Challenges in the Metabolomics-Based Biomarker Validation Pipeline.

As end-products of the intersection between the genome and environmental influences, metabolites represent a promising approach to the discovery of novel biomarkers for diseases. However, many potential biomarker candidates identified by metabolomics studies fail to progress beyond analytical validation for routine implementation in clinics. Awareness of the challenges present can facilitate the development and advancement of innovative strategies that allow improved and more efficient applications of metabolite-based markers in clinical settings. This minireview provides a comprehensive summary of the pre-analytical factors, required analytical validation studies, and kit development challenges that must be resolved before the successful translation of novel metabolite biomarkers originating from research. We discuss the necessity for strict protocols for sample collection, storage, and the regulatory requirements to be fulfilled for a bioanalytical method to be considered as analytically validated. We focus especially on the blood as a biological matrix and liquid chromatography coupled with tandem mass spectrometry as the analytical platform for biomarker validation. Furthermore, we examine the challenges of developing a commercially viable metabolomics kit for distribution. To bridge the gap between the research lab and clinical implementation and utility of relevant metabolites, the understanding of the translational challenges for a biomarker panel is crucial for more efficient development of metabolomics-based precision medicine.

Total serum pentosidine quantification using liquid chromatography-tandem mass spectrometry.

Pentosidine (PEN) is an Advanced Glycation End-product (AGE) that is known to accumulate in bone collagen with aging and contribute to fracture risk. The PEN content in bone is correlated with serum PEN, making it an attractive, potential osteoporosis biomarker. We sought to develop a method for quantifying PEN in stored serum. After conducting a systematic narrative review of PEN quantification methodologies, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying total serum PEN. Our method is both sensitive and precise (LOD 2 nM, LOQ 5 nM, %CV < 6.5 % and recovery 91.2-100.7 %). Our method is also equivalent or better than other methods identified in our review. Additionally, LC-MS/MS avoids the pitfalls and limitations of using fluorescence as a means of detection and could be adapted to investigate a broad range of AGE compounds.

Evaluation of Busulfan as a Third-Party Immunoassay on a Clinical Chemistry Analyzer.

BACKGROUND: Busulfan is widely used in conditioning regimens to prepare patients for hematopoietic stem cell transplantation. Therapeutic drug monitoring (TDM) is critical due to large inter- and intra-individual variability in busulfan pharmacokinetics, and the risk of adverse consequences of toxicity including hepatic veno-occlusive disease. Busulfan is most commonly measured by liquid chromatography-mass spectrometry (LC-MS/MS), which is not as widely available in clinical laboratories as automated routine clinical chemistry analyzers. The objective was to perform analytical verification of a busulfan immunoassay on the Abbott Alinity c platform. METHODS: The MyCare Oncology busulfan immunoassay was configured as a third-party reagent on the Abbott Alinity c. Imprecision, linearity, sample carryover, and onboard stability of reagent studies were evaluated. The performance of the busulfan immunoassay using the Abbott Alinity c was compared to the Beckman Coulter AU480 using sodium heparinized plasma, as well as to LC-MS/MS using lithium heparinized plasma. RESULTS: The imprecision goal of 8% was met, and linearity within the analytical measurement range of 240 to 1700 ng/mL was verified. Sample carryover was negligible, and the reagents were stable onboard for at least 84 days. The busulfan immunoassay correlated well with LC-MS/MS (slope = 0.949, y-intercept = -7.8 ng/mL, r2 = 0.9935) and the Beckman Coulter AU480 (slope = 1.090, y-intercept = -34.5 ng/mL, r2 = 0.9988). CONCLUSIONS: This study demonstrated successful analytical verification of a busulfan third-party immunoassay on the Abbott Alinity c platform. The ability to perform TDM of busulfan on a routine clinical chemistry analyzer will positively impact turnaround times to improve patient outcomes.

Evaluation of a New NT-proBNP Immunoassay on an Automated Core Laboratory System.

