Limited Contribution of Creatine Kinase-Myocardial Band Alongside High-Sensitivity Cardiac Troponin in Diagnosing Acute Myocardial Infarction in an Emergency Department.

Cardiac biomarkers, especially high-sensitivity cardiac troponin C or I (hs-cTnC or hs-cTnI, respectively), are vital for diagnosing acute myocardial infarction (AMI). Despite the specificity of hs-cTn as a biomarker, the creatine kinase-myocardial band (CK-MB) is commonly used alongside hs-cTn in emergency departments (EDs). We analyzed 23,771 simultaneous hs-cTn (hs-cTnT or hs-cTnI) and CK-MB requests for 17,185 patients in tertiary hospital ED in 2022. The objective of this study was to assess their practical value in diagnosing AMI in real-world settings. Among all 17,185 patients tested, 98.0% underwent hs-cTnT and CK-MB tests, and substantially fewer underwent hs-cTnI testing. We observed concordance between the initial hs-cTn and CK-MB results in 71.3% of patients. Of 131 AMI cases, 57 were positive for both biomarkers, 63 for hs-cTn only, and none for CK-MB alone. CK-MB positivity was often found in the absence of AMI. Discrepancies between the hs-cTnT and hs-cTnI results occurred in 30.0% of patients. Indiscriminate CK-MB testing for diagnosing AMI in EDs should be reconsidered. Efficient use of CK-MB is important for reducing costs and ensuring optimal patient care.

Comparative analysis of the quantitative point-of-care CareSTART™ total bilirubin with central laboratory total bilirubin assays in neonatal blood samples.

This study aimed to assess and compare the accuracy of point-of-care CareSTART™ S1 Total Bilirubin test with a central laboratory total bilirubin assay using neonatal samples. This study was conducted using 152 paired measurements obtained from 122 neonates admitted to the neonatal intensive care unit. Total serum bilirubin (TSB) levels assayed with the central laboratory assay, laboratory bilirubinomter, trancutaneous bliribubin (TcB) instrument and CareSTART were compared using Bland-Altman analysis. The mean difference between the CareSTART and TSB values was -1.43 mg/dL and the 95% limit of agreement (LoA) was -4.25 to 1.39 mg/dL. CareSTART tended to underestimate total bilirubin concentrations compared with TcB, however, the LoA was narrower due to the smaller SD of mean difference for CareSTART. The CareSTART Total Bilirubin test provides an accurate alternative to TcB for total serum bilirubin measurement. Given its low-cost, ease-of-use, and portability, the use of CareSTART is expected to provide point-of-care measurements, especially in low-resource settings.

A comprehensive Exdia TRF-LFIA for simultaneous quantification of GFAP and NT-proBNP in distinguishing ischemic and hemorrhagic stroke.

The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used. To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

Plasma Glial Fibrillary Acidic Protein and N-Terminal Pro B-Type Natriuretic Peptide: Potential Biomarkers to Differentiate Ischemic and Hemorrhagic Stroke.

Acute stroke management is critically time-sensitive and challenging. Blood-based biomarkers that can differentiate acute ischemic stroke (IS) from hemorrhagic stroke (HS) can greatly facilitate triage and early management. Admission blood samples obtained within 6 h of stroke symptom onset were analyzed in a derivation/validation design. GFAP, N-FL, NT-proBNP, copeptin, neutrophils (%), NLR, and platelet counts were assessed in the derivation cohort. The informative markers and the derived cutoff values were evaluated in the validation cohort. GFAP > 703 pg/mL showed a PPV of 76.9% and NPV of 95.8% for differentiating HS from IS. Multiple logistic regression analysis showed that GFAP and NT-proBNP were independent variables associated with IS and HS differentiation. Furthermore, applying a combined cutoff (GFAP > 703 pg/mL and NT-proBNP ≤ 125 pg/mL) for HS detection increased the PPV in both the derivation and validation cohorts (93.3% and 100%, respectively). GFAP and NT-proBNP levels were validated as informative blood biomarkers in the differentiation of IS and HS and using a combination of GFAP and NT-proBNP is suggested as a feasible strategy to differentiate stroke subtypes in the hyperacute phase of stroke.

