Performance of Three Anti-SARS-CoV-2 Anti-S and One Anti-N Immunoassays for the Monitoring of Immune Status and Vaccine Response.

This study aimed to evaluate and compare the performance of three anti-S and one anti-N assays that were available to the project in detecting antibody levels after three commonly used SARS-CoV-2 vaccines (Pfizer, Moderna, and Johnson & Johnson). It also aimed to assess the association of age, sex, race, ethnicity, vaccine timing, and vaccine side effects on antibody levels in a cohort of 827 individuals. In September 2021, 698 vaccinated individuals donated blood samples as part of the Association for Diagnostics & Laboratory Medicine (ADLM) COVID-19 Immunity Study. These individuals also participated in a comprehensive survey covering demographic information, vaccination status, and associated side effects. Additionally, 305 age- and gender-matched samples were obtained from the ADLM 2015 sample bank as pre-COVID-19-negative samples. All these samples underwent antibody level analysis using three anti-S assays, namely Beckman Access SARS-CoV-2 IgG (Beckman assay), Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG (Ortho assay), Siemens ADVIA Centaur SARS-CoV-2 IgG (Siemens assay), and one anti-N antibody assay: Bio-Rad Platelia SARS-CoV-2 Total Ab assay (BioRad assay). A total of 827 samples (580 COVID-19 samples and 247 pre-COVID-19 samples) received results for all four assays and underwent further analysis. Beckman, Ortho, and Siemens anti-S assays showed an overall sensitivity of 99.5%, 97.6%, and 96.9%, and specificity of 90%, 100%, and 99.6%, respectively. All three assays indicated 100% sensitivity for individuals who received the Moderna vaccine and boosters, and over 99% sensitivity for the Pfizer vaccine. Sensitivities varied from 70.4% (Siemens), 81.5% (Ortho), and 96.3% (Beckman) for individuals who received the Johnson & Johnson vaccine. BioRad anti-N assays demonstrated 46.2% sensitivity and 99.25% specificity based on results from individuals with self-reported infection. The highest median anti-S antibody levels were measured in individuals who received the Moderna vaccine, followed by Pfizer and then Johnson & Johnson vaccines. Higher anti-S antibody levels were significantly associated with younger age and closer proximity to the last vaccine dose but were not associated with gender, race, or ethnicity. Participants with higher anti-S levels experienced significantly more side effects as well as more severe side effects (e.g., muscle pain, chills, fever, and moderate limitations) (p < 0.05). Anti-N antibody levels only indicated a significant correlation with headache. This study indicated performance variations among different anti-S assays, both among themselves and when analyzing individuals with different SARS-CoV-2 vaccines. Caution should be exercised when conducting large-scale studies to ensure that the same platform and/or assays are used for the most effective interpretation of the data.

Large-Scale Scientific Study Led by a Professional Organization during the COVID-19 Pandemic: Operations, Best Practices, and Lessons Learned.

In 2021, the Association for Diagnostics & Laboratory Medicine (ADLM) (formerly the American Association for Clinical Chemistry [AACC]) developed a scientific study that aimed to contribute to the understanding of SARS-CoV-2 immunity during the evolving course of the pandemic. This study was led by a group of expert member volunteers and resulted in survey data from 975 individuals and blood collection from 698 of those participants. This paper describes the formulation and execution of this large-scale scientific study, encompassing best practices and insights gained throughout the endeavor.

Longitudinal impact of COVID-19 pandemic on the utilization of hemoglobin A1c testing in outpatients.

