Assay Cutoff Research Articles

8117 Late-night Salivary Cortisol in the Diagnosis of Cushing's Disease Recurrence

Abstract Disclosure: A.E. Alexandre: None. P.C. Elias: None. M. De Castro: None. L.M. Mermejo: None. D.C. Aragon: None. A.C. Moreira: None. Cushing’s Disease (CD) remission following transsphenoidal surgery (TSS) is most often defined by low morning cortisol levels and the requirement of glucocorticoid (GC) replacement therapy. However, CD recurrence is not a well studied event. There is no standard definition of remission criteria and no accurate cutoff for biochemical tests on CD recurrence. Late-night salivary cortisol (LNSC) has been shown to have higher accuracy in confirming CD recurrence than urinary free cortisol and dexamethasone suppression test (DST). However, few studies addressed LNSC performance following remission after TSS. The study aimed to establish cutoff values of LNSC with higher sensitivity (S) and specificity (E) in CD recurrence diagnosis.In a retrospective cohort study of 65 patients with remitted CD after TSS, remission was defined as improvement of sign and symptoms related to CD and a plasma cortisol <5mcg/dL in 30 days following TSS. Eleven patients progressed to permanent adrenal insufficiency and were excluded from the study. All 54 patients had at least four LNSC sampling and a minimal follow-up of 24 months after TSS. LNSC was determined by RIA. A cutoff value of 270 ng/dl for LNSC is used for the diagnosis of CD. Patients were divided in three different groups: Overt Recurrence (OR): elevated LNSC and worsening of CD`s signs and symptoms. Remission (RM): normal LNSC and no clinical signs of CD recurrence. Biochemical Recurrence (BR): at least 2 elevated biochemical parameters (LNSC or DST) but no worsening of signs and symptoms associated with CD. The mean of the 2 LNSC last values when OR was diagnosed and the mean of 2 LNSC last values of follow up for RM patients were used to estimate the accuracy of LNSC for diagnosing recurrence using a ROC curve (AUC) and its 95% confidence interval. Of the 54 patients included, 46 females; 8 males; mean age at diagnosis: 32 years, (5 – 58 yrs); mean time of follow-up: 12.8 years (4 – 31.7 yrs). Thirteen were classified as OR, 18 as RM and 23 as BR; and mean time of GC use after TSS was: 8.7; 34 and 17 yrs, respectively, significantly lower in OR compared to RM (p:0.046). A LNSC cutoff value of 272 mcg/dL had sensitivity of 85% and specificity of 95% (AUC: 0.97; 95% IC = [0.93; 1,00]) for CD recurrence diagnosis. BR patients presented LSNC fluctuation along follow-up.LNSC is a useful tool for CD recurrence screening. Setting LSNC specific assay cutoff values might be needed. LSNC cutoff values were similar for the initial CD diagnosis, as well as for CD recurrence diagnosis. Shorter time of GC replacement may be an important predictor of CD recurrence. Patients with BR should be followed more carefully as they may be at higher risk of recurrence. Multiple LNSC sampling on successive days can be easily performed and may detect subtle changes in cortisol rhythm. Presentation: 6/1/2024

Performance Validation of the NeoBase 2 Non-Derivatized MSMS Assay Kit and Cutoff Values Establishment of Term and Preterm Neonates

Objective NeoBase 2 Non-derivatized MSMS assay kit (NeoBase 2 kit) was used for newborn screening, the performance of the NeoBase 2 kit should be validated before its implementation in clinical diagnostic laboratories. Methods Leftover dried blood spot samples, quality control materials in the NeoBase 2 kit, and proficiency testing materials received from the NSQAP were used. Precision, accuracy, LOD, LLOQ, recovery, and stability were carried out to verify the performance of the Waters ACQUITY TQD MS/MS system with the NeoBase 2 kit for newborn screening. Cutoffs were determined and analytes requiring different cutoffs in preterm neonates were investigated. Results Within-run and between-run precisions ranged from 3.95% to 14.41%. The accuracy and stability were within 15%. All analytes demonstrated acceptable LOD, LLOQ, and recoveries. Cutoffs for term and preterm neonates were established. Conclusions The performance of the NeoBase 2 kit is acceptable and can be implemented in clinical diagnostic laboratories.