BACKGROUND: Heart failure remains a major cause of morbidity and mortality despite improvements in treatment. This study aimed to evaluate the Alere N-terminal pro B-type natriuretic peptide (NT-proBNP) immunoassay on the Abbott Alinity i platform. METHODS: The analytical performance including precision, linearity, limit of quantitation (LOQ), carryover, dilution-recovery, and stability was evaluated. A method comparison between the Abbott Alere NT-proBNP assay and Roche Elecsys proBNP II assay was performed using 70 residual plasma samples. RESULTS: Total imprecision was 4.1%, 3.5%, and 2.3% for low (120.9 ng/L), medium (333.9 ng/L), and high (4767.4 ng/L) QC levels, respectively. The manufacturer's claimed LOQ of 8.3 ng/L was verified. Method comparison between the Alere NT-proBNP assay and the Elecsys proBNP II assay showed good agreement between assays with an R value of 0.998, a slope of 1.05 (95% CI, 1.03-1.06), and an intercept of 45.81 (95% CI, -46.6.84 to 138.22). The Bland-Altman plot showed an absolute bias of 250 ng/L or 6.02%. Subrange analysis (NT-proBNP <2000 ng/L) showed good agreement with an R value of 0.998, a slope of 1.04 (95% CI, 1.02-1.06), and an intercept of -4.83 (95% CI, -26.95 to 17.28), with a mean bias of 26 ng/L or 3.2%. The stability of NT-proBNP was also verified in lithium heparin plasma samples stored at 4°C over a 7-day period. Hemolysis and lipemia interference thresholds were verified, but icterus impacted NT-proBNP recovery by >20% at low analyte concentrations. CONCLUSIONS: The Alere NT-proBNP assay demonstrated acceptable analytical performance and very good clinical concordance with the Elecsys proBNP II assay.

Classifying patients with psoriatic arthritis according to their disease activity status using serum metabolites and machine learning.

INTRODUCTION: Psoriatic arthritis (PsA) is a heterogeneous inflammatory arthritis, affecting approximately a quarter of patients with psoriasis. Accurate assessment of disease activity is difficult. There are currently no clinically validated biomarkers to stratify PsA patients based on their disease activity, which is important for improving clinical management. OBJECTIVES: To identify metabolites capable of classifying patients with PsA according to their disease activity. METHODS: An in-house solid-phase microextraction (SPME)-liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for lipid analysis was used to analyze serum samples obtained from patients classified as having low (n = 134), moderate (n = 134) or high (n = 104) disease activity, based on psoriatic arthritis disease activity scores (PASDAS). Metabolite data were analyzed using eight machine learning methods to predict disease activity levels. Top performing methods were selected based on area under the curve (AUC) and significance. RESULTS: The best model for predicting high disease activity from low disease activity achieved AUC 0.818. The best model for predicting high disease activity from moderate disease activity achieved AUC 0.74. The best model for classifying low disease activity from moderate and high disease activity achieved AUC 0.765. Compounds confirmed by MS/MS validation included metabolites from diverse compound classes such as sphingolipids, phosphatidylcholines and carboxylic acids. CONCLUSION: Several lipids and other metabolites when combined in classifying models predict high disease activity from both low and moderate disease activity. Lipids of key interest included lysophosphatidylcholine and sphingomyelin. Quantitative MS assays based on selected reaction monitoring, are required to quantify the candidate biomarkers identified.

Identifying Serum Metabolomic Markers Associated with Skin Disease Activity in Patients with Psoriatic Arthritis.

Psoriatic arthritis (PsA) is a chronic, systemic, immune-mediated inflammatory disease causing cutaneous and musculoskeletal inflammation that affects 25% of patients with psoriasis. Current methods for evaluating PsA disease activity are not accurate enough for precision medicine. A metabolomics-based approach can elucidate psoriatic disease pathogenesis, providing potential objective biomarkers. With the hypothesis that serum metabolites are associated with skin disease activity, we aimed to identify serum metabolites associated with skin activity in PsA patients. We obtained serum samples from patients with PsA (n = 150) who were classified into mild, moderate and high disease activity groups based on the Psoriasis Area Severity Index. We used solid-phase microextraction (SPME) for sample preparation, followed by data acquisition via an untargeted liquid chromatography-mass spectrometry (LC-MS) approach. Disease activity levels were predicted using identified metabolites and machine learning algorithms. Some metabolites tentatively identified include eicosanoids with anti- or pro-inflammatory properties, like 12-Hydroxyeicosatetraenoic acid, which was previously implicated in joint disease activity in PsA. Other metabolites of interest were associated with dysregulation of fatty acid metabolism and belonged to classes such as bile acids, oxidized phospholipids, and long-chain fatty acids. We have identified potential metabolites associated with skin disease activity in PsA patients.