BACKGROUND: During the COVID-19 pandemic, concerns arose about disparate access to health care and laboratory testing. There is limited information about the pandemic's impact on the frequency of diabetic laboratory testing across demographic subgroups (e.g., sex, age over 65 y, and race). METHODS: This retrospective study examined outpatient hemoglobin A1c (HbA1c) testing in a large academic medical center in Upstate New York between March 2019 and March 2021. Multivariate Poisson regression models were used to evaluate the pandemic's effects on HbA1c utilization. RESULTS: Over 190,000 HbA1c results from predominately white (76.1 %) and older (mean age, 60.6 y) outpatients were analyzed. Compared to pre-pandemic time period, the average number of HbA1c tests per patient during COVID time period experienced a small, though significant, drop (1.3 to 1.2; p < 0.001) on aggregate and in outpatients, males, females, and seniors. The modest reduction was not significant by race except for the white seniors (p < 0.001). However, the testing frequency remained within recommendations from the American Diabetes Association for monitoring prediabetic patients and patients with stable glycemic control. CONCLUSION: Given the propensity for healthcare disruptions to widen disparities, it is reassuring that we did not observe a worsening of disparities in rates of HbA1c testing during the COVID-19 pandemic.

Analytical Concordance of Total Vitamin D on a Fully Automated Random-Access LC-MS/MS Platform.

BACKGROUND: The adoption of LC-MS/MS laboratory developed tests in the clinical laboratory is limited by many factors including the lack of automation. Recently, the Cascadion™ clinical analyzer was introduced as a fully automated random-access LC-MS/MS platform. Here, the analytical concordance between the platform and a Roche immunoassay analyzer was investigated for vitamin D analysis in human serum, including samples selected for high triglyceride levels. METHODS: Analytical precision was evaluated on 3 levels of QC samples (10, 30, and 90 ng/mL) within days (n = 4, 5 days) and between days (20 days). Assay comparison to the Roche was performed using reference samples from the CDC and CAP programs for accuracy. Concordance was also monitored using routine patient samples, as well as samples selected for elevated triglyceride levels (>250 mg/dL). RESULTS: Precision met manufacturer specifications (<10% CV and <15% bias), whereas the accuracy evaluations showed a linear fit (y = 0.97x - 1.1, r = 0.995) with 1:1 correlation to reference samples, independent of C-3-epi-vitamin D levels. A mean positive bias (11%) was observed for the Roche measurements in normal patient samples, whereas a mean negative bias (-8%) was observed in samples selected for elevated triglyceride levels. CONCLUSIONS: Cascadion measurements of total vitamin D compared favorably with Roche results in our laboratory, although discordance was observed in the analysis of patient serum, which could be explained in terms of known differences between the 2 assays. However, operational issues need to be addressed to effect clinical adoption.

Tolerance for three commonly administered COVID-19 vaccines by healthcare professionals.

Importance: Most healthcare institutions require employees to be vaccinated against SARS-CoV-2 and many also require at least one booster. Objective: We determine the impact of vaccine type, demographics, and health conditions on COVID-19 vaccine side effects in healthcare professionals. Design: A COVID-19 immunity study was performed at the 2021 American Association for Clinical Chemistry Annual Scientific meeting. As part of this study, a REDCap survey with cascading questions was administered from September 9, 2021 to October 20, 2021. General questions included participant demographics, past and present health conditions, smoking, exercise, and medications. COVID-19 specific questions asked about SARS-CoV-2 vaccine status and type, vaccine-associated side effects after each dose including any boosters, previous infection with COVID-19, diagnostic testing performed, and type and severity symptoms of COVID-19. Results: There were 975 participants (47.1% male, median age of 50 years) who completed the survey. Pfizer was the most commonly administered vaccine (56.4%) followed by Moderna (32.0%) and Johnson & Johnson (7.1%). There were no significant differences in vaccine type received by age, health conditions, smoking, exercise, or type or number of prescription medications. Side effects were reported more frequently after second dose (e.g., Moderna or Pfizer) (54.1%) or single/only dose of Johnson & Johnson (47.8%). Males were significantly more likely to report no side effects (p < 0.001), while females were significantly more likely to report injection site reactions (p < 0.001), fatigue (p < 0.001), headache (p < 0.001), muscle pain (p < 0.001), chills (p = 0.001), fever (p = 0.007), and nausea (p < 0.001). There was a significant upward trend in participants reporting no side effects with increasing age (p < 0.001). There were no significant trends in side effects among different races, ethnicities, health conditions, medications, smoking status or exercise. In multivariate logistic regressions analyses, the second dose of Moderna was associated with a significantly higher risk of side effects than both the second dose of Pfizer and the single dose of Johnson & Johnson. Conclusions and relevance: Younger people, females, and those receiving the second dose of Moderna had more COVID-19 vaccine side effects that per self-report led to moderate to severe limitations. As reported in other studies, the increase in side effects from Moderna may be explained by higher viral mRNA concentrations but be associated with additional protective immunity.