#108 : Methods of Sperm DNA Fragmentation (SDF) Testing and Interpretation: Results from the Global Andrology Forum Survey

Background and Aims: Sperm DNA fragmentation (SDF) is caused by apoptosis, oxidative stress or incomplete chromatin maturation and can result in male infertility and poor reproductive outcomes. Despite growing trends, there are no specific recommendations on specific diagnostic tests used, assay cut-offs or abstinence periods prior to SDF testing. Therefore, we conducted a global survey to evaluate the approaches used to diagnose SDF, towards a consensus in the field of SDF research and clinical management. Method: The survey collected cross-sectional global data from 55 countries following the CHERRIES protocol. 436 respondents included clinicians, researchers and reproductive biologists from academic, public and private health sectors, all of whom utilize SDF testing in their routine patient care and practice (Fig. 1). Results: Survey results showed that TUNEL (28.6%), SCSA (24.1%), SCD (19.1%) and Comet (5.3%) were most frequently used to diagnose SD (Fig. 2A). Assays were chosen primarily due to their availability (70.0%), cost (44.05%) and accuracy (35.48%). Published evidence on assays (25.71%), training of staff (25.48%), cost to patients (21.67%), assay time (18.81%), ease of interpretation (18.33%) and performance (17.38%) were taken lesser into consideration when selecting SDF diagnostic tests (Fig. 2B). Assay cut-offs of “30% or higher” (33.7%) were most frequently used to diagnose SDF (Fig. 2C). Finally, an abstinence period of 3-5 days was mostly recommended (53.6%; Fig. 2D). The results of this survey provide valuable data on SDF assays used in clinical practice as well as factors affecting assay choices such as availability of resources, personnel and complexity in assay processing. As decreased abstinence period is associated with lower SDF, testing is recommended with an abstinence period of less than five days. We observed that there was a lack of standard cut-off values, and every laboratory should establish its own reference values based on predictive values for fertility outcomes. Conclusion: We present new data associated with SDF testing in real-world practice, on a global scale. The trends and conclusions derived from the study are recommended for inclusion into the 6[Formula: see text] Edition WHO Manual, towards optimized patient care.

Usefullness of Heparin Calibrated Anti-Xa Activity to Assess Anticoagulant Activity of Apixaban and Rivaroxaban in Emergency Patients Scheduled for Acute Interventions.

(1) Background: Direct oral anticoagulants (DOACs) require monitoring in some critical clinical situations. The specific tests for DOAC monitoring are not yet available in all labs. The aim of this study was to evaluate if a unique, more widespread heparin-calibrated anti-Xa assay could be suitable to estimate the concentrations of apixaban and rivaroxaban in order to establish an algorithm helping our clinicians in their therapeutic decision for patients treated with DOACs in emergencies. (2) Methods: A first retrospective part allowed us to determine of a conversion factor between the measured DOAC concentration and the deducted anti-Xa heparin activity based on optic density. During the second prospective part, both DOAC concentration (ng/mL) and anti-Xa activity heparin (UI/mL) were measured on the same sample, and the previously determined conversion factor was applied to each UI/mL value. We then compared the calculated and measured DOAC concentration values. (3) Results: The analysis of the derivation cohort confirmed a good correlation, especially between the anti-Xa heparin activity and the apixaban concentrations (r = 0.97). Additionally, we determined heparin-calibrated anti-Xa assay cut-offs for invasive procedures at 0.3 UI/mL and for intravenous thrombolysis at 0.51 UI/mL using ROC curves with a sensitivity at 98% and specificity at 95% for 0.3 UI/mL and a sensitivity at 97.7% and specificity at 88.2% for the cut-off of 0.51 UI/mL. In the validation cohort, we confirmed the agreement between measured and calculated DOAC concentrations for the low values, especially around cut-offs with an excellent negative predictive value for 0.51 UI/mL (94% for apixaban and 100% for rivaroxaban) and a good negative predictive value for 0.3 UI/mL (83.3% for apixaban and 85.7% for rivaroxaban). (4) Conclusions: Our results confirm that it is possible to correctly predict or exclude the presence of apixaban/rivaroxaban in emergency situations when specific tests are not readily available.

Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification

Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (SLC4A1) and menstrual fluid (STC1), saliva (HTN3) and vaginal material (CYP2B7P), and spermatozoa (PRM1) and seminal fluid (KLK2), all including a synthetic internal control RNA.Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R2 > 0.99), and high precision (most mean CV < 1 %). STC1 showed some instances of sporadic expression in blood, semen, and vaginal material at high CT values. CYP2B7P showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target CYP2B7P expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.

THU645 Comparison Of LC-MS/MS Testosterone Assays Towards Standardization Of Lower Testosterone Ranges

Abstract Disclosure: M.G. Cree: Consulting Fee; Self; Pollie, Inc. Research Investigator; Self; Eli-Lilly, Amino Co. S. Ottey: None. K.D. Sherif: None. O. Sugahara: None. F. Pokuah: None. A.N. Lyle: None. V. Hubert: None. Objective: Polycystic ovary syndrome (PCOS) is a common disorder in women. The diagnosis of PCOS often includes elevated concentrations of serum testosterone. Previous work has harmonized the higher range of the testosterone assay and normative cut-offs for low and high testosterone concentrations for the male gender. However, similar work is needed to better align existing Liquid Chromatography Tandem Mass Spectroscopy (LC-MS/MS) methods in the lower range of testosterone and determine upper limits of normal for females. Methods: This is a secondary analysis of a pooled cohort of 60 obese adolescents with and without PCOS, enrolled in 1 of three studies (NCT02157974; NCT03041129; NCT03717935) at the University of Colorado, Anschutz. None of the participants were taking hormonal medication to treat PCOS. Frozen plasma samples collected between 6-7 AM were analyzed by Esoterix hormone branch of LabCorp (Agoura Hills, CA) and by the Centers for Disease Control (CDC) Clinical Standardization Programs with LC-MS/MS. Samples were selected from the available participants to encompass the full range of testosterone concentrations available within the cohort. Results: Within the entire cohort (N=60, Age = 15.8, SD: ± 1.7 years, BMI 35.4, SD: ± 5.5 kg/m2), the means between the assays were statistically, but not clinically different (CDC 48.4 SD: ±24.4 ng/dL vs OL 45.4 SD: ±22.1, p=0.013).Deming regression indicated that the assays were not different with an intercept of 2.05 (-3.69 to 7.79, 95% confidence interval) and slope of 0.895 (0.753 to 1.04, 95% confidence interval). The two assays were highly correlated via linear regression (R2=0.859, p&amp;lt;0.001) and with a Bland-Altman comparison, the CDC assay only had a slight positive bias of 3.04, SD: ± 9.18 ng/dL relative to Esoterix. When divided by PCOS status (PCOS N=52, Hirsutism score 6.5±5.9, Control N=8, Hirsutism score 2.1±4.5), the CDC assay ROC demonstrated discrimination of PCOS status with an AUC of 0.903 (0.800-1.00), p&amp;lt;0.001 and the Youden index was &amp;lt;27.1 ng/dL with a sensitivity of 87.5% and a specificity of 86.5%. However, when the sensitivity was at 100%, the specificity was 57.7% and the cut-off &amp;lt;43.0 ng/dL. Conclusions: The Esoterix, a major commercial lab, assay is very similar to that of CDC LC-MS/MS, and able to discriminate PCOS status at 43.0 ng/dL. Future work is needed to 1) align more clinical assays to the CDC standard and 2) define a common cut-off to describe hyperandrogenism in females across the weight and age spectrum. Presentation: Thursday, June 15, 2023

