The kinetics and durability of antibody and T-cell responses to SARS-CoV-2 in children.

BACKGROUND: The kinetics and durability of T-cell responses to SARS-CoV-2 in children are not well-characterized. We studied a cohort of children aged 6 months to 20 years with COVID-19 in whom peripheral blood mononuclear cells (PBMC) and sera were archived at approximately 1, 6, and 12 months post-symptom onset. METHODS: We compared antibody (N = 85) and T-cell responses (N = 26) to nucleocapsid (N) and spike (S) glycoprotein over time across four age strata: 6 months to 5 years, 5-9, 10-14, and 15-20 years. RESULTS: N-specific antibody responses declined over time, becoming undetectable in 26/32 (81%) children by approximately one year post-infection. Functional breadth of anti-N CD4+ T-cell responses also declined over time and were positively correlated with N-antibody responses (Pearson's r = 0.31, p = 0.008). CD4+ T-cell responses to S displayed greater functional breadth than N in unvaccinated children, and, along with neutralization titers, were stable over time and similar across age strata. Functional profiles of CD4+ T-cell responses against S were not significantly modulated by vaccination. CONCLUSIONS: Our data reveal durable, age-independent T-cell immunity to SARS-CoV-2 structural proteins in children over time following COVID-19 infection as well as S-Ab responses overall, in comparison to declining antibody responses to N.

Tacrolimus and sirolimus in capillary dried blood spots allows for remote monitoring.

Therapeutic drug monitoring of tacrolimus and sirolimus plays a significant role in the clinical follow-up of transplant patients receiving IMS therapy. Success of transplant and favorable patient outcome relies on maintaining adequate therapeutic drug levels. The purpose of this research is to assess the clinical utility of remote collection of DBS for immunosuppressant monitoring and compare the IMS level in paired collections of venous whole blood and DBS. Sirolimus and tacrolimus levels were clinically correlated in capillary blood collected from a finger poke with venous whole blood from pediatric, post-transplant patients. The participants took the dried blood spot card home with them with a pre-addressed, postage-paid envelope and mailed it back to the laboratory. Overall, a small but statistically significant negative bias was observed (-0.6 ng/mL, p = 0.0011). A chart review was performed to assess whether clinical management would have changed, and none of the cases revealed a clinically significant change. Sirolimus in DBS also correlated with venous levels. Overall, a small but statistically negative bias was observed (-0.8 ng/mL, p = 0.029). In summary, analysis of IMS levels in DBS is possible, and the difference noted between capillary and venous blood is within the clinically acceptable limits.

Development of a Rapid Qualitative Screen for Anticoagulant Presence.

BACKGROUND: Direct oral anticoagulants (DOACs) and fondaparinux with stable pharmacokinetics are commonly used anticoagulants for outpatient care. Due to the lack of monitoring requirements, drug-specific assays are not available in most hospital laboratories, but drug levels are needed in some urgent/emergency situations. This study describes the development of a qualitative screen for the presence of DOAC or fondaparinux using coagulation tests found in most laboratories. METHODS: The DOAC screen is composed of a heparin anti-Xa activity assay and thrombin time (TT) assay. The STA®-Liquid-Anti-Xa assay calibrated with Stago Multi Hep® and STA®-TT were run on STA-R Max® analyzers. The anti-Xa activity and TT assays were repeated 5 times in samples of commercially available calibrators and controls for each drug: fondaparinux, dabigatran, rivaroxaban, apixaban, and edoxaban. Statistical analysis and correlations were performed for anti-Xa activity and TT results for each drug and pooled normal plasma. RESULTS: A significant correlation was found between heparin-calibrated anti-Xa levels and fondaparinux, rivaroxaban, apixiban, and edoxaban (r2 = 0.99-1.0). Dabigatran showed a strong linear correlation (r2 = 0.99) with TT. Anti-Xa levels >0.3 IU/mL and TT >25 seconds were determined as cutoffs at our lab for the detection of clinically relevant drug levels of factor Xa inhibitor and direct thrombin inhibitor, respectively. CONCLUSIONS: Our study demonstrates that commonly available heparin anti-Xa activity and TT assays can be used to qualitatively detect DOACs and fondaparinux and provides a method to establish a qualitative interpretation.

Paediatric healthcare and hospital worker SARS-CoV-2 IgG antibody: A longitudinal cohort study.