Introduction to Mass Spectrometry for Bimolecular Analysis in a Clinical Laboratory.

Mass spectrometry is a technique that identifies analytes based on mass-to-charge (m/z) ratio and structural fragments. Although this technique has been used in research and specialized clinical laboratories for decades, only in recent years has mass spectrometry become popular in routine clinical laboratories. Mass spectrometry, especially when coupled with gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of mass spectrometry include simultaneous analysis of multiple analytes (>100) and generally limited requirement for specialized reagents. Commonly measured analytes by mass spectrometry include metabolites, drugs, hormones, and proteins.

Sensitive and Robust LC-MS/MS Analysis of Salivary Cortisol in Negative Mode.

Cortisol is one of the most important glucocorticoids involved in the regulation of human metabolism and physiological stress. Monitoring of levels of cortisol is of immense clinical benefit. In particular, salivary cortisol levels have been shown to correlate well with diurnal changes in cortisol levels in serum and have been used widely for monitoring of cortisol levels for diagnosis and prognosis purposes. We present a sensitive, fast, and robust quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) assay for salivary cortisol in negative mode. This assay employs protein precipitation followed by reversed-phase liquid chromatographic separation, negative-mode electrospray ionization (ESI), and MS/MS detection. This assay has a total run time of 5.8 minutes and a limit of quantification of 0.5 ng/mL with a linear range up to 100 ng/mL. No carryover was observed at 10 µg/mL. This assay also incorporates the routine monitoring of prednisolone, a potential interferent to salivary cortisol.

High-Throughput Analysis of 25-OH-Vitamin D2 and D3 Using Multichannel Liquid Chromatography-Tandem Mass Spectrometry.

Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be an effective approach in the clinical analysis of 25-OH-vitamin D in patient serum. Test volumes vary among laboratories and different levels of throughput are required for different settings. LC-MS/MS assays with multiple LC channels can be beneficial for labs with the demand of large sample volume (e.g., 300 or more samples) to control costs and fulfill a reasonable turnaround time. We hereby present an assay that employs 4 LC channels (4-plex), which are coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument, for a high-throughput solution. Briefly, the pre- and postelution segments of the LC gradient are diverted to waste via solenoid valve controls, reserving the data acquisition for only the elution segment per injection per channel at a time. The multiplexing affords a manifold increase in throughput and the optimization of the duty cycle, without compromise in assay performance and precision.

AACC Practical Recommendations for Implementing and Interpreting SARS-CoV-2 Emergency Use Authorization and Laboratory-Developed Test Serologic Testing in Clinical Laboratories.

BACKGROUND: The clinical laboratory continues to play a critical role in managing the coronavirus pandemic. Numerous US Food and Drug Administration emergency use authorization (EUA) and laboratory-developed test (LDT) serologic assays have become available. The performance characteristics of these assays and their clinical utility continue to be defined in real time during this pandemic. The AACC convened a panel of experts from clinical chemistry, microbiology, and immunology laboratories; the in vitro diagnostics industry; and regulatory agencies to provide practical recommendations for implementation and interpretation of these serologic tests in clinical laboratories. CONTENT: The currently available EUA serologic tests and platforms, information on assay design, antibody classes including neutralizing antibodies, and the humoral immune responses to SARS-CoV-2 are discussed. Verification and validation of EUA and LDT assays are described, along with a quality management approach. Four indications for serologic testing are outlined. Recommendations for result interpretation, reporting comments, and the role of orthogonal testing are also presented. SUMMARY: This document aims to provide a comprehensive reference for laboratory professionals and healthcare workers to appropriately implement SARS-CoV-2 serologic assays in the clinical laboratory and to interpret test results during this pandemic. Given the more frequent occurrence of outbreaks associated with either vector-borne or respiratory pathogens, this document will be a useful resource in planning for similar scenarios in the future.