Identification of a frit-related sample carryover in newborn screening by tandem mass spectrometry.

The need for high-throughput analysis of multiple analytes for inborn errors of metabolism in newborn screening (NBS) has led to the introduction of tandem mass spectrometry (MS/MS) into the NBS laboratory. In a flow-injection analysis (FIA), the predominant MS/MS method utilized for NBS, samples are introduced directly into the mass spectrometer without chromatographic separation. When a high-throughput FIA-based MS/MS method is implemented on newer generations of mass spectrometers with increased sensitivity, the risk of carryover and contamination increases. In the present study, we report the carryover of ornithine identified during the implementation of the NeoBase™ 2 (PerkinElmer) non-derivatized kits on the Xevo-TQD platform (Waters Corporation) and describe the source of the carryover, which was traced to the stainless-steel frit-type inline filter. Furthermore, a possible compound-dependent interaction with the stainless-steel frit is suggested based on the structure of ornithine and its effect on separation techniques. Investigation and mitigation of carryover can be a time and resource consuming process, and to this end, our report on identification of a stainless-steel frit as the source of delayed elution and carryover of ornithine should be recognized as a rare, albeit possible source of carryover in FIA-MS/MS methods adopted for NST.

Indirect Method for Estimation of Reference Intervals of Inflammatory Markers.

Background: The direct method for reference interval (RI) estimating is limited due to the requirement of resources, difficulties in defining a non-diseased population, or ethical problems in obtaining samples. We estimated the RI for inflammatory biomarkers using an indirect method (RII). Methods: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and presepsin (PSEP) data of patients visiting a single hospital were retrieved from April 2009 to April 2021. Right-skewed data were transformed using the Box-Cox transformation method. A mixed population of non-diseased and diseased distributions was assumed, followed by latent profile analysis for the two classes. The intersection point of the distribution curve was estimated as the RI. The influence of measurement size was evaluated as the ratio of abnormal values and adjustment (n×bandwidth) of the distribution curve. Results: The RIs estimated by the proposed RII method (existing method) were as follows: CRP, 0-4.1 (0-4.7) mg/L; ESR, 0-10.2 (0-15) mm/hr and PSEP, 0-411 (0-300) pg/mL. Measurement sizes ≥2,500 showed stable results. An abnormal-to-normal value ratio of 0.5 showed the most accurate result for CRP. Adjustment values ≤5 or >5 were applicable for a measurement size <25,000 or ≥25,000, respectively. Conclusions: The proposed RII method could provide additional information for RI verification or estimation with some limitations.

Comparison of In Silico Tools for Splice-Altering Variant Prediction Using Established Spliceogenic Variants: An End-User's Point of View.

Assessing the impact of variants of unknown significance on splicing has become a critical issue and a bottleneck, especially with the widespread implementation of whole-genome or exome sequencing. Although multiple in silico tools are available, the interpretation and application of these tools are difficult and practical guidelines are still lacking. A streamlined decision-making process can facilitate the downstream RNA analysis in a more efficient manner. Therefore, we evaluated the performance of 8 in silico tools (Splice Site Finder, MaxEntScan, Splice-site prediction by neural network, GeneSplicer, Human Splicing Finder, SpliceAI, Splicing Predictions in Consensus Elements, and SpliceRover) using 114 NF1 spliceogenic variants, experimentally validated at the mRNA level. The change in the predicted score incurred by the variant of the nearest wild-type splice site was analyzed, and for type II, III, and IV splice variants, the change in the prediction score of de novo or cryptic splice site was also analyzed. SpliceAI and SpliceRover, tools based on deep learning, outperformed all other tools, with AUCs of 0.972 and 0.924, respectively. For de novo and cryptic splice sites, SpliceAI outperformed all other tools and showed a sensitivity of 95.7% at an optimal cut-off of 0.02 score change. Our results show that deep learning algorithms, especially those of SpliceAI, are validated at a significantly higher rate than other in silico tools for clinically relevant NF1 variants. This suggests that deep learning algorithms outperform traditional probabilistic approaches and classical machine learning tools in predicting the de novo and cryptic splice sites.