Reference Values for Videofluoroscopic Measures of Swallowing: An Update.

PURPOSE: It is essential that clinicians have evidence-based benchmarks to support accurate diagnosis and clinical decision making. Recent studies report poor reliability for diagnostic judgments and identifying mechanisms of impairment from videofluoroscopy (VFSS). Establishing VFSS reference values for healthy swallowing would help resolve such discrepancies. Steele et al. (2019) released preliminary reference data for quantitative VFSS measures in healthy adults aged < 60 years. Here, we extend that work to provide reference percentiles for VFSS measures across a larger age span. METHOD: Data for 16 VFSS parameters were collected from 78 healthy adults aged 21-82 years (39 male). Participants swallowed three comfortable sips each of thin, slightly, mildly, moderately, and extremely thick barium (20% w/v). VFSS recordings were analyzed in duplicate by trained raters, blind to participant and task, using the Analysis of Swallowing Physiology: Events, Kinematics and Timing (ASPEKT) Method. Reference percentiles (p2.5, 5, 25, 50, 75, 95, and 97.5) were determined as per Clinical and Laboratory Standards Institute EP28-A3c guidelines. RESULTS: We present VFSS reference percentile tables, by consistency, for (a) timing parameters (swallow reaction time; the hyoid burst-to-upper esophageal sphincter (UES)-opening interval; UES opening duration; time-to-laryngeal vestibule closure (LVC); and LVC duration) and (b) anatomically scaled pixel-based measures of maximum UES diameter, pharyngeal area at maximum pharyngeal constriction and rest, residue (vallecular, pyriform, other pharyngeal locations, total), and hyoid kinematics (X, Y, XY coordinates of peak position; speed). Clinical decision limits are proposed to demarcate atypical values of potential clinical concern. CONCLUSION: These updated reference percentiles and proposed clinical decision limits are intended to support interpretation and reliability for VFSS assessment data. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.24043041.

Immunogenicity and Safety of Booster SARS-CoV-2 mRNA Vaccine Dose in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Allogeneic hematopoietic stem cell transplantation (HSCT) recipients are susceptible to severe outcomes of Coronavirus disease 2019 (COVID-19). Most guidelines recommend a fourth dose (ie, booster) of COVID-19 vaccine to reduce the infection risk, and observational studies are needed to determine the immunogenicity and safety of the booster dose in this population. The primary outcome was to determine the quantitative anti-receptor-binding domain (RBD) antibody titers after the fourth dose of the COVID-19 vaccine. The secondary outcomes included adverse effects and all-cause mortality. This single-group prospective cohort included allogeneic HSCT recipients age ≥18 years who received their fourth dose of COVID-19 mRNA vaccine between December 15, 2021, and August 2, 2022. We excluded patients with a history of COVID-19 diagnosis and those who received i.v. Ig within 21 days of antibody testing or rituximab within 6 months before study entry. We used regression models to determine the contributing factors significantly associated with post-fourth dose anti-RBD titer. Sixty-seven patients (median age, 59.5 years; IQR, 53.5 to 65.5 years; 33 males [61%]) received the fourth dose of vaccine, and 54 were included in the anti-RBD titer analysis. The median anti-RBD titers at 4 to 6 weeks after the third and fourth doses differed significantly (13,350 U/mL [IQR, 2618 to 34,740 U/mL] and 44,500 U/mL [IQR, 11,163 to 84,330 U/mL], respectively; P < .0001). In univariate analysis, the post-third dose anti-RBD titer (ß = .70; 95% CI, .54 to .87; P < .001) and treatment with mycophenolate compounds (ß = -1.05; 95% CI, -1.97 to -1.12; P = .03) significantly predicted the antibody response to the fourth dose. In multivariate analysis, the inverse association between treatment with mycophenolate compounds and the post-fourth dose anti-RBD antibody titer was not significant (ß = -.57; 95% CI, -1.32 to .19; P = .14), whereas the significant association between the anti-RBD titers following the third and fourth doses did not change considerably (ß = .66; 95% CI, .47 to .86; P < .001). The most frequent adverse event was vaccination site soreness (44%), followed by fatigue (16%), myalgia (4%), and headache (2%). No recipient experienced new or worsened preexisting graft-versus-host disease within 40 days of vaccination, and no patient died. Six patients (11%) developed breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection not associated with hospitalization or severe outcomes. The fourth dose of the COVID-19 vaccine appears to be highly immunogenic and safe in allogeneic HSCT recipients. Further studies are needed to determine the neutralizing antibody titers against SARS-CoV-2 subvariants and the effectiveness and immunogenicity of bivalent vaccines in allogeneic HSCT recipients.

