributions of concordance correlation coefficient of gene expression levels in comparison to FF were comparable between CS_C and CS_E, but expression of genes enriched in stroma was lower in cytosmear samples than in FF or FFPE. Six signatures showed similar concordance to FF for all methods and two were slightly worse in CS_C and CS_E. Genomic signatures were highly concordant using targeted RNA-seq. The allele fraction of selected mutations calculated on cytosmear specimens was highly correlated with FF tissues using both RNA-seq methods. CONCLUSION: RNA can be reliably extracted from cytology smears and is suitable for transcriptome profiling or mutation detection, except for signatures of tumor stroma.

Quality Assessment of Proteins and RNA Following Storage in Archival Formalin-Fixed Paraffin-Embedded Human Breast Cancer Tissue Microarray Sections.

Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

OBJECTIVES: Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. METHODS: Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. CONCLUSIONS: The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.

In situ hybridization for the assessment of urokinase plasminogen activator and plasminogen activator inhibitor type­1 in formalin­fixed paraffin­embedded breast cancer specimens.

Urokinase plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI­1), have been reported as prognostic and predictive biomarkers in breast cancer, particularly in patients with node­negative tumors. uPA and PAI­1 expression levels classify patients into a poor­prognosis subgroup, requiring adjuvant chemotherapy and a favorable­prognosis subgroup, which can be considered for de­escalation. However, the clinical use of these two biomarkers remains limited, since fresh­frozen/fresh tumor samples are currently required for their quantification. The aim of the present study was to compare PLAU and SERPINE1 mRNA expression levels (corresponding to uPA and PAI­1 proteins, respectively), assessed using in situ hybridization in 83 formalin­fixed paraffin­embedded (FFPE) breast tumor samples, with uPA and PAI­1 protein expression assessed using immunometric assay with paired fresh­frozen breast cancer samples. The results from the two methods significantly correlated as regards uPA quantification; however, >30% of the samples were discordant, according to the clinically validated threshold. Concordance between the two analytical methods was less prominent for PAI­1 protein and SERPINE1 mRNA. Taken together, the results of the present study indicate that although PLAU and SERPINE1 mRNA may be reliably detected in FFPE samples using in situ hybridization, this technology cannot be used as a substitute for the replacement of the immunometric assay­derived quantification on fresh­frozen samples.

Fragmentation assessment of FFPE DNA helps in evaluating NGS library complexity and interpretation of NGS results.

Formalin-fixed paraffin-embedded (FFPE) tissue remains the most common source for DNA extraction from human tissue both in research and routine clinical practice. FFPE DNA can be considerably fragmented, and the amount of DNA measured in nanograms may not represent the amount of amplifiable DNA available for next-generation sequencing (NGS). Two samples with similar input DNA amounts in nanograms can yield NGS analyses of considerably different quality. Nevertheless, many protocols for NGS library preparation from FFPE DNA describe input DNA in nanograms without indication of a minimum requirement of amplifiable genome equivalent DNA. An important NGS quality metric is the library complexity, reflecting the number of DNA fragments from the original specimen represented in the final library. Aiming to illustrate the relationship between DNA fragmentation degree and library complexity, we assessed the fragmentation degree of 116 lung cancer FFPE DNA samples to calculate the amount of amplifiable input DNA used for library preparation. Mean unique coverage, coverage uniformity, and mean number of PCR duplicates with the same unique molecular identifier were used to evaluate library complexity. We showed that the amount of amplifiable input DNA predicted library complexity better than the input measured in nanograms. The frequent discrepancy between DNA amount in nanograms and the amount of amplifiable DNA indicate that the fragmentation degree should be considered when performing NGS of FFPE DNA. Importantly, the fragmentation assessment may help when interpreting NGS data and be a useful tool for evaluating library complexity in the absence of unique molecular identifiers.

Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†.

We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

Assessment of the Murine Tumor Microenvironment by Multiplex Immunofluorescence.

We describe the staining methods used for simultaneous detection of tumor microenvironment components as well as the automated quantification methodologies. This method uses mouse formalin-fixed paraffin-embedded tissues and multiplex immunofluorescence (Multiplex IF) followed by multispectral imaging. Currently, this methodology has shown to have a valuable role in murine immunoprofiling, and can be useful when evaluating the changes incurred on the tumor microenvironment upon various immunopreventive strategies.

Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together. Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes. Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues. Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.

Quantitative assessment of HER2 gene amplification of breast cancer using droplet digital PCR.

We previously reported the usefulness of droplet digital polymerase chain reaction (ddPCR) for the assessment of Human epithelial growth factor receptor 2 (HER2) gene amplification in breast cancer using formalin-fixed and paraffin-embedded sections. In our previous study, we combined HER2/CEP17 ratio (HER2 gene signals to chromosome 17 signals) with ddPCR and tumor content ratio (TCR) of each sample and determined the HER2 status by adopting a two-dimensional chart. This "ddPCR-TCR method" showed a high concordance with conventional HER2 status. In this study, we updated our method to assess the HER2 status of breast cancer in a more quantitative manner. We combined obtained data of the ddPCR ratio [Rx ] and TCR [x]; we calculated "(Rx - 1)/x + 1" for 41 samples with primary breast cancer and named the value led by this formula as "eHER2 (estimated HER2/CEP17 ratio of a tumor cell)". eHER2 was equivalent to conventional in situ hybridization (ISH) HER2/CEP17 ratio in most cases. eHER2 and ISH ratio showed a strong correlation (Spearman rank correlation ρ = 0.70, p < 0.0001). The obtained results indicated that eHER2 is a potential tool for HER2 status diagnosis in breast cancer.

Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Assessment of Lung Eosinophils In Situ Using Immunohistological Staining.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

Assessment of apoptotic index in various grades of oral epithelial dysplasia: A cross-sectional study.