Background: This study sought to determine the prevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) and spike (S) protein immunoglobulin G (IgG) antibodies in healthcare and hospital workers (HCHWs), and changes in IgG N antibody levels over time. Methods: Longitudinal study of HCHWs at a freestanding, urban paediatric tertiary care hospital. Asymptomatic HCHWs aged ≥18 years working in clinical areas were eligible to enrol. Participants completed four surveys and blood draws over 12 months. Specimens were tested for IgG N at four timepoints and IgG S at 12 months. Results: In total, 531 HCHWs enrolled in this study; of these, 481 (91%), 429 (81%) and 383 (72%) completed follow-up blood draws at 2, 6 and 12 months, respectively. Five of 531 (1%), 5/481 (1%), 6/429 (1%) and 5/383 (1.3%) participants were seropositive for IgG N at baseline, 2, 6 and 12 months, respectively. All (374/374; 100%) participants who received one or two doses of either mRNA COVID-19 vaccine were seropositive for IgG S. One of nine unvaccinated participants was seropositive for IgG S. Conclusions: In this paediatric hospital, IgG N and IgG S were detected in 1.9% and 97.9% of HCHWs, respectively. This study demonstrated low transmission of SARS-CoV-2 among HCHWs with appropriate infection prevention measures.

Higher Antibody Concentrations in U.S. Health Care Workers Associated with Greater Reactogenicity Post-Vaccination.

Multiple factors may be associated with immune responses to SARS-CoV-2 vaccines. Factors potentially related to magnitude and durability of response include age, time, and vaccine reactogenicity. This study analyzed SARS-CoV-2 IgG spike antibody responses following the second dose of vaccine in healthcare workers (HCWs). Data were collected from participants enrolled in a longitudinal SARS-CoV-2 serology study over a 12-month period. Participants completed a survey documenting symptoms post-vaccination. Serum specimens were tested for SARS-CoV-2 IgG antibodies using the Abbott Architect AdvisdeDx SARS-CoV-2 IgGII assay. Antibody levels were compared against time from second vaccine dose, and symptoms following vaccination. Altogether, 335 women (86.6%) and 52 men (13.4%) participated. Median age was 37 years (IQR 30-43). Overall median antibody level was 2150.80 [1246.12, 3556.98] AU/mL (IQR). Age was not associated with antibody concentration (p-value = 0.10). Higher antibody responses (2253 AU/mL vs. 1506 AU/mL; p = 0.008) were found in HCWs with one or more symptoms after the second dose of the vaccine (n = 311). Antibody responses persisted throughout the study period post-vaccination; statistically significant decreases in antibody responses were observed over time (p < 0.001). Higher antibody response was associated with reactogenicity post-vaccine. Age and sex were not associated with higher antibody responses.

Oral estrogen leads to falsely low concentrations of estradiol in a common immunoassay.

Objectives: Recently, an estradiol immunoassay manufacturer (Beckman Coulter, USA) issued an 'important product notice' alerting clinical laboratories that their assay (Access Sensitive Estradiol) was not indicated for patients undergoing exogenous estradiol treatment. The objective of this analysis was to evaluate immunoassay bias relative to liquid chromatography tandem mass spectrometry (LC-MS/MS) in transgender women and to examine the influence of unconjugated estrone on measurements. Design: Cross-sectional secondary analysis. Methods: Estradiol concentrations from 89 transgender women were determined by 3 immunoassays (Access Sensitive Estradiol ('New BC') and Access Estradiol assays ('Old BC'), Beckman Coulter; Estradiol III assay ('Roche'), Roche Diagnostics) and LC-MS/MS. Bias was evaluated with and without adjustment for estrone concentrations. The number of participants who shifted between three estradiol concentration ranges for each immunoassay vs LC-MS/MS (>300 pg/mL, 70-300 pg/mL, and <70 pg/mL) was calculated. Results: The New BC assay had the largest magnitude overall bias (median: -34%) and was -40%, -22%, and -10%, among participants receiving tablet, patch, or injection preparations, respectively. Overall bias was -12% and +17% for the Roche and Old BC assays, respectively. When measured with the New BC assay, 18 participants shifted to a lower estradiol concentration range (vs 9 and 10 participants based on Roche or Old BC assays, respectively). Adjustment for estrone did not minimize bias. Conclusions: Immunoassay measurement of estradiol in transgender women may lead to falsely decreased concentrations that have the potential to affect management. A multidisciplinary health care approach is needed to ensure if appropriate analytical methods are available.

