Pub Date : 2025-10-03DOI: 10.1093/clinchem/hvaf104
Adina Badea
{"title":"Commentary on Supratherapeutic Carbamazepine Concentration Following a Recent SARS-CoV-2 Infection.","authors":"Adina Badea","doi":"10.1093/clinchem/hvaf104","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf104","url":null,"abstract":"","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"157 1","pages":"1022"},"PeriodicalIF":9.3,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145209155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.131
Christina Higgins, John Zak, Michael Suster
Background Shelf-stable material to accurately replicate whole blood clotting for use as proficiency/external control, validation samples, and drug/biomarker discovery tools is not widely available. During clot formation, activated platelets change shape and aggregate, and erythrocytes (RBCs) are deformed by contractile forces; therefore, functional RBCs and platelets responsive to clotting pathway signaling are both fundamental to a representative clotting control. Standard storage methods preserve RBCs for up to 42 days and platelets up to 7 days, with functional deterioration during that period. Cryopreservation preserves clotting function for longer periods, but lot-to-lot performance is not well-characterized and cryopreserved material presents challenges for use as point-of-care external control. Lyophilization causes extensive membrane damage. Other membrane stabilization methods successfully prevent hemolysis but interfere with clotting. Here, we show that increasing RBC resistance to shear in conjunction with preserving RBC deformability and platelet responsiveness generates a refrigerator-stable product with acceptable clotting time precision and stability for 75 days post-draw, suitable as a proxy whole blood clotting sample. Methods Two lots of RBC/platelet material produced under ISO13485 were stored at 2–8°C during characterization, with periodic equilibration to room temperature, mimicking typical assay external control handling. The ClotChip System (IUO), intended for point-of-care use and utilizing dielectric spectroscopy to determine whole blood clotting time, was employed to assess clotting function. Both lots underwent 20-day precision assessment of clotting performance in the presence of normal plasma or clotting-factor deficient plasma (CLSI EP05-03). Material was also assessed for long-term and in-use stability assessments of clotting function (CLSI EP25). RBC deformability was assessed weekly via ektacytometry (LorrcaMaxSis) under normoxia and hypoxia. Complete blood counts (CBCs) were periodically measured (Abacus3CP). Responsiveness of RBCs and platelets to clotting factors was evaluated on ClotChip after combination with plasmas whose clotting factor levels varied. Results Clotting time repeatability was 3.3%-13.8% for both lots and total imprecision was 4.6%-15.0%. Mean clotting time for material combined with normal plasma remained within 20% of initial values for up to 75 days after blood draw; in-use stability at room temperature remained within 10% of initial values for the full period tested (5.5 hrs). RBC deformability (EImax) under normoxia or hypoxia fell within the fresh whole blood reference range and exhibited no significant change over 75 days (p>0.05) (see Figure). Half-maximal shear stress required to elongate cells was ∼30% elevated vs fresh blood at the start of storage, increased over time, and plateaued at 3-4x fresh blood levels. CBCs at baseline and 10 weeks indicated minimal changes in RBC o
{"title":"A-135 Characterization of refrigerator-stable erythrocyte and platelet product with utility for clotting assay external control and experimental applications","authors":"Christina Higgins, John Zak, Michael Suster","doi":"10.1093/clinchem/hvaf086.131","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.131","url":null,"abstract":"Background Shelf-stable material to accurately replicate whole blood clotting for use as proficiency/external control, validation samples, and drug/biomarker discovery tools is not widely available. During clot formation, activated platelets change shape and aggregate, and erythrocytes (RBCs) are deformed by contractile forces; therefore, functional RBCs and platelets responsive to clotting pathway signaling are both fundamental to a representative clotting control. Standard storage methods preserve RBCs for up to 42 days and platelets up to 7 days, with functional deterioration during that period. Cryopreservation preserves clotting function for longer periods, but lot-to-lot performance is not well-characterized and cryopreserved material presents challenges for use as point-of-care external control. Lyophilization causes extensive membrane damage. Other membrane stabilization methods successfully prevent hemolysis but interfere with clotting. Here, we show that increasing RBC resistance to shear in conjunction with preserving RBC deformability and platelet responsiveness generates a refrigerator-stable product with acceptable clotting time precision and stability for 75 days post-draw, suitable as a proxy whole blood clotting sample. Methods Two lots of RBC/platelet material produced under ISO13485 were stored at 2–8°C during characterization, with periodic equilibration to room temperature, mimicking typical assay external control handling. The ClotChip System (IUO), intended for point-of-care use and utilizing dielectric spectroscopy to determine whole blood clotting time, was employed to assess clotting function. Both lots underwent 20-day precision assessment of clotting performance in the presence of normal plasma or clotting-factor deficient plasma (CLSI EP05-03). Material was also assessed for long-term and in-use stability assessments of clotting function (CLSI EP25). RBC deformability was assessed weekly via ektacytometry (LorrcaMaxSis) under normoxia and hypoxia. Complete blood counts (CBCs) were periodically measured (Abacus3CP). Responsiveness of RBCs and platelets to clotting factors was evaluated on ClotChip after combination with plasmas whose clotting factor levels varied. Results Clotting time repeatability was 3.3%-13.8% for both lots and total imprecision was 4.6%-15.0%. Mean clotting time for material combined with normal plasma remained within 20% of initial values for up to 75 days after blood draw; in-use stability at room temperature remained within 10% of initial values for the full period tested (5.5 hrs). RBC deformability (EImax) under normoxia or hypoxia fell within the fresh whole blood reference range and exhibited no significant change over 75 days (p>0.05) (see Figure). Half-maximal shear stress required to elongate cells was ∼30% elevated vs fresh blood at the start of storage, increased over time, and plateaued at 3-4x fresh blood levels. CBCs at baseline and 10 weeks indicated minimal changes in RBC o","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"75 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145202929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.640
Wenying Zhang, Kevin Wen, Jack Bleesing, Mei Xin
