T Cell Responses Correlate with Self-Reported Disease Severity and Neutralizing Antibody Responses Predict Protection against SARS-CoV-2 Breakthrough Infection.

OBJECTIVES: The objective of this prospective study was to investigate the role of adaptive immunity in response to SARS-CoV-2 vaccines. DESIGN AND METHODS: A cohort of 677 vaccinated individuals participated in a comprehensive survey of their vaccination status and associated side effects, and donated blood to evaluate their adaptive immune responses by neutralizing antibody (NAb) and T cell responses. The cohort then completed a follow-up survey to investigate the occurrence of breakthrough infections. RESULTS: NAb levels were the highest in participants vaccinated with Moderna, followed by Pfizer and Johnson & Johnson. NAb levels decreased with time after vaccination with Pfizer and Johnson & Johnson. T cell responses showed no significant difference among the different vaccines and remained stable up to 10 months after the study period for all vaccine types. In multivariate analyses, NAb responses (<95 U/mL) predicted breakthrough infection, whereas previous infection, the type of vaccine, and T cell responses did not. T cell responses to viral epitopes (<0.120 IU/mL) showed a significant association with the self-reported severity of COVID-19 disease. CONCLUSION: This study provides evidence that NAb responses to SARS-CoV-2 vaccination correlate with protection against infection, whereas the T cell memory responses may contribute to protection against severe disease but not against infection.

Microfluidic systems in clinical diagnosis.

The use of microfluidic devices is highly attractive in the field of biomedical and clinical assessments, as their portability and fast response time have become crucial in providing opportune therapeutic treatments to patients. The applications of microfluidics in clinical diagnosis and point-of-care devices are continuously growing. The present review article discusses three main fields where miniaturized devices are successfully employed in clinical applications. The quantification of ions, sugars, and small metabolites is examined considering the analysis of bodily fluids samples and the quantification of this type of analytes employing real-time wearable devices. The discussion covers the level of maturity that the devices have reached as well as cost-effectiveness. The analysis of proteins with clinical relevance is presented and organized by the function of the proteins. The last section covers devices that can perform single-cell metabolomic and proteomic assessments. Each section discusses several strategically selected recent reports on microfluidic devices successfully employed for clinical assessments, to provide the reader with a wide overview of the plethora of novel systems and microdevices developed in the last 5 years. In each section, the novel aspects and main contributions of each reviewed report are highlighted. Finally, the conclusions and future outlook section present a summary and speculate on the future direction of the field of miniaturized devices for clinical applications.

New Diagnostic Cutoffs for Adrenal Insufficiency After Cosyntropin Stimulation Using Abbott Architect Cortisol Immunoassay.

INTRODUCTION: The accurate interpretation of the cosyntropin (adrenocorticotropic hormone [ACTH]) stimulation test requires method- and assay-specific cutoffs of the level of cortisol. Compared with a historical cutoff (18 µg/dL) for polyclonal antibody-based immunoassays, lower thresholds were proposed for the Roche Elecsys II assay, which uses a monoclonal antibody. However, cutoffs for other commonly adopted, monoclonal antibody-based cortisol assays were not yet available. Here, we established the thresholds for the level of cortisol specific to the Abbott Architect immunoassay by comparing the measurements of the level of cortisol using 3 immunoassays. METHODS: The ACTH stimulation test was performed in patients with suspected adrenal insufficiency (n = 50). The serum cortisol level was measured using the Abbott Architect, Roche Elecsys II, and Siemens Centaur assays. The results of the Abbott assay were also compared with those of liquid chromatography-tandem mass spectrometry. The receiver operating characteristic analysis was performed to derive new diagnostic thresholds for the Abbott assay using the polyclonal antibody-based Siemens assay as the reference method. RESULTS: The concentrations of cortisol measured using the Abbott assay were similar to those measured using liquid chromatography-tandem mass spectrometry and the Roche Elecsys II assay but significantly lower than those measured using the Siemens assay. The optimized threshold for cortisol using the Abbott assay was 14.6 µg/dL at 60 minutes after stimulation (sensitivity, 92%; specificity, 96%) and 13.2 µg/dL at 30 minutes after stimulation (sensitivity, 100%; specificity, 89%). CONCLUSION: We recommend a threshold of 14.6 µg/dL for the level of cortisol at 60 minutes after ACTH stimulation for the Abbott assay. In comparison with the historical threshold of 18 µg/dL, the application of the new cutoff may significantly decrease false-positive results due to ACTH stimulation testing. The use of assay-specific cutoffs will be essential for reducing misclassification and overtreatment in patients with suspected adrenal insufficiency.

