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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.

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.

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.

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.

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: 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 …

Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts, and most of the novel SAGE tags have low copy numbers. Further analysis indicated that these novel SAGE tags represent novel low-abundant transcripts expressed from loci outside of currently annotated exons including the intergenic and intronic regions, and antisense of the currently annotated exons in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies.

Background: Characterization of fusion gene transcripts in leukemia that result from chromosome translocations provides valuable information regarding appropriate treatment and prognosis. However, screening for multiple fusion gene transcripts is difficult with conventional PCR and state-of-the-art real-time PCR and high-density microarrays. Methods: We developed a multiplex reverse transcription-PCR (RT-PCR) assay for screening and quantification of fusion gene transcripts in human leukemia cells. Chimeric primers were used that contained gene-specific and universal sequences. PCR amplification of fusion and control gene transcripts was achieved with use of an excess of universal primers to allow the ratio of abundance of fusion gene to endogenous or exogenous controls to be maintained throughout PCR. Multiplex RT-PCR products analyzed by an ABI 310 Genetic Analyzer were consistent with those of duplex RT-PCR (single analytical sample plus control). In addition, multiplex RT-PCR results were analyzed by an assay using an oligonucleotide microarray that contained probes for the splice-junction sequences of various fusion transcripts. Results: The multiplex RT-PCR assay enabled screening of >10 different fusion gene transcripts in a single reaction. RT-PCR followed by analysis with the ABI Prism 310 Genetic Analyzer consistently detected 1 fusion-transcript-carrying leukemia cell in 100–10 000 cells. The assay covered a 1000-fold range. Preliminary results indicate that multiplex RT-PCR products can also be analyzed by hybridization-based microarray assay. Conclusions: The multiplex RT-PCR analyzed by either ABI Prism 310 Genetic Analyzer or microarray provides a sensitive and specific assay for screening of multiple fusion transcripts in leukemia, with the latter an assay that is adaptable to a high-throughput system for clinical screening.

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides sensitivity and specificity for monitoring tacrolimus drug level in blood, but it requires an LC system and sample preparation, which is not amenable to random access testing typical of immunoassays. Paper spray (PS) ionization generates gas phase analyte ions directly from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS method for tacrolimus drug monitoring in a clinical diagnostic laboratory. Methods Whole blood sample was mixed with stable isotope labeled internal standard ([13C, 2H2]-FK506) and spotted onto a cartridge containing triangular shaped card paper. After drying, samples were analyzed by PS MS/MS in the selected reaction monitoring (SRM) mode, with a run time of 3 min/sample. Results Analytical measurement range was 1.5–30 ng/ml. Assay inter-day imprecision was 13%, 8%, and 5% at tacrolimus concentrations of 4.5, 10.5, and 24.5 ng/ml, respectively. Accuracy was determined by pure tacrolimus solution and was confirmed by result correlation to an immunoassay (slope = 1.0, intercept = − 0.02; r2 = 0.99), and to a conventional LC-MS/MS method (slope = 0.90, intercept = 0.4; r2 = 0.94). Conclusions PS-MS/MS provides accurate results for tacrolimus with rapid turnaround time amenable to random access testing protocols.

A monoclonal antibody to homovanillic acid (HVA) was prepared by synthesis of a HVA-protein conjugate (HVA-ovalbumin) as an immunogen, immunization of mice, and the subsequent hybridization technique. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and accuracy. An indirect ELISA was developed for quantification of HVA in human urine. The assay was characterized and shown to have high specificity, with cross-reactivities to vanillylmandelic acid and normetanephrine at 0.18% and

Background Isolation of low-abundance transcripts expressed in a genome remains a serious challenge in transcriptome studies. The sensitivity of the methods used for analysis has a direct impact on the efficiency of the detection. We compared the EST method and the SAGE method to determine which one is more sensitive and to what extent the sensitivity is great for the detection of low-abundance transcripts. Results Using the same low-abundance transcripts detected by both methods as the targeted sequences, we observed that the SAGE method is 26 times more sensitive than the EST method for the detection of low-abundance transcripts. Conclusions The SAGE method is more efficient than the EST method in detecting the low-abundance transcripts.