Laboratory characteristics of IgG4-related disease: A retrospective study from a single tertiary medical center.

Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition with unique histopathological features that can affect most organs, making diagnosis challenging. This study characterized detailed laboratory characteristics of IgG4-RD. Baseline clinical and laboratory features of 33 patients with IgG4-RD were reviewed, including serum IgG4 concentrations, serum free light chains (sFLCs), IgGĸ- and IgGλ-heavy/light chains (HLCs), capillary serum protein electrophoresis (SPE), and immunofixation electrophoresis (IFE) of IgG4 subclass. The cohort of 33 patients showed male predominance (94%), with 8 (24%) exhibiting multiple organ involvement. Most patients (88%) had an elevated IgG4 concentration, and 67% had elevated erythrocyte sedimentation rate and IgE levels. Median IgG4 concentration at baseline was significantly higher in patients with >2 organs involved than those with ≤2. Furthermore, erythrocyte sedimentation rate was significantly correlated with serum IgG4 concentrations at baseline. SPE results demonstrated polyclonal gammopathy in most patients. Half of the patients had an increased κ/λ sFLC ratio, 42% had an increased IgGκ/IgGλ HLC ratio. Most patients exhibited hypergammaglobulinemia in the anodal end of the ɤ region on SPE. This study describes detailed laboratory features of IgG4-RD. Although none of these tests are considered diagnostically sufficient by itself, the provided laboratory characteristics can increase awareness of this disorder and help distinguish it from other IgG4-RD mimics.

Assessment of M-protein quantification using capillary electrophoresis and immunosubtraction-based integration in clinical samples with low M-protein concentrations.

OBJECTIVES: Quantification of monoclonal protein (M-protein) by serum protein electrophoresis (SPE) is indispensable for diagnosing and monitoring monoclonal gammopathies. However, quantification of small and beta migrating M-proteins is challenging because of overlapping non-immunoglobulin and/or polyclonal immunoglobulin protein fractions. We compared a new integration method based on immunosubtraction (IS-CE) using capillary zone electrophoresis (CZE) against the routine method, which includes a combination of perpendicular drop (0.4%), corrected perpendicular drop (1%) and tangent skimming (98.5%). DESIGN & METHODS: The proposed method of M-protein quantification involves calculating the difference in area under the curve between the SPE and a class-specific IS-CE trace. We analyzed the difference in estimated M-protein concentrations obtained with the new method and routine integration methods using 913 samples. For IgA M-proteins at < 10 g/L, the estimated M-protein concentrations were compared with the total IgA concentration. RESULTS: The median M-protein concentration of 913 consecutive samples was 6.2 g/L (IQR 2.1-14.3 g/L). The median and median % difference between the two integration methods was 0.68 g/L (IQR 0.01-1.55 g/L) and 10.9% (IQR 0.18-38.7%), showing a larger estimated M-protein concentration with the new method. More than 25% difference was observed in 38% of the samples and was associated with lower total protein concentration, lower M-protein concentration, IgA and IgM heavy chain isotypes, and beta- or beta-gamma migration. When 161 samples with IgA M-protein < 10 g/L were compared against total IgA concentration, the median bias of the new method was smaller compared to that of the existing method (-0.95 g/L vs. -1.3 g/L, P < 0.0001). CONCLUSIONS: The use of IS based integration using CZE and IS-CE is promising especially for small and beta migrating M-proteins.

A rare case of the IgE prozone phenomenon in IgE multiple myeloma.

IgE multiple myeloma is a rare subtype of myeloma, accounting for <0.1% of all myeloma cases. Difficulties in diagnosing and monitoring this rare myeloma type are recognized, including the need of heightened awareness for initial diagnosis, performing a reflex immunofixation test using an anti-IgE antisera, and recognizing the possibility of the prozone phenomenon. Here, we report a rare case of IgE multiple myeloma with the prozone phenomenon. This case was characterized by a paradoxical increase in IgE levels with a progressive increase in the dilution factor. Moreover, serial monitoring of IgE levels correlated with the trend in the serum free light chain ratio, especially when the monoclonal protein was no longer detectable. This case highlights the need for laboratory professionals to be vigilant about the occurrence of the prozone phenomenon in IgE multiple myeloma.