Environmental Lead Exposure and Influenza and Respiratory Syncytial Virus Diagnoses in Young Children: A Test-Negative Case-Control Study.

Experimental and epidemiological evidence suggests that environmental toxicants may influence susceptibility to influenza and respiratory syncytial virus (RSV). The objective of the present study was to estimate the association between blood lead concentrations and the odds of child influenza or RSV infection. A test-negative, case-control study was conducted among 617 children, <4 years of age, tested for influenza/RSV from 2012-2017 in Rochester, NY. There were 49 influenza cases (568 controls) and 123 RSV cases (494 controls). Blood lead concentrations reported in children's medical records were linked with influenza/RSV lab test results. Covariables were collected from medical records, birth certificates, and U.S. census data. In this sample, evidence of an association between blood lead levels and RSV or influenza diagnosis was not observed. Children with a lead level ≥1 µg/dL vs. <1 µg/dL had an adjusted odds ratio (aOR) and 95% confidence limit of 0.95 (0.60, 1.49) for RSV and 1.34 (0.65, 2.75) for influenza. In sex-specific analyses, boys with lead concentrations ≥1 µg/dL vs. <1 µg/dL had an aOR = 1.89 (1.25, 2.86) for influenza diagnosis, while the estimates were inconsistent for girls. These results are suggestive of sex-specific associations between blood lead levels and the risk of influenza, although the sample size was small.

Development of an accurate and sensitive method for lactate analysis in exhaled breath condensate by LC MS/MS.

Exhaled breath condensate (EBC) is easily obtained for clinical diagnosis and prognosis for pulmonary diseases and has gained much interest in biomarker discovery research and studies. Lactate, a physiological material, is found in EBC and has been demonstrated to be a potential indicator of chronic obstructive pulmonary disease and other lung diseases. Several assays are available to detect lactate in human body fluids, and yet none is suitable for detecting lactate in EBC. Due to the very low concentration of lactate in EBC and low volume of EBC, it is very important to develop an assay to measure lactate with high sensitivity, accuracy and easy sample processing. We report here a novel LC-MS/MS based assay to measure lactate using HILIC column separation. Sample preparation was simple and straightforward through a "dilute and shoot" approach with a separation of 4min. The limit of quantification was determined to be 0.5µM. This assay was linear from 0.5µM to at least 100µM. The inter- and intra- day precision at the levels of 1µM, 10µM, and 100µM were less than 3% with recovery within 5.4% of expected values. There was no ion suppression for the assay, and no carry-over was observed up to 500µM. Furthermore, we discovered that lactate is ubiquitously present in the lab environment, which can create significant challenges for accurate detection of lactate at low concentrations. We provided practical approaches in this paper to overcome the challenges and ensure the accuracy of the assay. In summary, this article presents an accurate and sensitive method using LC-MS/MS for measuring lactate in EBC, and this method is suitable for measuring lactate concentrations for non-invasive monitoring of pulmonary functions.

Liquid Chromatography-Tandem Mass Spectrometry: An Emerging Technology in the Toxicology Laboratory.

In the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in routine toxicology laboratories. LC-MS/MS offers significant advantages over other traditional testing, such as immunoassay and gas chromatography-mass spectrometry methodologies. Major strengths of LC-MS/MS include improvement in specificity, flexibility, and sample throughput when compared with other technologies. Here, the basic principles of LC-MS/MS technology are reviewed, followed by advantages and disadvantages of this technology compared with other traditional techniques. In addition, toxicology applications of LC-MS/MS for simultaneous detection of large panels of analytes are presented.

Mass Spectrometry in Clinical Laboratory: Applications in Biomolecular Analysis.