Background Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphadenopathy, splenomegaly, cytopenias, and an increased risk of lymphoma. Molecular genetic diagnosis is essential for the accurate diagnosis and management of ALPS, particularly due to its overlapping clinical features with autoimmune lymphoproliferative immunodeficiency (ALPID). About 80% of ALPS cases result from germline or somatic pathogenic variants in FAS (ALPS-FAS, ALPS-sFAS), primarily through dominant-negative interference or haploinsufficiency. The FAS mutation spectrum includes nonsense, frameshift, missense, splicing defect variants, and copy number variants. Notably, around 13% ALPS-FAS cases are attributed to splice site/region mutations in FAS that disrupt splicing. However, novel splice region or deep intronic FAS variants are often classified as variants of unknown significance (VUS) due to the lack of functional validation, making their precise classification difficult to determine. This study aims to identify and characterize novel intronic or splice region FAS VUS found in a cohort of 1,488 ALPS cases at a pediatric center, as well as those reported in the ClinVar database. Methods We retrospectively reviewed the records of 1,488 patients with suspected ALPS referred to our institution for either the FAS gene sequencing or ALPS next-generation sequencing (NGS) panel between 2005 and 2023. Previously unreported variants with potential splicing effect were identified. Additionally, we reviewed the ClinVar database for rare FAS variants with predicted effects on splicing. To functionally assess these variants, we plan to use a minigene assay, which involves constructing a simplified version of the gene containing the exon of interest and its flanking introns into a plasmid. This plasmid is then transfected into cells to analyze how the variant affects the resulting mRNA transcript. Results We identified 19 novel FAS variants with predicted splicing effect in 30 independent probands from our cohort. These variants were distributed across eight FAS intronic splicing regions, including one synonymous change in a deep exonic region predicted to activate a nearby cryptic splice donor. Additionally, a review of the ClinVar database revealed 19 previously unreported rare splice region variants in FAS, of which, 13 were predicted to affect splicing. We will use the minigene system to experimentally validate the functional consequences of these variants. Conclusion FAS pathogenic variants affecting splicing represent a significant contributor to ALP-FAS and ALP-sFAS. The minigene assay will provide crucial functional evidence to further characterize the pathogenicity of rare VUS with predicted splicing effects, thereby improving the molecular diagnosis for ALPS. This approach is particularly valuable when RNA samples are unavailable from affected patients, enabling a more comprehensive genetic evaluation of ALPS.
{"title":"B-252 Functional Validation of Novel FAS Intronic and Splice Region VUS Through Minigene Assays","authors":"Wenying Zhang, Kevin Wen, Jack Bleesing, Mei Xin","doi":"10.1093/clinchem/hvaf086.640","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.640","url":null,"abstract":"Background Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphadenopathy, splenomegaly, cytopenias, and an increased risk of lymphoma. Molecular genetic diagnosis is essential for the accurate diagnosis and management of ALPS, particularly due to its overlapping clinical features with autoimmune lymphoproliferative immunodeficiency (ALPID). About 80% of ALPS cases result from germline or somatic pathogenic variants in FAS (ALPS-FAS, ALPS-sFAS), primarily through dominant-negative interference or haploinsufficiency. The FAS mutation spectrum includes nonsense, frameshift, missense, splicing defect variants, and copy number variants. Notably, around 13% ALPS-FAS cases are attributed to splice site/region mutations in FAS that disrupt splicing. However, novel splice region or deep intronic FAS variants are often classified as variants of unknown significance (VUS) due to the lack of functional validation, making their precise classification difficult to determine. This study aims to identify and characterize novel intronic or splice region FAS VUS found in a cohort of 1,488 ALPS cases at a pediatric center, as well as those reported in the ClinVar database. Methods We retrospectively reviewed the records of 1,488 patients with suspected ALPS referred to our institution for either the FAS gene sequencing or ALPS next-generation sequencing (NGS) panel between 2005 and 2023. Previously unreported variants with potential splicing effect were identified. Additionally, we reviewed the ClinVar database for rare FAS variants with predicted effects on splicing. To functionally assess these variants, we plan to use a minigene assay, which involves constructing a simplified version of the gene containing the exon of interest and its flanking introns into a plasmid. This plasmid is then transfected into cells to analyze how the variant affects the resulting mRNA transcript. Results We identified 19 novel FAS variants with predicted splicing effect in 30 independent probands from our cohort. These variants were distributed across eight FAS intronic splicing regions, including one synonymous change in a deep exonic region predicted to activate a nearby cryptic splice donor. Additionally, a review of the ClinVar database revealed 19 previously unreported rare splice region variants in FAS, of which, 13 were predicted to affect splicing. We will use the minigene system to experimentally validate the functional consequences of these variants. Conclusion FAS pathogenic variants affecting splicing represent a significant contributor to ALP-FAS and ALP-sFAS. The minigene assay will provide crucial functional evidence to further characterize the pathogenicity of rare VUS with predicted splicing effects, thereby improving the molecular diagnosis for ALPS. This approach is particularly valuable when RNA samples are unavailable from affected patients, enabling a more comprehensive genetic evaluation of ALPS.","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"5 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.253
Marina Bezerra, Rachel Petrola, Edlâny Milanez
Background Urinary tract infections (UTIs) are among the leading causes of bacterial infections, affecting millions of people annually and impacting public health. These infections can affect any part of the urinary system, ranging from mild cases to complications like pyelonephritis and sepsis. Several factors can influence the development and severity of UTIs, including age, sex, and comorbidities such as diabetes mellitus. This study aims to analyze the epidemiological profile of UTIs in outpatients from a Brazilian population, investigating the distribution of etiological agents and their association with age, sex, diabetes, and bacterial colony counts. The use of big data for laboratory analysis provides a broad approach to understanding the behavior of these infections, their impact on clinical management, and corroboration with the literature. Methods This epidemiological, cross-sectional study analyzed the distribution of etiological agents in urine cultures from outpatients at a private laboratory in the Northeast region of Brazil. Data between January 2022 and February 2025 were extracted from the LIS system. Urinalysis and urine culture results were analyzed using Sysmex® and Vitek - 2 Compat ® equipment, considering bacterial colony counts and self-reported diabetes status. The population was stratified by sex and age. Results The population was mainly female (79%), with a mean age of 55 ± 22 years. Bacterial growth was observed in 22,326 (19.8%) of the urine cultures (N=112,506). The main pathogens identified were Escherichia coli (12%; N=13,259), Klebsiella pneumoniae (3.2%; N=3,652), Proteus mirabilis (0.9%; N=1,035), and Enterococcus faecalis (0.9%; N=1,002). Extended-spectrum beta-lactamase (ESBL)-positive cases accounted for 14% (N=1,450). Among the ESBL-positive cases, five patients had bacterial growth exceeding 1,000,000 CFU/mL (Klebsiella pneumoniae N=4, Proteus mirabilis N=1), with two cases of dual colonization (Klebsiella/E. coli and Proteus/E. coli). Among the patients, 15% (N=16,473) self-reported as diabetic. This group had a higher prevalence of ESBL-positive infections (18%, N=384), and in cases with more than one isolated microorganism, ESBL positivity was observed in 38%. Candida glabrata was more prevalent in diabetic patients. Conclusion The analysis revealed that most cases occurred in women, particularly those aged 18 to 50 years, with Escherichia coli as the most frequent pathogen. Diabetic patients had a higher prevalence of infections caused by ESBL-positive microorganisms, especially in mixed infections. Additionally, Candida glabrata was more prevalent in this group. The findings show that the prevalence of main UTI pathogens remains similar to that reported in recent years. This study emphasizes the importance of regularly updating epidemiological data to improve clinical management strategies for these infections.