Emergency Department Management of Chest Pain With a High-Sensitivity Troponin-Enabled 0/1-Hour Rule-Out Algorithm.

OBJECTIVES: The analytical sensitivity of high-sensitivity cardiac troponin T (hsTnT) assays has enabled rapid myocardial infarction rule-out algorithms for emergency department (ED) presentations. Few studies have analyzed the real-world impact of hsTnT algorithms on outcomes and operations. METHODS: Comparison of ED length of stay (LOS) and 30-day outcomes (return to ED, inpatient admission, and mortality) for patients presenting with chest pain during 2 separate 208-day periods using a 0/1-hour hsTnT-enabled algorithm or fourth-generation TnT. RESULTS: Discharge, 30-day readmission, and 30-day mortality rates were not significantly different with fourth-generation TnT vs hsTnT. Thirty-day return rates were significantly decreased with hsTnT (17.4% vs 14.9%; P < .01). For encounters with TnT measured at least twice and resulting in discharge, median ED LOS decreased by 61 minutes with the use of hsTnT (488 vs 427 minutes; P < .0001). Median time between first and second TnT results decreased by 82 minutes with hsTnT (202 vs 120 minutes; P < .0001), suggesting that the 0/1-hour algorithm was incompletely adopted. CONCLUSIONS: Implementation of the hsTnT algorithm was associated with decreased 30-day return rates and decreased ED LOS for a subset of patients, despite incomplete adoption of the 0/1-hour algorithm.

Diagnostic Characteristics of Urinary Red Blood Cell Distribution Incorporated in UF-5000 for Differentiation of Glomerular and Non-Glomerular Hematuria.

BACKGROUND: Automated urine sediment analysis has been developed to address the limitations of microscopic examination of dysmorphic red blood cells (RBCs). We evaluated the urinary RBC distribution (URD) parameter of a recently launched automated urinary flow cytometry analyzer, UF-5000 (Sysmex, Kobe, Japan), to differentiate glomerular hematuria (GH) from non-GH (NGH). METHODS: Samples submitted for urine sediment analysis from patients with hematuria (>20 RBCs/µL) were divided into derivation (N=156; 101 GH, 55 NGH) and validation cohorts (N=107; 60 GH, 47 NGH). The clinical diagnosis of GH or NGH was established based on clinical data review. Differences in UF-5000 parameters (URD, small RBC, lysed RBC, RBC-P70FSC, RBC-SF-FSC-W, mean forward-scattered light, and mean side-scattered light) between GH and NGH, and areas under the ROC curves (AUC) were analyzed in the derivation cohort. The derived ideal cut-off value was evaluated in the validation cohort. We applied the Kitasato criteria to compare the diagnostic performance. RESULTS: URD (%), differed significantly between GH and NGH (P<0.001) in the two cohorts. The AUC of URD was 0.814 and 0.806 in the derivation and validation cohorts, respectively. Using a cut-off of >20.1%, the sensitivity was 99.0%/89.4% and the specificity was 50.9%/63.3% in the derivation/validation cohort. When the Kitasato criteria were applied, the sensitivity and specificity were 80.2% and 52.7%, respectively. CONCLUSIONS: URD is a rapid, objective, and quantitative measure that can be used to differentiate GH and NGH.

Diagnostic Value of Procalcitonin in Transplant Patients Receiving Immunosuppressant Drugs: A Retrospective Electronic Medical Record-Based Analysis.

OBJECTIVES: To evaluate concentrations of procalcitonin (PCT) in transplant recipients receiving immunosuppressive therapy compared with nonimmunosuppressed patients. METHODS: We analyzed a data set of 9,500 inpatient encounters to compare levels of PCT and other biomarkers of infection (C-reactive protein [CRP], WBC count, and absolute neutrophil count [ANC]) between immunosuppressed and nonimmunosuppressed cohorts. We also assessed the correlation between PCT and clinical variables in immunosuppressed patients. RESULTS: Patients receiving immunosuppressive drugs had significantly higher levels of maximal and minimal PCT compared with the nonimmunosuppressed patients (P < .0001 and P = .0019, respectively). However, CRP levels, WBC count, and ANC were significantly lower in immunosuppressed patients compared with the nonimmunosuppressed patients (P = .0003, P < .0019, and P = .0001, respectively). CONCLUSIONS: Our results from real-world data demonstrated that PCT dynamics remain intact despite immunosuppressive therapy, in contrast to other biomarkers such as CRP, WBC, and ANC. In addition, higher PCT levels are associated with systemic infections and reflect disease severity.