A-384 Laboratory Evaluation of the SeptiCyte Rapid Host Response Assay In Differentiating Infection-Positive Versus Infection-Negative Systemic Inflammation Patients Suspected of Sepsis

Abstract Background Early diagnosis of sepsis is critical for timely treatment but remains challenging. A rapid host response assay, SeptiCyte, was recently approved by the FDA for aiding early differentiation of infection-positive from infection-negative systemic inflammation in patients suspected of sepsis. This test is a fully automated reverse transcription polymerase chain reaction (RT-qPCR) assay that quantifies changes in the expression of two host immune response genes and results are available in about 1 hour. The present study aimed to evaluate the SeptiCyte assay performance in a clinical setting at a large medical center. Methods Eighty-two patients admitted to the University Hospitals Cleveland Medical Center with suspected sepsis from July to September 2022 were included in the study. SeptiCyte samples were obtained at the time of initial evaluation. In addition, healthy donor blood samples and extracted RNA samples were also utilized for assay validation. The SeptiCyte assay was performed according to the manufacturer's instructions using multi-chambered fluidic cartridges and the Idylla Console (Immunexpress, Seattle, WA). The assay result was reported as the SeptiScore (range 0–15), a calculated ratio of the expression of two host immune response genes, and the SeptiCyte band (ranging from 1 to 4, with 1 indicating the lowest risk of sepsis and 4 indicating the highest risk). The assay cutoff values are 5.0, 6.2 and 7.4 respectively for the band 2, 3 and 4. Results Intra- and inter-assay precision was 0.2–6.2% and 0–3.2%, respectively. % Recovery at the cutoff for band 3 (SeptiScore 6.0) was 102.4% with a CV of 2.4%. Method comparison with the Immunexpress lab demonstrated equivalent performance, as evidenced by a slope of 0.99 and an intercept of 0.27 by Deming regression. SeptiScore showed a weak positive correlation with CRP (Correlation Coefficient 0.389, P &amp;lt; 0.01) but no association with procalcitonin (n = 76), lactate (n = 78), international normalized ratio (INR), platelet, neutrophil, lymphocyte, or white blood cell (WBC) count (n = 82). CRP (n = 71) median concentration in patients with band 4 (16.8) was significantly higher than in patients who were band 1 (3.6). The SeptiScore of blood culture positive cases (n = 22) was 7.2 ± 2.0 (mean ± SD) and that of pathogen negative non-sepsis (by sepsis retrospective review of event only, n = 10) cases was 5.6 ± 1.6 (P &amp;lt; 0.05). In addition, the median SeptiScore was notably higher in patients with viral infection compared to those without infection (9.2 vs 6.2, P &amp;lt; 0.05). Further analysis showed 92% (11/12) of patients with hematologic cancer were categorized as band 4 including one non-sepsis case per the retrospective review. SeptiScore was similar between patients with non-hematologic cancer and those without malignancy (P &amp;gt; 0.05). Conclusion The SeptiCyte RAPID assay showed good analytical performance. SeptiScore represents an independent factor to quantify host response in patients suspected of sepsis. It may add value in aiding differentiation between infection-positive from infection-negative systemic inflammation with promising results in patients with viral infections or those with positive blood cultures. The negative predictive value of low SeptiScore within band 1 or 2 may need further investigation.

Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis

Pleural tuberculosis (pTB) is a grave clinical challenge. A novel cell-free M. tuberculosis DNA (cfM.tb-DNA) probe-based-qPCR assay was developed for the diagnosis of pTB. Total cell-free DNA was extracted from pleural fluid (PF) and paired plasma samples and cfM.tb-DNA was quantified by probe-based qPCR targeting devR (109-bp) gene of M. tuberculosis in patients with pleural effusion. Patient categorization was done using ‘Composite-Reference-Standard’ formulated for the study. Assay cut-offs were determined from samples in the ‘Development set’ (n = 17; ‘Definite & Probable’ pTB; n = 9 and ‘Non-TB’; n = 8) by ROC-curve analysis and applied to ‘Validation set’ (n = 112; ‘Definite’ pTB; n = 8, ‘Probable’ pTB; n = 34, ‘Possible’ pTB; n = 28 and ‘Non-TB’; n = 42). cfM.tb-DNA qPCR had a sensitivity of 62.5% (95%CI; 24.4,91.4) in ‘Definite’ pTB category and 59.5% (95%CI; 43.2,74.3) in ‘Definite & Probable’ pTB category with 95.2% (95%CI; 83.8,99.4) specificity using PF. In plasma (n = 85), the assay had a sub-optimal sensitivity of 7.6% (95%CI; 0.95,25.1) with 88.2% (95%CI; 72.5,96.7) specificity in ‘Definite & Probable’ pTB group. Xpert MTB/RIF assay detected only six-samples in the ‘Validation set’. Logistic regression analysis indicated that PF-cfM.tb-DNA qPCR provided incremental advantage over existing pTB diagnostic algorithms. To the best of our knowledge, this is the first report describing the utility of cfM.tb-DNA for pTB diagnosis in India.

Early evaluation of the safety, reactogenicity, and immune response after a single dose of modified vaccinia Ankara–Bavaria Nordic vaccine against mpox in children: a national outbreak response

In response to a national mpox (formerly known as monkeypox) outbreak in England, children exposed to a confirmed mpox case were offered modified vaccinia Ankara-Bavaria Nordic (MVA-BN), a third-generation smallpox vaccine, for post-exposure prophylaxis. We aimed to assess the safety and reactogenicity and humoral and cellular immune response, following the first reported use of MVA-BN in children. This is an assessment of children receiving MVA-BN for post-exposure prophylaxis in response to a national mpox outbreak in England. All children receiving MVA-BN were asked to complete a post-vaccination questionnaire online and provide a blood sample 1 month and 3 months after vaccination. Outcome measures for the questionnaire included reactogenicity and adverse events after vaccination. Blood samples were tested for humoural, cellular, and cytokine responses and compared with unvaccinated paediatric controls who had never been exposed to mpox. Between June 1 and Nov 30, 2022, 87 children had one MVA-BN dose and none developed any serious adverse events or developed mpox disease after vaccination. Post-vaccination reactogenicity questionnaires were completed by 45 (52%) of 87 children. Their median age was 5 years (IQR 5-9), 25 (56%) of 45 were male, and 22 (49%) of 45 were White. 16 (36%) reported no symptoms, 18 (40%) reported local reaction only, and 11 (24%) reported systemic symptoms with or without local reactions. Seven (8%) of 87 children provided a first blood sample a median of 6 weeks (IQR 6·0-6·5) after vaccination and five (6%) provided a second blood sample at a median of 15 weeks (14-15). All children had poxvirus IgG antibodies with titres well above the assay cutoff of OD450nm 0·1926 with mean absorbances of 1·380 at six weeks and 0·9826 at 15 weeks post-vaccination. Assessment of reactivity to 27 recombinant vaccina virus and monkeypox virus proteins showed humoral antigen recognition, primarily to monkeypox virus antigens B6, B2, and vaccina virus antigen B5, with waning of humoral responses observed between the two timepoints. All children had a robust T-cell response to whole modified vaccinia Ankara virus and a select pool of conserved pan-Poxviridae peptides. A balanced CD4+ and CD8+ T-cell response was evident at 6 weeks, which was retained at 15 weeks after vaccination. A single dose of MVA-BN for post-exposure prophylaxis was well-tolerated in children and induced robust antibody and cellular immune responses up to 15 weeks after vaccination. Larger studies are needed to fully assess the safety, immunogenicity, and effectiveness of MVA-BN in children. Our findings, however, support its on-going use to prevent mpox in children as part of an emergency public health response. UK Health Security Agency.