{"title":"A-264 Urine Culture Trends from 2022 to 2025: A Review of Microorganism Prevalence","authors":"Marina Bezerra, Rachel Petrola, Edlâny Milanez","doi":"10.1093/clinchem/hvaf086.253","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.253","url":null,"abstract":"Background Urinary tract infections (UTIs) are among the leading causes of bacterial infections, affecting millions of people annually and impacting public health. These infections can affect any part of the urinary system, ranging from mild cases to complications like pyelonephritis and sepsis. Several factors can influence the development and severity of UTIs, including age, sex, and comorbidities such as diabetes mellitus. This study aims to analyze the epidemiological profile of UTIs in outpatients from a Brazilian population, investigating the distribution of etiological agents and their association with age, sex, diabetes, and bacterial colony counts. The use of big data for laboratory analysis provides a broad approach to understanding the behavior of these infections, their impact on clinical management, and corroboration with the literature. Methods This epidemiological, cross-sectional study analyzed the distribution of etiological agents in urine cultures from outpatients at a private laboratory in the Northeast region of Brazil. Data between January 2022 and February 2025 were extracted from the LIS system. Urinalysis and urine culture results were analyzed using Sysmex® and Vitek - 2 Compat ® equipment, considering bacterial colony counts and self-reported diabetes status. The population was stratified by sex and age. Results The population was mainly female (79%), with a mean age of 55 ± 22 years. Bacterial growth was observed in 22,326 (19.8%) of the urine cultures (N=112,506). The main pathogens identified were Escherichia coli (12%; N=13,259), Klebsiella pneumoniae (3.2%; N=3,652), Proteus mirabilis (0.9%; N=1,035), and Enterococcus faecalis (0.9%; N=1,002). Extended-spectrum beta-lactamase (ESBL)-positive cases accounted for 14% (N=1,450). Among the ESBL-positive cases, five patients had bacterial growth exceeding 1,000,000 CFU/mL (Klebsiella pneumoniae N=4, Proteus mirabilis N=1), with two cases of dual colonization (Klebsiella/E. coli and Proteus/E. coli). Among the patients, 15% (N=16,473) self-reported as diabetic. This group had a higher prevalence of ESBL-positive infections (18%, N=384), and in cases with more than one isolated microorganism, ESBL positivity was observed in 38%. Candida glabrata was more prevalent in diabetic patients. Conclusion The analysis revealed that most cases occurred in women, particularly those aged 18 to 50 years, with Escherichia coli as the most frequent pathogen. Diabetic patients had a higher prevalence of infections caused by ESBL-positive microorganisms, especially in mixed infections. Additionally, Candida glabrata was more prevalent in this group. The findings show that the prevalence of main UTI pathogens remains similar to that reported in recent years. This study emphasizes the importance of regularly updating epidemiological data to improve clinical management strategies for these infections.","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"62 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.710
Grace Williams
Background America continues to be plagued by an opioid epidemic, and the third wave, predominated by fentanyl analogs has yet to abate. The detectability of fentanyl analogs and synthetic opioids is of critical concern to clinical and forensic laboratories. The US Centers for Disease Control and Prevention has coordinated production of a Traceable Opioid Material® Kits (TOM Kits®) to support laboratory detection of current and potentially emerging opioids as well as common co-drugs found in fentanyl-containing samples. Previously published studies by this group have focused on analyzing compound structures in relation to assay detectability to predict the likely target epitope. The final data analysis of these over 1,800 measurements will also include this prediction. Methods From the TOM Kits®, 215 opioids were evaluated. These were analyzed using four commercially available fentanyl immunoassays: ARK™ Fentanyl II, Immunalysis Fentanyl Urine HEIA®, Immunalysis Fentanyl Urine SEFRIA® Drug Screening Kit, and the Lin-Zhi Fentanyl Enzyme Immunoassay. The detectability of the opioids was initially evaluated by preparing each opioid in certified drug-free human urine at 1 ng/mL, then analyzing the sample using the four immunoassays to determine if the analog screened positive in singlicate. Testing of that opioid was discontinued if the initial results was positive. If the result was negative, the resultant value was evaluated to determine whether a concentration of 10 ng/mL or 100 ng/mL would be appropriate for the second analysis. Opioids that did not screen positive at a concentration of 100 ng/mL were considered undetected by that immunoassay. Results The difference in reactivity of the immunoassay’s reagents was evaluated in conjunction with the chemical structure of each opioid. All four immunoassays detected 106 of the opioids at the concentrations tested. Eighty-two opioids had variable cross-reactivity with the four immunoassays determined by the unique epitope for each reagent antibody. 28 opioids were not detected by any assay and 17 of these were the emerging synthetic opioids. At the lowest concentration tested (1 ng/mL), the ARK II assay detected 36 compounds, the SEFRIA detected 74, LZI detected 5, and the HEIA detected 18. Higher concentration testing was conducted on the other compounds. The undetected opioids included the surgical anesthetic parent compounds remifentanil and alfentanil, and sufentanil metabolite norsufentanil. Additionally, three carfentanil analogs were not detected by the four immunoassays. Of the 34 compounds only detected by one assay, 27 of those positive results were from the Lin-Zhi assay. Conclusion This information will be of use in both clinical and forensic settings in evaluation of the potential false-positive or false negatives in urine fentanyl screening. The detectability of fentanyl analogs and synthetic opioids varies by assay and it may be possible to predict the detection or lack thereof for a par