A Solid-Phase Microextraction-Liquid Chromatography-Mass Spectrometry Method for Analyzing Serum Lipids in Psoriatic Disease.

Approximately 25% of psoriasis patients have an inflammatory arthritis termed psoriatic arthritis (PsA). There is strong interest in identifying and validating biomarkers that can accurately and reliably predict conversion from psoriasis to PsA using novel technologies such as metabolomics. Lipids, in particular, are of key interest in psoriatic disease. We sought to develop a liquid chromatography-mass spectrometry (LC-MS) method to be used in conjunction with solid-phase microextraction (SPME) for analyzing fatty acids and similar molecules. A total of 25 chromatographic methods based on published lipid studies were tested on two LC columns. As a proof of concept, serum samples from psoriatic disease patients (n = 27 psoriasis and n = 26 PsA) were processed using SPME and run on the selected LC-MS method. The method that was best for analyzing fatty acids and fatty acid-like molecules was optimized and applied to serum samples. A total of 18 tentatively annotated features classified as fatty acids and other lipid compounds were statistically significant between psoriasis and PsA groups using both multivariate and univariate approaches. The SPME-LC-MS method developed and optimized was capable of detecting fatty acids and similar lipids that may aid in differentiating psoriasis and PsA patients.

Role of EBUS-TBNA/EUS-FNA and mass spectrometry for diagnosis and typing of lymph node amyloidosis: 10-year experience in two tertiary care academic centers.

BACKGROUND: The objectives of this study were to investigate the utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)/endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for the diagnosis of amyloidosis coupled with the feasibility of mass spectrometry (MS) for amyloid subtyping. METHODS: All patients who had amyloid diagnosed by EBUS-TBNA/EUS-FNA at two tertiary care centers from 2011 to 2020 were retrieved along with the MS subtype, clinical findings, and outcomes. RESULTS: Eight patients were included: seven underwent EBUS-TBNA of mediastinal lymph nodes, and one underwent EUS-FNA of a periportal lymph node. Ages ranged from 37 to 79 years (median, 69 years), with equal numbers of men and women. Presenting clinical history included one case each of follicular lymphoma, lymphoplasmacytic lymphoma, rheumatoid arthritis, possible sarcoid, cirrhosis, and chronic renal insufficiency, and one case each of suspected pulmonary and cardiac amyloidosis. All cases showed waxy, amorphous material on direct smears (n = 5) or ThinPrep slides (n = 3), which were confirmed as amyloid on Congo Red staining. Immunohistochemistry showed dominant lambda staining in two of three cases. MS was performed in all cases and identified five of the light-chain (AL) type, one of the heavy-chain/AL type, and two suggestive of AL amyloidosis. Bone marrow biopsy performed in seven patients demonstrated that three had monoclonal plasma cells and one had lymphoplasmacytic lymphoma. Two of four patients with systemic amyloidosis received chemotherapy and remained alive, whereas three with localized disease remained stable under observation. CONCLUSIONS: EBUS-TBNA/EUS-FNA is effective for amyloidosis diagnosis and provides adequate material for ancillary tests, including MS, which can identify the precursor amyloidogenic protein, leading to appropriate patient management.