CONTEXT: : Oral cancer is a major health problem worldwide. In cancer, the equilibrium between cell proliferation and apoptosis is disturbed. The defect in the apoptotic pathway allows cells to proliferate with genetic abnormalities. Thus, the apoptotic index (AI) can be used to assess the significance of apoptosis as a proliferative marker in oral epithelial dysplasia. AIMS: To assess the apoptotic index in various grades of epithelial dysplasia. OBJECTIVES: 1) To calculate the apoptotic index in various grades of oral epithelial dysplasia, 2) To compare the apoptotic index between various grades of oral epithelial dysplasia, 3) To predict the biologic behavior of oral epithelial dysplasia based on an apoptotic index. SETTINGS AND DESIGN: Cross-sectional tissue analyzing study. METHODS AND MATERIALS: This study constituted 30 cases, previously diagnosed with various grades of oral epithelial dysplasia (OED). AI was calculated as the number of apoptotic bodies/cells expressed as a percentage of the total number of cells counted in each case. STATISTICAL ANALYSIS USED: Statistical analysis was carried out using ANOVA test. RESULTS: A statistically significant difference was observed between mild dysplasia and severe dysplasia where P = 0.002. The mean AI was increased progressively with increasing grades of OED. CONCLUSIONS: This study demonstrated the clinical significance of apoptosis in assessing disease progression in Oral Potentially Malignant Disorder (OPMD) which may be used as a prognostic indicator in OED. This would, in turn, help in knowing the prognosis of the disease and to develop targeted drug therapy.

Detection and assessment of capsular invasion, vascular invasion and lymph node metastasis volume in thyroid carcinoma using microCT scanning of paraffin tissue blocks (3D whole block imaging): a proof of concept.

In the modern era, detailed pathologic characteristics of a thyroid tumor are crucial to achieve accurate diagnosis and guide treatment. The presence of capsular invasion (CI) is diagnostic for carcinoma, whereas vascular invasion (VI) and nodal metastasis (NM) are included in risk stratification. However, the very definition of CI and VI is surrounded by controversies and an accurate assessment of NM is lacking. Whole Block Imaging (WBI) by microCT is a new imaging modality to create 3D reconstruction of whole tissue block with microscopic level resolution without the need for tissue sectioning. In this study, we aimed to define CI, VI, and NM volume using WBI by microCT. Twenty-eight paraffin blocks (PBs) from 26 thyroid tumors were scanned. Ten PBs contained CI, whereas 7 had VI. 3D microCT images were compared with whole slide images (WSI) of corresponding H&E slides. In 2 cases with VI and/or CI, WSI of serial H&E slides were obtained and underwent 3D-reconstruction to be compared with the WBI. Satellite tumor nodules beyond tumor capsule were shown to be CI by demonstrating the point of penetration using microCT and 3D reconstruction. Additional foci of CI were detected using microCT. VI was seen using microCT. Fibrin associated with tumor thrombus was not always present on serially sectioned H&E slides. WBI by microCT scanner was able to assess the volume of NM. In conclusion, WBI is able to detect CI, VI, and assess the volume of NM in thyroid carcinoma without tissue sectioning. It is the ultimate method for the complete sampling of the tumor capsule. It has the potential to increase the detection rate of CI, better define criteria for CI and VI, and provide an accurate assessment of the volume of nodal disease. This technology may impact the future practice of surgical pathology.

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment.

To maximize data obtainment from valuable cardiac tissue, we hypothesized that myocardium fixed in optimal cutting temperature (OCT) medium for histology could also be used to investigate the function of myofilament proteins in situ. We compared tissue prepared via conventional liquid nitrogen (LN) snap freezing with tissue fixed in OCT and then sectioned in fiber-parallel orientation. We found that actin-myosin Ca2+ sensitivity, activation rate by Ca2+, cooperativity along the thin filament, as well as cross-bridge cycling rate were unaffected by OCT storage and could reliably be interpreted after sectioning. Absolute values in maximum force generation per cross-sectional area, as well as passive strain, are difficult to investigate after sectioning, as myofibrillar continuity along the preparation cannot be guaranteed. We have shown that myocardial tissue stored in OCT and sectioned before analysis is available for functional analysis, a valuable means of maximizing usage of precious cardiac biopsies.NEW & NOTEWORTHY Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

External Quality Assessment Schemes for Biomarker Testing in Oncology: Comparison of Performance between Formalin-Fixed, Paraffin-Embedded-Tissue and Cell-Free Tumor DNA in Plasma.

Liquid biopsies have emerged as a useful addition to tissue biopsies in molecular pathology. Literature has shown lower laboratory performances when a new method of variant analysis is introduced. This study evaluated the differences in variant analysis between tissue and plasma samples after the introduction of liquid biopsy in molecular analysis. Data from a pilot external quality assessment scheme for the detection of molecular variants in plasma samples and from external quality assessment schemes for the detection of molecular variants in tissue samples were collected. Laboratory performance and error rates by sample were compared between matrices for variants present in both scheme types. Results showed lower overall performance [65.6% (n = 276) versus 89.2% (n = 1607)] and higher error rates [21.0% to 43.5% (n = 138) versus 8.7% to 16.7% (n = 234 to 689)] for the detection of variants in plasma compared to tissue, respectively. In the plasma samples, performance was decreased for variants with an allele frequency of 1% compared to 5% [56.5% (n = 138) versus 74.6% (n = 138)]. The implementation of liquid biopsy in the detection of circulating tumor DNA in plasma was associated with poor laboratory performance. It is important both to apply optimal detection methods and to extensively validate new methods for testing circulating tumor DNA before treatment decisions are made.