{"title":"B-323 Evaluating the Detection of 215 Fentanyl Analogs and Synthetic Opioids in Urine Using Four Commercial Immunoassays","authors":"Grace Williams","doi":"10.1093/clinchem/hvaf086.710","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.710","url":null,"abstract":"Background America continues to be plagued by an opioid epidemic, and the third wave, predominated by fentanyl analogs has yet to abate. The detectability of fentanyl analogs and synthetic opioids is of critical concern to clinical and forensic laboratories. The US Centers for Disease Control and Prevention has coordinated production of a Traceable Opioid Material® Kits (TOM Kits®) to support laboratory detection of current and potentially emerging opioids as well as common co-drugs found in fentanyl-containing samples. Previously published studies by this group have focused on analyzing compound structures in relation to assay detectability to predict the likely target epitope. The final data analysis of these over 1,800 measurements will also include this prediction. Methods From the TOM Kits®, 215 opioids were evaluated. These were analyzed using four commercially available fentanyl immunoassays: ARK™ Fentanyl II, Immunalysis Fentanyl Urine HEIA®, Immunalysis Fentanyl Urine SEFRIA® Drug Screening Kit, and the Lin-Zhi Fentanyl Enzyme Immunoassay. The detectability of the opioids was initially evaluated by preparing each opioid in certified drug-free human urine at 1 ng/mL, then analyzing the sample using the four immunoassays to determine if the analog screened positive in singlicate. Testing of that opioid was discontinued if the initial results was positive. If the result was negative, the resultant value was evaluated to determine whether a concentration of 10 ng/mL or 100 ng/mL would be appropriate for the second analysis. Opioids that did not screen positive at a concentration of 100 ng/mL were considered undetected by that immunoassay. Results The difference in reactivity of the immunoassay’s reagents was evaluated in conjunction with the chemical structure of each opioid. All four immunoassays detected 106 of the opioids at the concentrations tested. Eighty-two opioids had variable cross-reactivity with the four immunoassays determined by the unique epitope for each reagent antibody. 28 opioids were not detected by any assay and 17 of these were the emerging synthetic opioids. At the lowest concentration tested (1 ng/mL), the ARK II assay detected 36 compounds, the SEFRIA detected 74, LZI detected 5, and the HEIA detected 18. Higher concentration testing was conducted on the other compounds. The undetected opioids included the surgical anesthetic parent compounds remifentanil and alfentanil, and sufentanil metabolite norsufentanil. Additionally, three carfentanil analogs were not detected by the four immunoassays. Of the 34 compounds only detected by one assay, 27 of those positive results were from the Lin-Zhi assay. Conclusion This information will be of use in both clinical and forensic settings in evaluation of the potential false-positive or false negatives in urine fentanyl screening. The detectability of fentanyl analogs and synthetic opioids varies by assay and it may be possible to predict the detection or lack thereof for a par","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"101 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.365
Kenneth Harlow, Karin Blume, Felicia Andersson, Liu Guanghui, Søren Echwald, Niklas Mårtensson
Background The ability to make rapid and early clinical decisions regarding diagnosis and therapy is often highly correlated with the ability to detect one or more specific disease-relevant biomarkers early and at very low concentrations during the onset of a particular illness. High-sensitivity immunoassays employing various methodologies have been instrumental in pushing the limits of detection for immunoassay techniques to lower values for many disease-relevant biomarkers. This in turn has allowed clinicians to make relevant diagnostic and therapeutic decisions earlier than possible when using more conventional immunoassay methodology. The unique ability of semiconductor nanowires of specific diameters to enhance and amplify the fluorescence of fluorescent reporter molecules provides a generic way to increase the sensitivity of fluorescent immunoassays (FLIA) and has allowed us to demonstrate a generically applicable mode of FLIA enhancement using semiconductor nanowire arrays. Nanowire enhanced fluorescence thus provides a general way to extend the utility of FLIAs to achieve earlier clinical decision points and improve treatment and clinical outcome. Methods Arrays containing silicon nanowires with dimensions designed to interact with specific wavelengths of light were fabricated and used as substrates for performing sandwich type FLIAs for a variety of protein biomarkers. A range of biomarkers including CEA, Troponin and IL-6 were employed in order to evaluate how general the sensitivity enhancement effects were across different analyte assays. Commercially available antibodies and reagents were used to set up the immunoassays used in these studies. FLIA assays were performed on these nanowire array substrates using standard immunochemical procedures. Following assay for a particular biomarker analyte on the nanowire arrays, the arrays were imaged using low magnification in an inverted fluorescence microscope to record the spatial distribution and intensity of fluorescent signals present on the arrays. These images were further analyzed using a proprietary analysis algorithm to extract values for total fluorescence intensities, and to locate and enumerate the number of fluorescent nanowires and to determine the average fluorescence intensity per nanowire. These values were then used to calculate concentrations of analytes present in calibrator solutions. Results In general, we were able to demonstrate an enhancement in sensitivity of from 20 to 200-fold over the same assay conducted on a planar material such as plastic or glass and using the same reagents. In addition, we observed extended dynamic ranges compared to assays run on planar surfaces often with dynamic ranges of 6-7 orders of magnitude. Fluorescent intensity measurements of individual nanowires at low concentrations were constant over a range of concentrations while the number of fluorescing nanowires increased with increasing concentrations at these low levels. This suggests si