Appropriateness of tacrolimus therapeutic drug monitoring timing in the emergency department.

PURPOSE: Evaluate the appropriateness of the timing of serum samples collected in the emergency department (ED) for tacrolimus (TAC) measurement. METHODS: Single-center, retrospective review of TAC samples collected in the ED from September 1 to October 31, 2017. The primary evaluation was incidence of inappropriate sample collection for TAC measurement, defined as samples not collected 12 h (±2 h; 10-14 h) after the last dose, or within 2 h of the next dose if last known dose time was not documented. Incidence of repeat TAC measurements obtained within 24 h of ED presentation (if initial sample collection inappropriate), inappropriate TAC regimen adjustments, and healthcare costs of inappropriate TAC measurements was evaluated. Data collection included patient demographics, ED visit information, TAC measurement and timing related to last or next dose, changes to TAC regimen, and ED disposition. Descriptive data are reported. RESULTS: Sixty-two patients were included. Forty-one (66%) initial TAC measurements were collected inappropriately in the ED. No patients had a regimen adjustment as a result of inappropriate concentration collection, but 32 patients (78%) did require a repeat measurement within 24 h of ED presentation due to initial inappropriate collection. Costs associated with incorrectly collected TAC measurements were $2,647.78 for the two-month time period and this is extrapolated to an estimated $15,886.68 annual expense for patients. CONCLUSIONS: Inappropriate sample collection for TAC measurements was common in the ED, resulting in frequent repeat laboratory draws and increased healthcare costs.

Persistently elevated hCG in a patient with no clear evidence of pregnancy.

BACKGROUND: We describe a case of a female patient with a complex medical history, including squamous cell carcinoma of the tongue, and persistent elevation in serum hCG and no confirmed intrauterine or extrauterine pregnancy. METHODS: The clinical chemistry laboratory was called upon to help clarify the situation and reach a diagnosis. RESULTS: Multiple potential sources of elevated hCG were considered and excluded. CONCLUSIONS: This case shows a rare example of paraneoplastic hCG expression and highlights the role of the clinical chemistry laboratory in resolving diagnostic dilemmas and interpreting laboratory results in patients with multiple medical comorbidities.

Effect of hemolysis, icterus, and lipemia on three acetaminophen assays: Potential medical consequences of false positive results.

BACKGROUND: We evaluated the effect of hemolysis, icterus and lipemia on 3 acetaminophen assays: namely the Syva® EMIT®, the Microgenics DRI® assay, and the Roche assay on a Roche Cobas® c501 or an Integra 800 analyzer. METHODS: Discarded acetaminophen - free serum samples (blank pool) and patient serum with acetaminophen overdose were used to prepare samples. Three levels of acetaminophen (5, 10, and 30 µg/ml) were evaluated for interference: hemolysis (H index range: 0-1000), icterus (I index range: 0-40), and lipemia (L index range: 0-1000). RESULTS: Measurements showed that the EMIT® assay was not significantly affected by hemolysis or icterus at all 3concentrations evaluated, but was negatively affected by lipemia at all three levels at 1000 mg/dl intralipids. The DRI® assay was similarly affected by hemolysis and icterus, but lipemia (at 1000 mg/dl intralipids) only affected the 5 µg/ml level. The Roche acetaminophen assay was significantly affected by hemolysis at all three concentrations. It was significantly affected by icterus at 20 mg/dl bilirubin and > 5 µg/ml and at icterus levels of 30 and 40 mg/dl bilirubin at 10 and 30 µg/ml acetaminophen concentrations, respectively. However, the Roche assay was least affected by lipemia. CONCLUSION: Hemolysis and icterus had insignificant interference on the Syva EMIT® and the DRI® assays for the analysis of acetaminophen, but significant interference effect on the Roche assay. On the other hand lipemia interfered less markedly with the Roche assay. The effect of hemolysis, icterus and lipemia should always be considered. Cautions are warranted when interpreting results for the potential false positive results in the presence of hemolysis and icterus at the concentrations evaluated in this study.