Analytical Unreliability of 25 Hydroxy Vitamin D Measurements in Pre-Term Neonates.

BACKGROUND: Vitamin D supplementation is common practice for neonates and infants due to limited stores of vitamin D at birth. Although not commonly encountered, vitamin D toxicity can occur due to over-supplementation. However, toxic concentrations are often not included in method validation experiments, and assays often are not validated in the neonatal population. METHODS: We compared serial 25 hydroxy vitamin D [25(OH)D] measurements in pre-term neonates receiving 25(OH)D supplementation and identified 12 patients wherein concentrations of 25(OH)D were above 50 ng/mL (125 nM) that required additional investigations as the 25(OH)D results did not match the clinical picture. Available samples were compared across 4 immunoassay platforms (LIAISON XL, Roche Cobas e602, Abbott Alinity i, and Siemens Centaur XP) and LC-MS/MS. RESULTS: Concentrations of 25(OH)D observed on one individual immunoassay platform (LIAISON XL) fluctuated substantially between subsequent blood draws in select neonates with elevated concentrations. Serum samples from these patients showed variable agreement between LC-MS/MS and other immunoassay platforms. These fluctuations were not explained by the presence of 3-epimer-25(OH)D or 24,25(OH)2D. CONCLUSIONS: Although we were unable to identify a cause for the variable elevated results, our findings suggest that neonatal 25(OH)D measurements alone should not be used for assessment of nutritional monitoring, and that clinical correlation and other laboratory parameters including ionized calcium should be considered.

Immunogenicity, Safety, and Breakthrough Severe Acute Respiratory Syndrome Coronavirus 2 Infections After Coronavirus Disease 2019 Vaccination in Organ Transplant Recipients: A Prospective Multicenter Canadian Study.

Background: Solid organ transplant (SOT) recipients are at risk for severe coronavirus disease 2019 (COVID-19), despite vaccination. Our study aimed to elucidate COVID-19 vaccine immunogenicity and evaluate adverse events such as hospitalization, rejection, and breakthrough infection in a SOT cohort. Methods: We performed a prospective, observational study on 539 adult SOT recipients (age ≥18 years old) recruited from 7 Canadian transplant centers. Demographics including transplant characteristics, vaccine types, and immunosuppression and events such as hospitalization, infection, and rejection were recorded. Follow ups occurred every 4-6 weeks postvaccination and at 6 and 12 months from first dose. Serum was processed from whole blood to measure anti-receptor binding domain (RBD) antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to assess immunogenicity. Results: The COVID-19 vaccines were found to be safe in SOT recipients with low rates of rejection requiring therapy (0.7%). Immunogenicity improved after the third vaccine dose, yet 21% developed no anti-RBD response. Factors such as older age, lung transplantation, chronic kidney disease, and shorter duration from transplant were associated with decreased immunogenicity. Patients with at least 3 doses were protected from hospitalization when experiencing breakthrough infections. Significantly increased anti-RBD levels were observed in patients who received 3 doses and had breakthrough infection. Conclusions: Three or four doses of COVID-19 vaccines were safe, increased immunogenicity, and protected against severe disease requiring hospitalization. Infection paired with multiple vaccinations significantly increased anti-RBD response. However, SOT populations should continue to practice infection prevention measures, and they should be prioritized for SARS-CoV-2 pre-exposure prophylactics and early therapeutics.

Maternal Anti-Ro Antibody Titers Obtained With Commercially Available Immunoassays Are Strongly Associated With Immune-Mediated Fetal Heart Disease.