{"title":"A-381 Nanowire assisted fluorescence immunoassay (FLIA): Enabling low-cost, high-sensitivity biomarker assays for expanded clinical utility","authors":"Kenneth Harlow, Karin Blume, Felicia Andersson, Liu Guanghui, Søren Echwald, Niklas Mårtensson","doi":"10.1093/clinchem/hvaf086.365","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.365","url":null,"abstract":"Background The ability to make rapid and early clinical decisions regarding diagnosis and therapy is often highly correlated with the ability to detect one or more specific disease-relevant biomarkers early and at very low concentrations during the onset of a particular illness. High-sensitivity immunoassays employing various methodologies have been instrumental in pushing the limits of detection for immunoassay techniques to lower values for many disease-relevant biomarkers. This in turn has allowed clinicians to make relevant diagnostic and therapeutic decisions earlier than possible when using more conventional immunoassay methodology. The unique ability of semiconductor nanowires of specific diameters to enhance and amplify the fluorescence of fluorescent reporter molecules provides a generic way to increase the sensitivity of fluorescent immunoassays (FLIA) and has allowed us to demonstrate a generically applicable mode of FLIA enhancement using semiconductor nanowire arrays. Nanowire enhanced fluorescence thus provides a general way to extend the utility of FLIAs to achieve earlier clinical decision points and improve treatment and clinical outcome. Methods Arrays containing silicon nanowires with dimensions designed to interact with specific wavelengths of light were fabricated and used as substrates for performing sandwich type FLIAs for a variety of protein biomarkers. A range of biomarkers including CEA, Troponin and IL-6 were employed in order to evaluate how general the sensitivity enhancement effects were across different analyte assays. Commercially available antibodies and reagents were used to set up the immunoassays used in these studies. FLIA assays were performed on these nanowire array substrates using standard immunochemical procedures. Following assay for a particular biomarker analyte on the nanowire arrays, the arrays were imaged using low magnification in an inverted fluorescence microscope to record the spatial distribution and intensity of fluorescent signals present on the arrays. These images were further analyzed using a proprietary analysis algorithm to extract values for total fluorescence intensities, and to locate and enumerate the number of fluorescent nanowires and to determine the average fluorescence intensity per nanowire. These values were then used to calculate concentrations of analytes present in calibrator solutions. Results In general, we were able to demonstrate an enhancement in sensitivity of from 20 to 200-fold over the same assay conducted on a planar material such as plastic or glass and using the same reagents. In addition, we observed extended dynamic ranges compared to assays run on planar surfaces often with dynamic ranges of 6-7 orders of magnitude. Fluorescent intensity measurements of individual nanowires at low concentrations were constant over a range of concentrations while the number of fluorescing nanowires increased with increasing concentrations at these low levels. This suggests si","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"39 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.456
Lisa Salvucci
Background Accurate and timely serum, plasma, and urine osmolality measurements are crucial for diagnosing and treating conditions such as hyponatremia, toxic alcohol ingestion, and diabetes insipidus. Most clinical osmometers rely on manual methods which require pipetting and testing one sample, control, or calibration standard at a time. These manual steps are time-consuming and increase the risk of human error. A new osmometer, the OsmoPRO® MAX, saves time and reduces these risks by automating freezing-point depression measurements (Advanced Instruments, LLC). The purpose of this study was to evaluate the analytical performance of the OsmoPRO MAX in measuring the osmolality of serum, plasma, and urine. Methods OsmoPRO MAX analytical performance was evaluated at Boston Medical Center in Boston, Massachusetts using four criteria: (1) Linearity was assessed with an allowable systematic error of <2 mOsm/kg H2O and an allowable total error of <6.0 mOsm/kg H2O over a range of 0-2000 mOsm/kg H2O using an Osmolality Linearity Set. (2) Simple Accuracy was evaluated over a range of 240 to 800 mOsm/kg H2O using Clinitrol™ 290 with an acceptable range of +/- 4 mOsm/kg H2O as well as Protinol™ 240, 280, and 320, each with an acceptable range of +/- 7 mOsm/kg H2O, and Renol™ 300 and 800, both with an acceptable range of +/- 10 mOsm/kg H2O. (3) Simple Precision was measured using Clinitrol 290 with an acceptable within-run standard deviation (SD) of <2.0 mOsm/kg H2O as well as Protinol 240, 280, and 320, each with an acceptable within-run SD of <3 mOsm/kg H2O, and Renol 300 and 800, with an acceptable within-run SD of <3 mOsm/kg H2O and <4.7 mOsm/kg H2O, respectively. (4) Alternative Method Comparison was conducted against the OsmoPRO® Multi-Sample Micro-Osmometer on 80 patient samples analyzed by Passing-Bablok regression analysis. Results OsmoPRO MAX passed all criteria. Linearity testing produced a negligible observable error of 0.1 mOsm/kg H2O, accurate within the allowable systematic error and all results were accurate within the total allowable error. Simple Accuracy testing produced replicates all within their specified target range. Simple Precision testing of all replicates for each standard tested fell within their respective within-run SD. Alternative Quantitative Method Comparison demonstrated strong agreement between the two methods with a correlation coefficient of 0.9998 and a bias of -2.1 (-0.6%). Conclusion This study demonstrates that the OsmoPRO MAX Automated Osmometer meets all acceptance criteria for analytical performance including linearity, simple accuracy, simple precision, and alternative quantitative method comparison against the OsmoPRO Multi-Sample Micro-Osmometer.
