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Background Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathy. However, conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of structural characterization. We describe the use of neutral-coating capillary electrophoresis coupled with high-resolution mass spectrometry (CE-HR-MS) to achieve high-performance top-down identification of Hb variants. Methods An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 capillary electrophoresis (CE) unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Deconvolution of raw mass spectrometry data was carried out to merge multiple charge states and isotopic peaks of an analyte to obtain its monoisotopic mass. Results The neutral-coating CE could baseline separate individual Hb subunits dissociated from intact Hb forms, and the HR-MS could achieve both intact-protein analysis and top-down analysis of analytes. A number of patient samples that contain Hb subunit variants were analyzed, and the variants were successfully identified using the CE-HR-MS method. Conclusions The CE-HR-MS method has been demonstrated as a useful tool for top-down identification of Hb variants. With the ability to characterize the primary structures of Hb subunits, the CE-HR-MS method has significant advantages to complement or partially replace the conventional methods for the identification of Hb variants.

Background An immunodiagnostic assay that sensitively detects a cell-mediated immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed for epidemiological investigation and for clinical assessment of T- cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses. Methods The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in coronavirus disease 2019 (COVID-19) convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen. Results The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI]: 79.0–89.0) and 86.6% (123/142; 95% CI: 80.0–91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI: 80.6–100) at 0.5-month post-infection to 79.5% (31/39; 95% CI: 64.4–89.2) at 10 months post-infection (P Conclusions The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

BackgroundCerebrospinal fluid (CSF) leak is typically diagnosed by detecting a protein marker β2-transferrin (β2-Tf) in secretion samples. β2-Tf and β1-transferrin (β1-Tf) are glycoforms of human transferrin (Tf). A novel affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to analyze the Tf glycoforms and elucidate the structures of β1-Tf and β2-Tf.MethodsTo implement MPIE, an analyte is first captured on the surface of a microprobe, and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via next-generation biolayer interferometry (BLI). When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for HR-MS analysis. Serum, CSF, and secretion samples were analyzed using MPIE-ESI-MS.ResultsBased on the MPIE-ESI-MS results, the structures of β1-Tf and β2-Tf were solved. As Tf glycoforms, β1-Tf and β2-Tf share the amino acid sequence but have varying N-glycans. β1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611. β2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611.ConclusionsThe structures of β1-Tf and β2-Tf were successfully elucidated by MPIE-ESI-MS analysis. The resolving power of the novel MPIE-ESI-MS method was demonstrated in this study. On the other hand, knowing the N-glycan structures on β2-Tf allows for the design of other novel test methods for β2-Tf in the future.

Objective Severe hypertriglyceridemia (SHTG; plasma triglycerides >1000 mg/dL) is a rare but serious complication in children who develop diabetic ketoacidosis (DKA) from uncontrolled or new-onset type 1 diabetes. Methods We present the case of a severely malnourished 16-year-old with a 10-month history of presumed type 2 diabetes managed with lifestyle modifications and metformin, who presented with SHTG, acute pancreatitis, and DKA. On examination, there was no evidence of lipemia retinalis, cutaneous xanthomas, or xanthelasma. He was initially treated with an insulin infusion and intravenous fluids. Despite this treatment, his pancreatitis symptoms worseneed and lipase level increased, necessitating 2 courses of plasmapheresis that immediately resolved his symptoms and dramatically improved his clinical status. He was discharged on hospital day 5. During his hospital admission, islet cell antigen 512, insulin, glutamic acid decarboxylase 65, and zinc transporter 8 autoantibodies were positive in the presence of insulinopenia, consistent with type 1 diabetes. Results Hypertriglyceridemia and hypercholesterolemia did not recur during follow-up, suggesting that the underlying mechanism for SHTG was insulin deficiency. Conclusion This report of SHTG, DKA, and pancreatitis in an adolescent highlights the safe, early initiation of plasmapheresis as an effective treatment. To our knowledge, plasmapheresis has rarely been used so early in the course of treatment for an adolescent with SHTG, DKA, and acute pancreatitis.

Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April–June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (

Highlights • Anti-SARS-CoV-2 IgG specificity is critical for Covid-19 spread and prevalence studies. • A large collection of pre-pandemic samples is necessary for the assessment of assay specificity. • Specificity of Abbott Architect anti-SARS-CoV-2 IgG assay is high. • Sensitivity of both anti-SARS-CoV-2 IgG assays evaluated are high., Background We report our findings of test performance especially specificity of a fully automated Abbott Architect anti-SARS-CoV-2 CMIA IgG and Euroimmun anti-SARS-CoV-2 ELISA IgA/IgG in human plasma. Methods We used positive cohort of 97 samples from Covid-19 patients or healthcare workers, collected at late time points from symptom onsets. We also included another cohort of 215 samples as negative controls, 78 of which had positive serology test results of other infectious diseases or autoimmunity. Assay specificity was assessed by using a total of 847 anonymized samples which were collected before the Covid-19 pandemic from local patient populations seeking clinical care for rheumatoid diseases, thyroid cancer, and therapeutic drug monitoring. Results Abbott IgG, Euroimmun IgG/IgA had high precision, demonstrated by both intra- and inter-day CVs of

BackgroundAn immunodiagnostic assay that sensitively detects a cell-mediated immune response to SARS-CoV-2 is needed for epidemiological investigation and for clinical assessment of T cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses.MethodsThe performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific CD4 and CD8 T cells was evaluated in COVID-19 convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen.ResultsThe overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI] 79.0-89.0) and 86.6% (123/142; 95% CI;80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI 64.4-89.2) at 10 months post-infection (PConclusionsThe SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.Key pointsSARS-CoV-2 immunodiagnostics are needed to identify infected individuals in order to understand the transmission dynamics of emerging variants and to assess vaccine response. Interferon-gamma release assay maintains sensitivity 10 months post-infection in convalescents and detects more household contacts than IgG.

Serum cortisol measurements after ACTH stimulation are currently used to evaluate for adrenal insufficiency in children. We aim to determine if salivary cortisol measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) can confirm or replace serum cortisol during high dose ACTH stimulation test to improve test compliance and interpretation. We also aim to gain preliminary understanding of normal ranges of salivary cortisol in normal children at am, bedtime, and midnight. Children aged 6–17 years meeting study criteria and tested for adrenal insufficiency were recruited to concomitantly collect saliva and serum samples during high dose ACTH stimulation test. Normal children aged 3–18 years were recruited to collect morning, bedtime, and midnight saliva samples. Salivary cortisol was measured using LC–MS/MS while serum cortisol was determined by an immunoassay. Salivary cortisol in normal children were higher at am and lower at bedtime and midnight (p value

Background: The quantification of hepatitis B (HBV) DNA in serum is critical to identify patients requiring antiviral therapy and to monitor the response to treatment. Method: This study describes the evaluation of the Aptima HBV Quant Dx assay (Aptima HBV) performed on the automated Panther system. Results: Aptima HBV was linear from 1.70 to 7.70 log10 IU/mL with a commercial reference panel, as well as clinical specimens representing genotypes B and C, and total imprecision, as measured by the percentage coefficient of variation (%CV) at 2.0 log10 IU/mL was Conclusions: The Aptima HBV test affords a suitable alternative to CAP/CTM for serum virus load testing and provides a key component of the diagnostic algorithm for the global eradication of viral hepatitis.

Background Neonatal severe hyperparathyroidism (NSHPT) is commonly treated with either parathyroidectomy or pharmacologic agents with varying efficacy and numerous side effects. Reports of using cinacalcet for NSHPT have increased, however, the effective dose for pediatric patients from the onset of symptoms through infancy has not been established. Case presentation We describe the clinical course of a newborn with a de novo R185Q mutation in the calcium-sensing receptor (CASR) gene, causing NSHPT. The infant received cinacalcet from the first days of life until 1 year of age. Conclusions Cinacalcet therapy effectively controlled the patient’s serum calcium, phosphorus, and parathyroid hormone (PTH) levels without side effects.

Background Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results. Methods Over a 2-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis. Results There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] vs 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] vs 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6. Conclusion Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.

To evaluate the significance of antithyroglobulin and antithyroid peroxidase antibody levels associated with locoregional metastatic disease in patients with well-differentiated thyroid cancer.Patients underwent initial treatment for well-differentiated thyroid cancer at our institution between 2014 and 2018. The following variables were collected: age, sex, pre-operative thyroid-stimulating hormone, thyroglobulin, antithyroglobulin antibody (TgAb), antithyroid peroxidase antibody (TPOAb), the extent of surgery, T-stage, N-stage, extrathyroidal extension (ETE), extranodal extension (ENE), lymphovascular invasion, and multifocal disease. The relationships between disease status and pre-operative TPOAb, TgAb, thyroglobulin, and thyroid-stimulating hormone were analyzed.A total of 405 patients (mean age, 52 years) were included in the study, of which 66.4% were women. Elevated TgAb was associated with the presence of lymph node metastases (LNM) in both the central and lateral neck (P.01), with a stronger correlation to N1b versus N1a disease (P = .03). The presence of ETE was inversely related to the TgAb titer (P = .03). TPOAb was associated with a lower T-stage (P = .04), fewer LNM (P = .04), and a lower likelihood of ETE (P = .02). From multivariable analysis, TgAb ≥40 IU/mL was an independent predictive factor for a higher N-stage (P.01 for N0 vs N1; P = .01 for N1a vs N1b), and ENE (P.01). TPOAb ≥60 IU/mL was associated with a lower T-stage (P = .04 for T3) and absence of ETE (P = .01).Elevated pre-operative TgAb was an independent predictor of nodal metastases and ENE, while elevated TPOAb was associated with a lower pathologic T- and N-stage. Pre-operative antithyroid antibody titers may be useful to inform the disease extent and features.