Evaluation of the quick-clotting serum separator tube, VQ-Tube™, for clinical chemistry and thyroid hormone assays.

BACKGROUND: The type of blood collection tube affects specimen quality and laboratory results. Because plasma specimens have a shorter processing time compared with serum specimens, emergency biochemistry tests use plasma. However, serum specimens remain stable after centrifugation and show more accurate results than plasma. Therefore, a quick-clotting serum separator tube is expected to be useful for shorter turnaround times and accurate results. We evaluated a new quick-clotting serum separator tube VQ-Tube™ (AB Medical, Korea) for clinical chemistry and thyroid hormone assays. METHODS: One hundred volunteers from four university hospitals were recruited, and peripheral blood samples were collected in quick-clotting serum separator tube VQ-Tubes™ and the commonly used serum separator tube V-Tubes™. The obtained specimens were used for 16 clinical chemistry assays and three thyroid hormone assays. RESULTS: The differences (%) in the test results obtained from the samples in each tube satisfied the allowable difference ranges (19 assays). The differences in the test results between the tubes satisfied the desired specifications for accuracy except for the glucose results (2.75%). The paired t-test revealed significant differences between the results of six assays, but each set of results showed a good correlation. Samples were visually inspected for serum clarity and gel barrier integrity, and incomplete clotting reactions and haemolysed serum were not observed. CONCLUSIONS: The new quick-clotting VQ-Tube™ demonstrated reliable test results compared with the commonly used serum separator tube V-Tube™. This quick-clotting tube will provide fast test results with adequately separated serum specimens, especially for patients who need fast tests.

Body fluid matrix effect evaluation on the Hitachi Labospect 008 system.

BACKGROUND: Non-standard body fluids (NSBFs) can provide essential clinical information otherwise unobtainable with conventional biological specimens. However, as most commercial chemistry reagents are only validated for serum, plasma, and urine by manufacturers, individual laboratories have to validate testing with NSBF to comply with regulatory standards. However, the heightened level of oversight and uncertainty of validation requirements to comply with regulatory standards pose a significant challenge for NSBF testing in clinical laboratories. METHODS: 28 combinations of high-volume chemistry tests requested on NSBF with established clinical utility were selected from retrospective data analysis. Specimens were analyzed with both closed and open channel chemistry reagents on a LABOSPECT 008AS platform (Hitachi High-Tech Co., Tokyo, Japan). Recovery studies were performed using a high concentration serum sample and 5 clinical NSBF samples at varying concentrations for each analyte. Acceptable performance limits were defined as 100 ± 10% of expected recovery. RESULTS: The average percent recovery ranged from 94.5% to 106.6% depending on the analyte/NSBF combination evaluated, and for each of the 28 combinations, the average percent recovery was within the predefined acceptable limits of ± 10%. CONCLUSIONS: The recovery results from this study on the LABOSPECT 008AS platform demonstrates that any systematic matrix interference of high-volume chemistry testing on NSBF samples is well within the defined limits of acceptability. This work also demonstrates recovery studies performed by an individual laboratory are practial and it is feasible to demonstrate compliance with regulatory requirements for accuracy of chemistry testing on NSBF samples.

The factors influencing clinical outcomes after leukapheresis in acute leukaemia.