{"title":"B-058 Evaluating the analytical performance of the OsmoPRO® MAX Automated Osmometer","authors":"Lisa Salvucci","doi":"10.1093/clinchem/hvaf086.456","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.456","url":null,"abstract":"Background Accurate and timely serum, plasma, and urine osmolality measurements are crucial for diagnosing and treating conditions such as hyponatremia, toxic alcohol ingestion, and diabetes insipidus. Most clinical osmometers rely on manual methods which require pipetting and testing one sample, control, or calibration standard at a time. These manual steps are time-consuming and increase the risk of human error. A new osmometer, the OsmoPRO® MAX, saves time and reduces these risks by automating freezing-point depression measurements (Advanced Instruments, LLC). The purpose of this study was to evaluate the analytical performance of the OsmoPRO MAX in measuring the osmolality of serum, plasma, and urine. Methods OsmoPRO MAX analytical performance was evaluated at Boston Medical Center in Boston, Massachusetts using four criteria: (1) Linearity was assessed with an allowable systematic error of &lt;2 mOsm/kg H2O and an allowable total error of &lt;6.0 mOsm/kg H2O over a range of 0-2000 mOsm/kg H2O using an Osmolality Linearity Set. (2) Simple Accuracy was evaluated over a range of 240 to 800 mOsm/kg H2O using Clinitrol™ 290 with an acceptable range of +/- 4 mOsm/kg H2O as well as Protinol™ 240, 280, and 320, each with an acceptable range of +/- 7 mOsm/kg H2O, and Renol™ 300 and 800, both with an acceptable range of +/- 10 mOsm/kg H2O. (3) Simple Precision was measured using Clinitrol 290 with an acceptable within-run standard deviation (SD) of &lt;2.0 mOsm/kg H2O as well as Protinol 240, 280, and 320, each with an acceptable within-run SD of &lt;3 mOsm/kg H2O, and Renol 300 and 800, with an acceptable within-run SD of &lt;3 mOsm/kg H2O and &lt;4.7 mOsm/kg H2O, respectively. (4) Alternative Method Comparison was conducted against the OsmoPRO® Multi-Sample Micro-Osmometer on 80 patient samples analyzed by Passing-Bablok regression analysis. Results OsmoPRO MAX passed all criteria. Linearity testing produced a negligible observable error of 0.1 mOsm/kg H2O, accurate within the allowable systematic error and all results were accurate within the total allowable error. Simple Accuracy testing produced replicates all within their specified target range. Simple Precision testing of all replicates for each standard tested fell within their respective within-run SD. Alternative Quantitative Method Comparison demonstrated strong agreement between the two methods with a correlation coefficient of 0.9998 and a bias of -2.1 (-0.6%). Conclusion This study demonstrates that the OsmoPRO MAX Automated Osmometer meets all acceptance criteria for analytical performance including linearity, simple accuracy, simple precision, and alternative quantitative method comparison against the OsmoPRO Multi-Sample Micro-Osmometer.","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"109 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.119
Shayan Askari, Lawrence Goldfinger
Background Platelet concentrates stored at room temperature have a shelf life of five days. During storage, platelets undergo glycoprotein cleavage by metalloproteases, notably ADAM17 cleavage of GPIba, which leads to decreased post-transfusion reactivity and recovery. Aim: To investigate putative ADAM17 synthesis and its contributions to GPIba proteolysis in stored platelets. Methods Human platelets maintained in autologous plasma were treated on day 1 of storage with naked endonuclease-resistant ADAM17 siRNA and monitored for molecular and cellular effects. Platelet-specific Adam17-deleted mice were generated and dynamics of GpIba cleavage were assessed. Results Platelets translated nascent ADAM17 during storage as evidenced by increased expression blocked by puromycin, and by metabolic labeling with azidohomoalanine, coinciding with progressive GPIba ectodomain cleavage. SiRNA treatment suppressed ADAM17 translation, and rescued total but not surface levels of full-length GPIba in resting platelets during storage. Flow cytometry and confocal microscopy in permeabilized platelets confirmed the existence of an internal pool of GPIba as suggested by prior EM studies. Stimulation of washed platelets with thrombin or calcium ionophore led to 27.3 ± 4.7% and 47.5 ± 0.96% decrease in surface GPIba, respectively. Platelet pre-treatment with cell-permeable ADAM17 inhibitor KP-457 protected GPIba from ectodomain cleavage following platelet stimulation. However, cell-impermeable 5G6 antibody against the scissile domain of GPIba failed to protect GPIba upon platelet stimulation. ADAM17 siRNA did not alter thrombin-induced decrease in surface GPIba across 5 days in storage. However, surface GpIba was increased in resting and thrombin-stimulated Adam17-deleted murine platelets. Adam17-deficient platelets showed similar lifespan to wild type platelets as assessed by in vivo pulse chase labeling. Conclusions: An internal pool of GPIba, possibly in the open canalicular system (OCS), is exposed upon platelet stimulation but subject to rapid cleavage by ADAM17. Chemical inhibition or genetic deletion of ADAM17 allows the internal reservoir of GPIba to reach the surface in stimulated platelets. However, the pool of existing, siRNA-resistant ADAM17 is sufficient to cleave GPIba upon stimulation in stored platelets.