Evaluation of Exome Sequencing Criteria for Hospital Stewardship and Insurance Authorization at a Pediatric Hospital.

CONTEXT.­: Genomic molecular testing practices in a pediatric tertiary care institution can vary in utility by patient indication. OBJECTIVE.­: To evaluate exome sequencing (ES) ordering practices and the effects of applying criteria to support ES stewardship. Exome sequencing can provide molecular diagnostic information for patients with known or suspected genetic diseases, but it is relatively expensive, and the cost is often borne by patients, institutions, and payers. DESIGN.­: We examined ordering patterns of ES approved by board-certified geneticists at our tertiary pediatric care center, as well as preauthorization outcomes for ES requests. We compared positivity rates among patients by patient phenotype, composite insurance coverage criteria, and insurance preauthorization outcome. RESULTS.­: Patients who met composite coverage criteria were more likely to receive a positive result from ES compared to patients who did not meet composite coverage criteria, though this trend was not statistically significant. There was no significant difference in ES results between patients who were denied or not denied preauthorization by insurance payers. CONCLUSIONS.­: Insurance payers should consider implementing and/or expanding coverage criteria for ES, and institutions should implement stewardship programs to support appropriate ES practices.

Small volume retinol binding protein measurement by liquid chromatography-tandem mass spectrometry.

BACKGROUND: The measurement of plasma concentrations of retinol binding protein is a component of nutritional assessment in neonatal intensive care. However, serial testing in newborns is hampered by the limited amount of blood that can be sampled. Limitations are most severe with preterm infants, for whom close monitoring may be most important. METHODS: We developed an assay to quantify retinol binding protein using trypsin digestion and liquid chromatography-tandem mass spectrometry, which requires a serum or plasma volume of 5 µl. Additionally, we validated the method according to current recommendations and performed comparison with a standard nephelometry platform in clinical use. RESULTS: The assay demonstrated linearity from below 1 mg/dL (0.48 µM) to more than 20 mg/dL (9.7 µM), and an imprecision of 11.8% at 0.43 mg/dL (0.21 µM). The distribution of results observed with the new method was different when compared with nephelometry. CONCLUSION: Liquid chromatography-tandem mass spectrometry facilitated testing a smaller sample volume, thereby increasing the ability to monitor key nutritional markers in premature infants. The differences in results compared with a commercially-available nephelometric assay revealed questionable results for lower concentrations by immunoassay.

Adapting to telemedicine in the COVID-19 era: Feasibility of dried blood spot testing for hemoglobin A1c.

BACKGROUND AND AIMS: The COVID-19 pandemic has led to a rapid growth in the use of telemedicine for delivery of ambulatory diabetes care. This study evaluated the feasibility of remote HbA1c monitoring via dried blood spot (DBS) testing to support assessment of glycemic control for telemedicine visits and examined clinical and demographic characteristics associated with patient completion of DBS testing. METHODS: Providers could place orders for DBS HbA1c 3 weeks prior to telemedicine visits. Feasibility was assessed by examining DBS completion rates, time to completion, and availability of DBS results prior to telemedicine visits. Chi-square tests and Mann Whitney tests were used to assess whether completion rates were associated with participant characteristics. RESULTS: Of 303 DBS orders placed for telemedicine visits in June 2020, 162 patients completed the DBS test for a completion rate of (53.4%). Average time from collection at home to result being reported was 6.9 (3.8) days. The DBS result was available in 67.6% of patients who completed successful DBS, before the telemedicine clinic visit. HbA1c was lower in the DBS completion group as compared to the non-completion group (8.2% vs. 8.9%, p = 0.01). No other clinical or demographic characteristics were significantly different between the two groups. CONCLUSION: Remote HbA1c monitoring via DBS is feasible and offers an avenue to support assessment of glycemic control for patients seen via telemedicine. Future work should focus on improving clinic and laboratory processes to support remote DBS collection.

Acute ANCA Vasculitis and Asymptomatic COVID-19.