We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.

Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC–MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4–1500 pmol/L for E1 and 4–2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC–MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC–MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.

Background Our institution utilizes an antigen/antibody screening test followed by a confirmatory antibody assay for preliminary positive results. Given the low prevalence for HIV infections in our institution's county, we suspect that a substantial portion of the reactive screens are false positives. Objectives We aimed to characterize the false positivity rate of the HIV screening test performed at Stanford Health Care. In parallel, we modified our reporting workflow to release both the screening and confirmatory results simultaneously to mitigate the stress of a presumptive positive test. Study Design We reviewed 45,296 eligible HIV screen specimens that underwent the Abbott ARCHITECT™ 4th generation HIV antigen/antibody combination assay between August 5, 2016 and March 16, 2021. Final sample signal/cutoff (S/CO) ratios ≥ 1 were deemed positive, which triggers a reflex order for the confirmatory Bio-Rad Geenius™ HIV 1/2 Supplemental Assay. Additional chart review was performed for positive screen cases with negative or indeterminate confirmatory results. Results Our institution demonstrated a 0.28% (128/45,296) positive screen rate, with 12.5% (16/128) of these samples confirmed as false positives based on a negative HIV-1 RNA test. Median S/CO ratios of true positive screens were significantly higher than those with negative or indeterminate confirmatory tests (602.27vs 2.98; p = 0.0000323). We implemented a new synchronized reporting system for positive screens, which co-releases screen and confirmatory reports without compromise in the overall turnaround time. Conclusions Our study demonstrates a relatively high percentage of false positive screens. Subsequently, by providing a more complete picture up front, our new reporting pipeline may reduce anxiety of a stand-alone positive screen and optimize downstream clinical decision-making.

Hepatitis B virus (HBV) infection has a global reach with high prevalence in resource-limited areas like China and Africa. HBV patients in these areas have limited access to the currently used, costly HBV assays, which are performed in centralized clinical laboratories using single-plexed assays with bulky and expensive instruments. We aim to overcome these limitations by developing a simple and affordable HBV diagnostic platform to allow for timelier diagnosis and intervention of HBV infection. Using giant magnetoresistive (GMR) biosensor chips, we developed an automated and multiplexed quantitative platform for the measurement of a panel of HBV serology markers, including HBV “e” antigen (HBeAg), HBV surface antigen (HBsAg), and the antibody against HBsAg (anti-HBs). Our assay platform was able to detect each HBV marker with high specificity and sensitivity (with three orders of magnitude in dynamic range for each marker). Blinded analysis of HBV patient sera showed excellent correlation between our multiplexed quantitative HBsAg results and the qualitative results obtained using FDA-approved immunoassays, as well as those obtained using quantitative, single-plexed, enzyme-linked immunosorbent assays (ELISAs). The portable, automated, multiplexed, quantitative HBV serology assay platform we designed shows great promise as a more accessible alternative for HBV screening, diagnosis, and treatment monitoring.

Hepatitis C virus (HCV) RNA quantitation is the primary method by which active HCV infections are identified and the response to direct-acting antiviral therapy is monitored. This study describes the evaluation of the Aptima HCV Quant Dx assay (Aptima HCV) performed on the Panther system. The clinical performance of Aptima HCV was compared to that of the Cobas AmpliPrep/Cobas TaqMan HCV test v2.0 (CAP/CTM). Overall agreement was 84.9% (186/219) with a kappa statistic of 0.755 (standard error, 0.037; 95% confidence interval [CI], 0.682 to 0.828). Passing-Bablok regression of log10 IU/ml values revealed a regression line of Y = 1.163 × X − 0.991 (95% CI of the slope, 1.103 to 1.221, and intercept, −1.341 to −0.642). The 95% lower limit of detection (LLOD) for Aptima HCV on dried blood spot (DBS) samples was calculated to be 2.43 log10 IU/ml (267 IU/ml; 95% CI, 2.31 to 2.73 log10 IU/ml [204 to 540 IU/ml]). A comparison of Aptima HCV testing on paired DBS and serum specimens collected from patients at the time of routine blood collection for CAP/CTM demonstrated an overall agreement of 90.1% (82/91) with a kappa statistic of 0.657 (standard error, 0.101; 95% CI, 0.458 to 0.855). In conclusion, Aptima HCV provides a suitable alternative for HCV RNA testing on serum and DBS samples.