{"title":"A-123 Nascent ADAM17 synthesis potentiates GPIba cleavage in resting and stimulated stored platelets","authors":"Shayan Askari, Lawrence Goldfinger","doi":"10.1093/clinchem/hvaf086.119","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.119","url":null,"abstract":"Background Platelet concentrates stored at room temperature have a shelf life of five days. During storage, platelets undergo glycoprotein cleavage by metalloproteases, notably ADAM17 cleavage of GPIba, which leads to decreased post-transfusion reactivity and recovery. Aim: To investigate putative ADAM17 synthesis and its contributions to GPIba proteolysis in stored platelets. Methods Human platelets maintained in autologous plasma were treated on day 1 of storage with naked endonuclease-resistant ADAM17 siRNA and monitored for molecular and cellular effects. Platelet-specific Adam17-deleted mice were generated and dynamics of GpIba cleavage were assessed. Results Platelets translated nascent ADAM17 during storage as evidenced by increased expression blocked by puromycin, and by metabolic labeling with azidohomoalanine, coinciding with progressive GPIba ectodomain cleavage. SiRNA treatment suppressed ADAM17 translation, and rescued total but not surface levels of full-length GPIba in resting platelets during storage. Flow cytometry and confocal microscopy in permeabilized platelets confirmed the existence of an internal pool of GPIba as suggested by prior EM studies. Stimulation of washed platelets with thrombin or calcium ionophore led to 27.3 ± 4.7% and 47.5 ± 0.96% decrease in surface GPIba, respectively. Platelet pre-treatment with cell-permeable ADAM17 inhibitor KP-457 protected GPIba from ectodomain cleavage following platelet stimulation. However, cell-impermeable 5G6 antibody against the scissile domain of GPIba failed to protect GPIba upon platelet stimulation. ADAM17 siRNA did not alter thrombin-induced decrease in surface GPIba across 5 days in storage. However, surface GpIba was increased in resting and thrombin-stimulated Adam17-deleted murine platelets. Adam17-deficient platelets showed similar lifespan to wild type platelets as assessed by in vivo pulse chase labeling. Conclusions: An internal pool of GPIba, possibly in the open canalicular system (OCS), is exposed upon platelet stimulation but subject to rapid cleavage by ADAM17. Chemical inhibition or genetic deletion of ADAM17 allows the internal reservoir of GPIba to reach the surface in stimulated platelets. However, the pool of existing, siRNA-resistant ADAM17 is sufficient to cleave GPIba upon stimulation in stored platelets.","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"24 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.757
Jiawei Zhang, Qishui Ou, Can Liu, Junjie Lai, Fuguo Zhan
Background Hepatocellular carcinoma (HCC) is a malignant neoplastic disease characterized by high clinical incidence and mortality. The prognosis for HCC remains poor mainly due to the challenging in early diagnosis. It is urgent to identify reliable biomarkers for early detection to improve prognosis of HCC. N6-methyladenine (m6A) modification of RNA has emerged as an important regulatory mechanism in various cancers, including HCC. It alters the stability, translation, and splicing of RNA and further influences development and progression of cancers. The microRNAs are involved in the initiation and progression of HCC through regulating gene expression. Abnormal expression of specific microRNAs can serve as biomarkers for HCC diagnosis and prognosis. This study aims to investigate the role of m6A modification in HCC development and identify microRNAs with significant expression differences in HCC patients. A nomogram model based on serum expression levels was developed to predict HCC risk and assess its diagnostic efficacy, providing valuable insights for early detection and more personalized management of HCC. Methods Twenty-four HCC patients and 22 matched healthy individuals were enrolled. The overall level of serum m6A was measured using an RNA m6A level detection kit. The expression levels of m6A modifying enzymes METTL3, BCDIN3D, as well as liver cancer-related microRNAs (microRNA-122, microRNA-198, microRNA-361, microRNA-378 and microRNA-532) were assessed by qRT-PCR. Each index was compared between patients and controls. Risk factors related to the incidence of HCC were identified using multivariate logistic regression analysis, and a nomogram model was constructed to predict HCC risk. Results Univariate analysis revealed that the overall level of m6A modification was down-regulated in HCC patients (0.0015 vs 0.0030, p=0.0015). The expression of microRNA-122, microRNA-198 and microRNA-532 was significantly higher in HCC patients (p<0.05). Multivariate logistic regression analysis identified m6A level (OR=0.288, 95%CI:0.123-0.676, P=0.004), microRNA-122 expression (OR=1.338, 95%CI:1.006-1.779, P=0.045) and microRNA-532 expression (OR=1.403, 95%CI:1.011-1.947, P=0.043) as independent risk factors for HCC. Two prediction models for HCC based on these indicators were developed: the m6A + microRNA-122 model (AUC=0.849, 95%CI=0.738-0.961) and the m6A + microRNA-532 model (AUC=0.847, 95%CI=0.730-0.963). Both models were internally verified by Bootstrap self-sampling method with the C-index value of 0.85, indicating good discrimination. Calibration curves showed a good fit, and decision curve analysis confirmed that the nomogram model provided greater clinical benefit than using single risk factors. Conclusion Serum RNA m6A modification is significantly decreased in HCC patients, while the expression of microRNA-122, microRNA-198, and microRNA-532 is elevated, which could be of value for the diagnosis of HCC. In addition, RNA m6A, microRN
{"title":"B-372 The diagnostic value of serum RNA m6A methylation and microRNAs in hepatocellular carcinoma","authors":"Jiawei Zhang, Qishui Ou, Can Liu, Junjie Lai, Fuguo Zhan","doi":"10.1093/clinchem/hvaf086.757","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.757","url":null,"abstract":"Background Hepatocellular carcinoma (HCC) is a malignant neoplastic disease characterized by high clinical incidence and mortality. The prognosis for HCC remains poor mainly due to the challenging in early diagnosis. It is urgent to identify reliable biomarkers for early detection to improve prognosis of HCC. N6-methyladenine (m6A) modification of RNA has emerged as an important regulatory mechanism in various cancers, including HCC. It alters the stability, translation, and splicing of RNA and further influences development and progression of cancers. The microRNAs are involved in the initiation and progression of HCC through regulating gene expression. Abnormal expression of specific microRNAs can serve as biomarkers for HCC diagnosis and prognosis. This study aims to investigate the role of m6A modification in HCC development and identify microRNAs with significant expression differences in HCC patients. A nomogram model based on serum expression levels was developed to predict HCC risk and assess its diagnostic efficacy, providing valuable insights for early detection and more personalized management of HCC. Methods Twenty-four HCC patients and 22 matched healthy individuals were enrolled. The overall level of serum m6A was measured using an RNA m6A level detection kit. The expression levels of m6A modifying enzymes METTL3, BCDIN3D, as well as liver cancer-related microRNAs (microRNA-122, microRNA-198, microRNA-361, microRNA-378 and microRNA-532) were assessed by qRT-PCR. Each index was compared between patients and controls. Risk factors related to the incidence of HCC were identified using multivariate logistic regression analysis, and a nomogram model was constructed to predict HCC risk. Results Univariate analysis revealed that the overall level of m6A modification was down-regulated in HCC patients (0.0015 vs 