We describe the presentation and diagnosis of a child with newly diagnosed antineutrophil cytoplasmic antibody-associated vasculitis and associated diffuse alveolar hemorrhage who was positive for coronavirus disease 2019 immunoglobulin G antibodies, indicative of a previous asymptomatic infection. Results of multiple polymerase chain reaction tests coinciding with the start of symptoms were negative, indicating that acute infection was not the cause of the patient's symptoms. Coronavirus disease 2019-induced autoimmune diseases have been described in adults, but this case report represents the first case described in a pediatric patient.

Sublingual Estradiol Is Associated with Higher Estrone Concentrations than Transdermal or Injectable Preparations in Transgender Women and Gender Nonbinary Adults.

Purpose: Serum hormone profiles among different feminizing gender-affirming hormone therapies (GAHT) are poorly characterized. To address this gap, we described the serum estrogen profiles of three 17ß-estradiol preparations, taken with or without an antiandrogen, using a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay in adults taking feminizing GAHT. Methods: This was a secondary analysis of 93 healthy transgender women and gender nonbinary adults taking feminizing GAHT in a prospective cross-sectional study. Eligible participants took 17ß-estradiol (sublingual tablet, transdermal patch, or intramuscular/subcutaneous injection) with or without oral spironolactone for ≥12 months before study entry. We determined serum estrone and estradiol concentrations for each hormone preparation and described the association between estrone and (1) clinically relevant estradiol concentration ranges (≤200 and >200 pg/mL) and (2) antiandrogen use. To achieve our objectives, we described our protocol for developing an LC-MS/MS assay to measure estrone and estradiol concentrations. Results: Estrone concentrations were higher among participants taking sublingual 17ß-estradiol tablets compared with transdermal or injectable preparations (p < 0.0001). Estradiol concentrations were higher for injectable versus transdermal preparations (p = 0.0201), but both were similar to sublingual tablet concentrations (p > 0.05). Estradiol >200 pg/mL (vs. ≤200 pg/mL) was associated with higher estrone concentrations among participants taking sublingual 17ß-estradiol, but not transdermal or injectable 17ß-estradiol. We observed no association between spironolactone and estrone concentrations (p > 0.5). Conclusion: Estrone concentrations were higher among transgender women and gender nonbinary adults taking sublingual 17ß-estradiol compared with transdermal or injectable preparations. The role of estrone in clinical monitoring and the influence of other antiandrogens (e.g., cyproterone acetate) on the estrogen profile remain to be determined.

Prevalence of Health Care and Hospital Worker SARS-CoV-2 IgG Antibody in a Pediatric Hospital.

OBJECTIVES: Asymptomatic transmission of coronavirus disease 2019 (COVID-19) in health care settings is not well understood. In this study, we aimed to determine the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) antibodies in health care and hospital workers (HCHWs) and assess how antibody levels change over time. METHODS: Cross-sectional study of employed HCHWs at a freestanding, urban pediatric tertiary care hospital. Employed HCHWs ≥18 years old who were asymptomatic and worked in clinical hospital locations were eligible to participate. Participants completed blood draws and surveys at baseline (between May 4, 2020, and June 2, 2020) and 2 months later (between July 6, 2020, and August 7, 2020). Surveys collected demographic information, SARS-CoV-2 exposures, and previous COVID-19 diagnosis. RESULTS: In total, 530 participants enrolled in and completed baseline study activities. The median age was 37 years (range 19-67 years); 86% identified as female, and 80% identified as white. Two months later, 481 (91%) HCHWs completed another survey and blood draw. Four of 5 (0.9%) seropositive subjects at baseline remained seropositive at 2 months, although 3 had decreasing IgG indices. Five (1.0%) seropositive individuals, including 4 who were previously seropositive and 1 newly seropositive, were detected 2 months later. History of positive SARS-CoV-2 polymerase chain reaction testing results (P < .001) and history of COVID-19 exposure (P < .001) were associated with presence of SARS-CoV-2 antibodies. CONCLUSIONS: SARS-CoV-2 antibodies were detected in 1% of HCHWs in an urban pediatric hospital in a city with moderate SARS-CoV-2 prevalence. Participants with a known previous COVID-19 diagnosis showed a decline or loss of IgG antibodies over 2 months. These results have implications for identifying those with previous exposure and for ongoing public health recommendations for ensuring workplace safety.

Reproductive Endocrinology Reference Intervals for Transgender Men on Stable Hormone Therapy.