Background 24 h urinary free cortisol measurement is a clinically important first-line screening test for Cushing’s syndrome (CS). Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve and validate a LC-MS/MS method to measure urinary free cortisol in both adult and pediatric patients and to characterize its clinical diagnostic performance of CS by chart review. Methods We improved a LC-MS/MS method previously reported for urinary free cortisol to be able to measure urinary and salivary cortisol in the same batch for increased efficiency. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay’s analytical performance was characterized, and a retrospective patient chart review was conducted to evaluate the assay’s clinical performance in diagnosing CS. Results The LC-MS/MS assay demonstrated enhanced sensitivity and was linear within an analytical measurement range of 10–10,000 ng/dL. Assay accuracy was satisfactory as determined by spike and recovery studies and highly correlated with a reference LC-MS/MS method. The assay’s clinical diagnostic sensitivity and specificity in detecting CS was 96% and 91%, respectively, when compared to a urinary cortisol excretion of at least 50 µg/24 h. Conclusions The improved LC-MS/MS method is both sensitive and specific with enhanced analytical performance and clinical diagnostic utility to screen for CS. The clinical diagnostic sensitivity and specificity were superior based on retrospective patient chart review.

Background: Implementing a laboratory-developed test sometimes requires incorporating an unconventional device into the laboratory information system (LIS) and customizing an interface to reduce transcription error and improve turnaround time. Such a custom interface is a necessity for complicated high-volume tests such as 25-OH Vitamin D by liquid chromatography-tandem mass spectrometry (LC-MS/MS) when there is no vendor-or LIS-supplied interface available. Here, we describe our work and experience interfacing a API 5000 LC-MS/MS instrument with our newly implemented LIS, Epic Beaker, using a combination of in-house scripting software and a middleware vendor, Data Innovations. Materials and Methods: For input interfacing, custom scripting software was developed to transcribe batched order lists generated by Epic into files usable by the instrument software, Analyst®. For output interfacing, results from the LC-MS/MS system were fed to a unidirectional instrument driver made by Data Innovations and selected data were transferred to the LIS. Results: Creation and validation of a new driver by Data Innovations took approximately 6 months. The interface was adopted for 25-OH Vitamin D and testosterone testing during periods of increasing test volume (4.5-fold over 8 years and 1.25-fold over 5 years). The amount of time spent reporting 25-OH Vitamin D results decreased 82% per order resulting in a savings of 1370 technician work hours and the amount of time spent reporting testosterone results decreased 75% per order resulting in a savings of 400 technician work hours. Conclusions: A mixed model using custom scripting and curated commercial middleware serve as a durable interface solution for laboratory instrumentation such as an LC-MS/MS and are flexible to future changes in instrument software, networking protocols, and the scope of LISs and work area managers.

To the Editor: Paper spray (PS)1 is a recently described method for the direct mass spectrometry (MS) analysis of blood and other biological samples (1–3). Paper spray is performed by depositing a sample such as whole blood onto a paper substrate contained within a disposable cartridge; extraction and ionization occur directly from the disposable cartridge with an automated MS attachment (Fig. 1). No punching or offline extraction is required. Total analysis time, including sample extraction, is several minutes. Fig. 1. Paper spray for direct analysis of blood spots by MS. Ion chronograms from the lowest calibrator in whole blood, overlaid with representative chronograms from blank blood, are shown for cyclosporine and sirolimus. Quantification was performed with the entire area under the curve for each analyte, normalized by SIL-IS, to obtain calibration curves as shown. Because of its simplicity, paper spray could lower the barrier for implementation of MS-based assays in clinical laboratories. We previously used PS-MS/MS to monitor tacrolimus in clinical samples and showed good correlation with 2 immunoassays and an external HPLC-MS/MS method (4). Here we extend this approach by demonstrating the simultaneous quantification of cyclosporine and sirolimus by PS-MS/MS. Therapeutic drug monitoring of these drugs is important for organ transplant patient care. The method used was similar to our …