0.0030, p=0.0015). The expression of microRNA-122, microRNA-198 and microRNA-532 was significantly higher in HCC patients (p&lt;0.05). Multivariate logistic regression analysis identified m6A level (OR=0.288, 95%CI:0.123-0.676, P=0.004), microRNA-122 expression (OR=1.338, 95%CI:1.006-1.779, P=0.045) and microRNA-532 expression (OR=1.403, 95%CI:1.011-1.947, P=0.043) as independent risk factors for HCC. Two prediction models for HCC based on these indicators were developed: the m6A + microRNA-122 model (AUC=0.849, 95%CI=0.738-0.961) and the m6A + microRNA-532 model (AUC=0.847, 95%CI=0.730-0.963). Both models were internally verified by Bootstrap self-sampling method with the C-index value of 0.85, indicating good discrimination. Calibration curves showed a good fit, and decision curve analysis confirmed that the nomogram model provided greater clinical benefit than using single risk factors. Conclusion Serum RNA m6A modification is significantly decreased in HCC patients, while the expression of microRNA-122, microRNA-198, and microRNA-532 is elevated, which could be of value for the diagnosis of HCC. In addition, RNA m6A, microRN","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"17 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-02DOI: 10.1093/clinchem/hvaf086.503
Audrianna Kern, Adam Okerlund, Charlotte Kunkler, Christopher Warner
Background Human serum albumin (HSA) and bovine serum albumin (BSA) are commonly used as a blocking agents in immunoassays to prevent non-specific surface binding, thereby reducing background signal and increasing assay sensitivity. However, these native albumins present challenges such as donor dependency, lot-to-lot variability, and contamination with plasma-derived molecules (e.g., vitamins, hormones, growth factors, antibodies/Ig), which can affect assay performance and reproducibility. Recombinant human albumin (rHA) offers a potential alternative by providing the same binding and blocking characteristics as native albumin without the limitations. A new rHA produced in Thermothelomyces heterothallica (C1) and purified using a scalable proprietary process may mitigate these challenges. This study aims to evaluate the amount of binding on different surfaces of this new rHA compared to native albumins to determine the viability of rHA as a surface blocker in immunoassays. Methods The amount of albumin binding to different surfaces was assessed using a bead binding assay. Here, albumin (rHA, HSA, BSA) was incubated with commercially available Dynabeads: hydrophilic (carboxylic acid or amine functionalized) and hydrophobic (tosylactivated). The amount of protein bound to the beads was determined by heating the coated beads in a denaturing buffer and quantifying via densitometry on an SDS-PAGE gel against a standard curve. Statistical analysis was performed using GraphPad Prism, with data presented as the mean of at least three independent replicates with standard deviations. A two-tailed t-test was used to determine statistical significance at the 95% confidence interval. Results All tested albumin products demonstrate 10-fold more protein binding to the hydrophobic beads than either of the hydrophilic beads, highlighting the chemical similarities of albumin from different sources. For both hydrophilic bead types, all tested albumins demonstrated statistically the same amount of binding, ranging from 0.10-0.23 µg and 0.19-0.23 µg for the carboxylic acid and amine beads, respectively, with rHA at 0.15 ± 0.06 µg and 0.23 ± 0.07 µg. The hydrophobic beads had a much larger range of albumin binding, from 1.4-3.7 µg, with rHA being at the lower end of the range at 1.4 ± 0.2 µg, but statistically the same as BSA and two HSA products. The large differences in binding of the native albumins showcase the variability of natively derived products from different producers. Even with these slight differences, these data demonstrate rHA has comparable surface binding chemistry to native albumins. Conclusion This new rHA produced by C1 and purified in a scalable process binds surfaces comparably to commercially available native albumin products on all surfaces tested. These results support the use of rHA as an alternative to native albumins in diagnostic applications, providing effective surface binding without the limitations associated with plasma-derived prod
{"title":"B-105 A New Recombinant Human Albumin Produced in Thermothelomyces heterothallica (C1) Demonstrates Comparable Surface Binding to Native Serum Albumin","authors":"Audrianna Kern, Adam Okerlund, Charlotte Kunkler, Christopher Warner","doi":"10.1093/clinchem/hvaf086.503","DOIUrl":"https://doi.org/10.1093/clinchem/hvaf086.503","url":null,"abstract":"Background Human serum albumin (HSA) and bovine serum albumin (BSA) are commonly used as a blocking agents in immunoassays to prevent non-specific surface binding, thereby reducing background signal and increasing assay sensitivity. However, these native albumins present challenges such as donor dependency, lot-to-lot variability, and contamination with plasma-derived molecules (e.g., vitamins, hormones, growth factors, antibodies/Ig), which can affect assay performance and reproducibility. Recombinant human albumin (rHA) offers a potential alternative by providing the same binding and blocking characteristics as native albumin without the limitations. A new rHA produced in Thermothelomyces heterothallica (C1) and purified using a scalable proprietary process may mitigate these challenges. This study aims to evaluate the amount of binding on different surfaces of this new rHA compared to native albumins to determine the viability of rHA as a surface blocker in immunoassays. Methods The amount of albumin binding to different surfaces was assessed using a bead binding assay. Here, albumin (rHA, HSA, BSA) was incubated with commercially available Dynabeads: hydrophilic (carboxylic acid or amine functionalized) and hydrophobic (tosylactivated). The amount of protein bound to the beads was determined by heating the coated beads in a denaturing buffer and quantifying via densitometry on an SDS-PAGE gel against a standard curve. Statistical analysis was performed using GraphPad Prism, with data presented as the mean of at least three independent replicates with standard deviations. A two-tailed t-test was used to determine statistical significance at the 95% confidence interval. Results All tested albumin products demonstrate 10-fold more protein binding to the hydrophobic beads than either of the hydrophilic beads, highlighting the chemical similarities of albumin from different sources. For both hydrophilic bead types, all tested albumins demonstrated statistically the same amount of binding, ranging from 0.10-0.23 µg and 0.19-0.23 µg for the carboxylic acid and amine beads, respectively, with rHA at 0.15 ± 0.06 µg and 0.23 ± 0.07 µg. The hydrophobic beads had a much larger range of albumin binding, from 1.4-3.7 µg, with rHA being at the lower end of the range at 1.4 ± 0.2 µg, but statistically the same as BSA and two HSA products. The large differences in binding of the native albumins showcase the variability of natively derived products from different producers. Even with these slight differences, these data demonstrate rHA has comparable surface binding chemistry to native albumins. Conclusion This new rHA produced by C1 and purified in a scalable process binds surfaces comparably to commercially available native albumin products on all surfaces tested. These results support the use of rHA as an alternative to native albumins in diagnostic applications, providing effective surface binding without the limitations associated with plasma-derived prod","PeriodicalId":10690,"journal":{"name":"Clinical chemistry","volume":"101 1","pages":""},"PeriodicalIF":9.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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