BACKGROUND: Gender-affirming therapy with testosterone is commonly prescribed to aid in the masculinization of transgender men. Sex-hormone concentrations are routinely measured, but interpretation of results can be difficult due to the lack of published reference intervals. METHODS: Healthy transgender individuals who had been prescribed testosterone (n = 82) for at least a year were recruited from internal medicine and primary care clinics that specialize in transgender medical care. Total testosterone and estradiol were measured using immunoassay and mass spectrometry; LH, FSH, SHBG, prolactin, progesterone, anti-Müllerian hormone (AMH), and dehydroepiandrosterone sulfate (DHEAS) were measured using immunoassay; free testosterone was calculated. Reference intervals (central 95%) were calculated according to Clinical Laboratory Standards Institute guidelines. RESULTS: When evaluating general endocrine laboratory tests in people using masculinizing hormones, reference intervals for cisgender men can be applied for total and free testosterone and SHBG and reference intervals for cisgender women can be applied for prolactin. Reference intervals for estradiol, LH, FSH, AMH, and DHEAS differ from those used for cisgender men and cisgender women, and therefore should be interpreted using intervals specific to the transmasculine population. For testosterone and estradiol, results from immunoassays were clinically equivalent to mass spectrometry. CONCLUSION: Masculinizing hormones will alter the concentrations of commonly evaluated endocrine hormones. Providers and laboratories should use appropriate reference intervals to interpret the results of these tests.

Reproductive Endocrinology Reference Intervals for Transgender Women on Stable Hormone Therapy.

BACKGROUND: Transgender women and nonbinary people seeking feminizing therapy are often prescribed estrogen as a gender-affirming hormone, which will alter their reproductive hormone axis. Testosterone, estradiol, and other reproductive hormones are commonly evaluated to assess therapy, but reference intervals specific to transgender women have not been established. The objective of this study was to derive reference intervals for commonly measured analytes related to reproductive endocrinology in a cohort of healthy gender nonconforming individuals on stable feminizing hormone therapy. METHODS: Healthy transgender individuals who had been prescribed estrogen (n = 93) for at least a year were recruited from internal medicine and primary care clinics that specialize in transgender medical care. Total testosterone and estradiol were measured using immunoassay and mass spectrometry; LH, FSH, sex hormone binding globulin, prolactin, progesterone, anti-mullerian hormone (AMH), and dehydroepiandrosterone sulfate (DHEAS) were measured using immunoassay; free testosterone was calculated. Reference intervals (central 95%) were calculated according to Clinical Laboratory Standards Institute guidelines. RESULTS: The distribution of results for transgender women was different than what would be expected from cisgender men or women across all measurements. Use of spironolactone was associated with changes in the result distribution of AMH, FSH, LH, and progesterone. Compared to liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), immunoassay was sufficient for the majority of estradiol and total testosterone measurements; free testosterone added little clinical value beyond total testosterone. CONCLUSION: Reference intervals specific to transgender women should be applied when evaluating reproductive endocrine analytes. Spironolactone is a significant variable for result interpretation of some tests.

Capillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digests.

We perform 2-D capillary electrophoresis on fluorescently labeled proteins and peptides. Capillary sieving electrophoresis (CSE) was performed in the first dimension and MEKC was performed in the second. A cellular homogenate was labeled with the fluorogenic reagent FQ and separated using the system. This homogenate generated a pair of ridges; the first had essentially constant migration time in the CSE dimension, while the second had essentially constant migration time in the MEKC dimension. In addition, a few spots were scattered through the electropherogram. The same homogenate was digested using trypsin, and then labeled and subjected to the 2-D separation. In this case, the two ridges observed from the original 2-D separation disappeared and were replaced by a set of spots that fell along the diagonal. Those spots were identified using a local-maximum algorithm and each was fit using a 2-D Gaussian surface by an unsupervised nonlinear least squares regression algorithm. The migration times of the tryptic digest components were highly correlated (r=0.862). When the slowest migrating components were eliminated from the analysis, the correlation coefficient improved to r=0.956.

Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection.

CIEF and CZE are coupled with LIF detection to create an ultrasensitive 2-D separation method for proteins. In this method, two capillaries are joined through a buffer-filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first-dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second-dimension separation. A fraction was transferred to the second-dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.