Leukapheresis is used for the mechanical removal of leukaemic cells in hyperleukocytosis. However, the effectiveness of leukapheresis remains unclear due to selection and confounding factors in the cohorts. We compared the effectiveness of leukapheresis among the subgroups according to either the 2016 World Health Organization classification or the number of cytogenetic abnormalities with a retrospective, single-centre study from January 2009 to December 2018. Acute myeloid leukaemia (AML, n = 212) and acute lymphoblastic leukaemia (ALL, n = 97) were included. The 30-day survival rates (95% confidence interval, 95% CI) for AML and ALL were 86.3% (81.6-90.9%) and 94.8% (90.3-99.2%), respectively. For AML, 'primary AML with myelodysplasia-related changes' and 'AML with biallelic mutation of CEBPA' showed better 30-day survival outcomes (P = 0.026) than the other subgroups. A higher platelet count after leukapheresis was associated with better 30-day survival in AML patients (P = 0.029). A decrease in blast percentage count after leukapheresis was associated with better 30-day survival in ALL patients (P = 0.034). Our study suggested that prophylactic platelet transfusion to raise the platelet count to 50 × 109/L or greater might improve clinical outcome in AML patients undergoing leukapheresis.

Immunosuppressive Drug Measurement by Liquid Chromatography Coupled to Tandem Mass Spectrometry: Interlaboratory Comparison in the Korean Clinical Laboratories.

BACKGROUND: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is increasingly used for immunosuppressive drug tests. However, most LC-MS/MS tests are laboratory-developed and their agreement is unknown in different Korean laboratories. This interlaboratory comparison study evaluated test reproducibility and identified potential error sources. METHODS: Test samples containing three concentrations of tacrolimus, sirolimus, everolimus, cyclosporine, and mycophenolic acid were prepared by pooling surplus samples from patients undergoing routine therapeutic drug monitoring and tested in duplicate in the participating 10 clinical laboratories. Reconstitution and storage experiments were conducted for the commonly used commercial calibrator set. The robust estimators of reproducibility parameters were calculated. Spearman's rank correlation coefficient (rho, ρ) was used to evaluate the correlation between drugs. Multiple linear regression was used to determine whether the experimental conditions alter the calibration curves. RESULTS: The reproducibility coefficient of variation exceeded 10% only for sirolimus concentrations 1 and 2 (10.8% and 12.5%, respectively) and everolimus concentrations 1 and 2 (12.3% and 11.4%, respectively). The percent difference values showed weak correlations between sirolimus and everolimus (ρ=0.334, P =0.175). The everolimus calibration curve slope was significantly altered after reconstitution following prolonged 5°C storage (P =0.015 for 14 days; P =0.025 for 28 days); the expected differences at 6 ng/mL were 0.598% for 14 days and 0.384% for 28 days. CONCLUSIONS: LC-MS/MS test reproducibility for immunosuppressive drugs seems to be good in the Korean clinical laboratories. Continuous efforts are required to achieve test standardization and harmonization, especially for sirolimus and everolimus.

Targeted Next-Generation Sequencing of Plasma Cell-Free DNA in Korean Patients with Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second-most-common cause of cancer-related deaths worldwide, and an accurate and non-invasive biomarker for the early detection and monitoring of HCC is required. We assessed pathogenic variants of HCC driver genes in cell-free DNA (cfDNA) from HCC patients who had not undergone systemic therapy. METHODS: Plasma cfDNA was collected from 20 HCC patients, and deep sequencing was performed using a customized cfDNA next-generation sequencing panel, targeting the major HCC driver genes (TP53, CTNNB1, TERT) that incorporates molecular barcoding. RESULTS: In 13/20 (65%) patients, we identified at least one pathogenic variant of two major HCC driver genes (TP53 and CTNNB1), including 16 variants of TP53 and nine variants of CTNNB1. The TP53 and CTNNB1 variants showed low allele frequencies, with median values of 0.17% (range: 0.06%-6.99%) and 0.07% (range: 0.05%-0.96%), respectively. However, the molecular coverage of variants was sufficient, with median values of 5,543 (range: 2,317-9,088) and 7,568 (range: 2,400-9,633) for TP53 and CTNNB1 variants, respectively. CONCLUSIONS: Our targeted DNA sequencing successfully identified low-frequency pathogenic variants in the cfDNA from HCC patients by achieving high coverage of unique molecular families. Our results support the utility of cfDNA analysis to identify somatic gene variants in HCC patients.