Mass spectrometry (MS) is a technique that can identify analytes on the basis of mass-to-charge (m/z) ratio. Although this technique has been used in research and specialized clinical laboratories for decades, however, in recent years, MS has been increasingly used in routine clinical laboratories. MS, especially when coupled to gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of MS include simultaneous analysis of multiple analytes (>100) and generally without need for specialized reagents. Commonly measured analytes by MS include drugs, hormones, and proteins.

Quantitative Analysis of Salivary Cortisol Using LC-MS/MS.

Cortisol is one of the most important glucocorticoids and plays important roles in regulating human metabolism. Midnight salivary cortisol has been shown to correlate well with free cortisol concentration in serum and is one of the first tests recommended for the diagnosis of Cushing's syndrome.The procedure described here involves centrifugation of the saliva samples to remove solids and mucus strands before they are diluted with buffer and mixed with deuterated internal standard D4-cortisol. The samples are then subjected to reverse phase separation on a C18 column and analyzed by a tandem mass spectrometry method (LC-MS/MS). Quantification is achieved by comparing the responses of a given sample to the responses of the calibrators of known concentrations. The calibrators are prepared and analyzed along with the patient samples. Analytical specificity is ensured by using multiple reaction monitoring with fragment ions that are unique to cortisol and deuterated internal standard.

Mass Spectrometry in Clinical Laboratory: Applications in Therapeutic Drug Monitoring and Toxicology.

Mass spectrometry (MS) has been used in research and specialized clinical laboratories for decades as a very powerful technology to identify and quantify compounds. In recent years, application of MS in routine clinical laboratories has increased significantly. This is mainly due to the ability of MS to provide very specific identification, high sensitivity, and simultaneous analysis of multiple analytes (>100). The coupling of tandem mass spectrometry with gas chromatography (GC) or liquid chromatography (LC) has enabled the rapid expansion of this technology. While applications of MS are used in many clinical areas, therapeutic drug monitoring, drugs of abuse, and clinical toxicology are still the primary focuses of the field. It is not uncommon to see mass spectrometry being used in routine clinical practices for those applications.

Impact of automation on mass spectrometry.

Mass spectrometry coupled to liquid chromatography (LC-MS and LC-MS/MS) is an analytical technique that has rapidly grown in popularity in clinical practice. In contrast to traditional technology, mass spectrometry is superior in many respects including resolution, specificity, multiplex capability and has the ability to measure analytes in various matrices. Despite these advantages, LC-MS/MS remains high cost, labor intensive and has limited throughput. This specialized technology requires highly trained personnel and therefore has largely been limited to large institutions, academic organizations and reference laboratories. Advances in automation will be paramount to break through this bottleneck and increase its appeal for routine use. This article reviews these challenges, shares perspectives on essential features for LC-MS/MS total automation and proposes a step-wise and incremental approach to achieve total automation through reducing human intervention, increasing throughput and eventually integrating the LC-MS/MS system into the automated clinical laboratory operations.

Prevalence of 25-hydroxyvitamin D2 in Western New York: a 3-year study.

BACKGROUND: We evaluated the distribution of 25OH-D2 and 25OH-D3 in a general patient population in Western New York to provide insights into how common detectable vitamin D2 is among samples from a general patient population. METHODS: Serum 25OH-D2 and 25OH-D3 results measured by LC-MS/MS from June 2009 to December 2012 were retrospectively analyzed. RESULTS: A total of 266,269 serum tests were included for analysis. The percentage of tests with 25OH-D2 levels above the assay limit of quantitation (LoQ) decreased from 32% to 17% over the course of the study period. The percentage of tests with 25OH-D2 levels higher than those of 25OH-D3 decreased from 21% to 12%. Sixty-seven percent of the test results with 25OH-D2 levels above the LoQ had serum concentrations of 25OH-D2 higher than those of 25OH-D3. CONCLUSION: Prevalence of tests with quantifiable 25OH-D2 decreased over time and yet 17% of them still had detectable levels of 25OH-D2, 67% of which had 25OH-D2 levels higher than 25OH-D3. To achieve accurate 25-hydroxyvitamin D measurement, clinical laboratories should assess the accuracy of their assays, and if necessary, determine the local prevalence of 25OH-D2 to determine if mass spectrometry is the platform of choice to assess vitamin D deficiency.