OBJECTIVE: Anti-Ro antibody-positive mothers are frequently referred for serial echocardiography due to the fetal risk of developing heart block and endocardial fibroelastosis. Little is known why only some and not all offspring develop these cardiac manifestations of neonatal lupus (CNL). This prospective study examined associations between anti-Ro antibody titers and fetal CNL. METHODS: Antibody-positive mothers referred since 2018 for fetal echocardiography at risk of CNL (group 1; n = 240) or with CNL (group 2; n = 18) were included. Maternal antibody titers were measured with a chemiluminescent immunoassay (CIA). Additional testing on diluted serum samples was used to quantify anti-Ro 60 antibody titers above the analytical measuring range (AMR) of the standard CIA (≥1,375 chemiluminescent units [CU]). RESULTS: Among 27 total mothers with a fetal diagnosis of CNL, all displayed anti-Ro 60 antibody titers that exceeded the AMR of the CIA at least 10-fold. Of 122 mothers in group 1 who underwent additional anti-Ro 60 antibody testing, event rates of CNL (n = 9) were 0% (0 of 45) among mothers with anti-Ro 60 antibody titers from 1,375-10,000 CU, 5% (3 of 56) among mothers with titers from 10,000-50,000 CU, but 29% (6 of 21) among mothers with titers >50,000 CU (odds ratio 13.1, P = 0.0008). Of mothers in group 2 with a primary diagnosis of CNL, 0% (0 of 18 mothers) had anti-Ro 60 antibody titers <10,000 CU, 44% (8 of 18 mothers) had titers from 10,000-50,000 CU, and 56% (10 of 18 mothers) had titers >50,000 CU. CONCLUSION: CNL is associated with substantially higher anti-Ro antibody titers than are obtained using a standard CIA. Enhancing the assay measuring range allows an improved specificity of identifying pregnancies at risk of CNL.

Analytical evaluation and Sigma metrics of 6 next generation chemistry assays on the Abbott Architect system.

BACKGROUND: We evaluated analytical and Sigma performance for 6 next generation chemistry assays on the Abbott Architect c8000 system. METHODS: Albumin with bromocresol purple or green, amylase, cholesterol, total protein, and urea nitrogen were analyzed using photometric technology. Analytical performance goals were defined based on Accreditation Canada Diagnostics (ACD) and Clinical Laboratory Improvement Amendments (CLIA). Precision study consisted of testing 2 quality control concentrations and 3 patient serum sample pools, twice a day in quintuplicate over 5 days. Linearity testing consisted of 5-6 concentrations of commercial linearity materials. We tested a minimum of 120 serum/plasma specimens on the new and current Architect methods for comparison. We assessed accuracy with reference materials for 5 assays, and a calibration standard for cholesterol. Bias from the reference standard target value was used for Sigma metric analysis. RESULTS: Observed total imprecision of the assays ranged from 0.5 to 4%, meeting pre-defined goals. Linearity was acceptable over the tested range. Measurements on the new and current Architect methods were comparable. Accuracy ranged from 0 to 2.0% absolute mean difference from target value. All 6 next generation clinical chemistry assays demonstrated Six Sigma quality, using CLIA standards. CONCLUSION: Applying ACD recommendations, 5 assays showed Six Sigma, while cholesterol showed Five Sigma performance.

Evaluation and comparison of four quantitative SARS-CoV-2 serological assays in COVID-19 patients and immunized healthy individuals, cancer patients, and patients with immunosuppressive therapy.

BACKGROUND: Semi-quantitative and quantitative immunoassays are the most commonly used methodology to evaluate immunity post immunization. OBJECTIVES: To compare four quantitative SARS-CoV-2 serological assays in COVID-19 patients and immunized healthy individuals, cancer patients, and patients with immunosuppressive therapy. STUDY DESIGN: 210 serological samples from COVID-19 infection and vaccination cohorts were used to create a serological sample repository. Serological methods from four manufacturers, namely Euroimmun, Roche, Abbott, and DiaSorin, were evaluated for quantitative, semi-quantitative, and qualitative antibody measurements. All four methods measure IgG antibodies against the SARS-CoV-2 spike receptor-binding domain and report the results in Binding Antibody Unit/mL (BAU/mL). A Total Error Allowable (TEa) of ±25% was chosen as the criteria to determine whether two methods are clinically equivalent quantitatively. Semi-quantitative results (titers) were derived using numeric antibody concentration divided by the cut-off value for each method. RESULTS: All paired quantitative comparisons demonstrated unacceptable performance. With ±25% as TEa, the best agreement was 74 (35.2% out of 210 samples) between Euroimmun and DiaSorin, whereas the lowest agreement was 11 (5.2% out of 210 samples) between Euroimmun and Roche. Antibody titers amongst all four methods were significantly different (p < 0.001). The highest titer difference from the same sample is between Roche and DiaSorin with a 1392-fold difference. On qualitative comparison, none of the paired comparison showed acceptable comparison (p < 0.001). CONCLUSIONS: Poor correlation exists between four evaluated assays, quantitatively, semi-quantitatively, and qualitatively. Further harmonization of assays is required to achieve comparable measurements.

Longitudinal efficacy and toxicity of SARS-CoV-2 vaccination in cancer patients treated with immunotherapy.

Despite more than 2 years having elapsed since the onset of SARS-CoV-2 pandemic, a level of hesitation around increased SARS-CoV-2 vaccine toxicity in cancer patients receiving immunotherapy (IO) remains. This hesitation stems from the idea that IO agents could elicit an overwhelming immune stimulation post vaccination and therefore increase the risk of vaccine-related toxicity. The aim of our study was to explore serological responses to SARS-CoV-2 vaccination in patients treated with IO and describe the level of immune stimulation using parameters such as blood cytokines, autoantibody levels and immune related adverse events (irAEs) post vaccination. Fifty-one evaluable patients were enrolled in this longitudinal study. Absolute levels and neutralization potential of anti-SARS-CoV-2 antibodies were not significantly different in the IO group compared to non-IO. Chemotherapy adversely affected seroconversion when compared to IO and/or targeted treatment. Following vaccination, the prevalence of grade ≥2 irAEs in patients treated with IO was not higher than the usual reported IO toxicity. We report, for the first time, that anti-SARS-CoV-2 vaccination, elicited the generation of five autoantibodies. The significantly increased autoantibodies were IgM autoantibodies against beta-2 glycoprotein (p = 0.02), myeloperoxidase (p = 0.03), nucleosome (p = 0.041), SPLUNC2 (p < 0.001) and IgG autoantibody against Myosin Heavy Chain 6 (MYH6) (p < 0.001). Overall, comprehensive analysis of a small cohort showed that co-administration of SARS-CoV-2 vaccine and IO is not associated with increased irAEs. Nevertheless, the detection of autoantibodies post anti-SARS-CoV-2 vaccination warrants further investigation (NCT03702309).

Evaluation of hemolysis, lipemia, and icterus interference with common clinical immunoassays.

OBJECTIVES: Hemolysis, icterus, and lipemia (HIL) are common sources of endogenous interference in clinical laboratory testing. Defining the threshold of interference for immunoassays enables appropriate reporting of their results when they are affected by HIL. METHODS: Pools of residual patient serum samples were spiked with a known amount of interferent to create samples with varying concentrations of hemolysate, bilirubin, and Intralipid that mimicked the effects of endogenous HIL. Samples were analysed on the Alinity i analyser (Abbott Diagnostics) for more than 25 immunoassays. The average recovery relative to the non-spiked sample was calculated for each interference level and was compared to a predefined allowable bias. RESULTS: C-peptide, estradiol, serum folate, free T4, homocysteine, insulin, and vitamin B12 were found to be affected by hemolysis, at hemoglobin concentrations between 0.3 to 20 g/L. Immunoassays for BNP, estradiol, free T3, and homocysteine were affected by icterus at conjugated bilirubin concentrations between 50 to 1,044 µmol/L. BNP, serum folate, and homocysteine were affected by Intralipid with measured triglyceride concentrations between 0.8 to 10 mmol/L. Lastly, serological immunoassays for HIV and hepatitis A, B and C were also affected by interferences. CONCLUSIONS: Immunoassays are impacted by varying degrees of HIL interference. Some measurands, in the presence of interference, are affected in a manner not previously indicated. The data presented herein provide an independent evaluation of HIL thresholds and will be of aid to resource-limited clinical laboratories that are unable to internally verify endogenous interferences when implementing the Alinity i analyser.