A Novel Enzymatic Hydrolysis Method for Urine Aldosterone Quantification: A Case for Reassessing Clinical Cut-Offs of Primary Aldosteronism.

Primary aldosteronism (PA) is a common endocrine cause of secondary hypertension. The aldosterone/renin ratio is an important tool for PA screening, and dynamic testing in serum or urine is used to confirm the diagnosis. While LC-MS/MS is considered the gold standard for testing, there is significant interlaboratory variability between the extraction procedures, which can impact diagnostic interpretation. To help overcome this, we present a simple and accurate LC-MS/MS method for the quantification of both serum and urine aldosterone using a novel enzymatic hydrolysis procedure. Serum and urine aldosterone was extracted and measured by LC-MS/MS. Urine-conjugated aldosterone glucuronide was hydrolyzed using a genetically modified glucuronidase enzyme. The assay precision, accuracy, limit of quantification, recovery, and carryover were evaluated and the new assay cut-offs were proposed. The liquid chromatography method allowed for adequate separation of the aldosterone peak from closely eluting peaks. Significant in vitro aldosterone loss was observed during acid-catalyzed hydrolysis of urine, which was corrected with the addition of the internal standard to the urine before the hydrolysis step. Glucuronidase catalyzed hydrolysis of urine aldosterone glucuronide displays good correlation with the corrected acid-catalyzed hydrolysis. Serum aldosterone showed good agreement with reference values and the consensus range reported for external quality assessment specimens. A simple, fast, and highly accurate method for the detection of serum and urine aldosterone has been developed. The proposed novel enzymatic procedure allows for short hydrolysis time and compensates for urine aldosterone loss during the hydrolysis step.

Development, Validation, and Utilization of a Luminex-Based SARS-CoV-2 Multiplex Serology Assay.

SARS-CoV-2 antibody testing is important for seroprevalence studies and for evaluating vaccine immune responses. We developed and validated a Luminex bead-based multiplex serology assay for measuring IgG levels of anti-SARS-CoV-2 antibodies against full-length spike (S), nucleocapsid (N), and receptor-binding domains (RBDs) of wild-type, RBD N501Y mutant, RBD E484K mutant, RBD triple mutant SARS-CoV-2 proteins, Sars-CoV-1, MERS-CoV, and common human coronaviruses, including SARS-CoV-2, OC43, 229E, HKU1, and NL63. Assay cutoff values, sensitivity, and specificity were determined using samples from 160 negative controls and 60 PCR-confirmed, SARS-CoV-2-infected individuals. The assay demonstrated sensitivities of 98.3%, 95%, and 100% and specificities of 100%, 99.4%, and 98.8% for anti-(S), -N, and -RBD, respectively. Results are expressed as IgG antibody concentrations in BAU/mL, using the WHO international standard (NIBSC code 20/136) for anti-SARS-CoV-2 IgG antibodies. When the multiplex assay was performed and compared with singleplex assays, the IgG antibody measurement geometric mean ratios were between 0.895 and 1.122, and no evidence of interference was observed between antigens. Lower and upper IgG concentration limits, based on accuracy (between 80% and 120%), precision (percent relative standard deviation, ≤25%), and sample dilutional linearity (between 75% and 125%), were used to establish the assay range. Precision was established by evaluating 24 individual human serum samples obtained from vaccinated and SARS-CoV-2-infected individuals. The assay provided reproducible, consistent results with typical coefficients of variation of ≤20% for all assays, irrespective of the run, day, or analyst. Results indicate the assay has high sensitivity and specificity and thus is appropriate for use in measuring SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. IMPORTANCE The SARS-CoV-2 pandemic resulted in the development and validation of multiple serology tests with variable performance. While there are multiple SARS-CoV-2 serology tests to detect SARS-CoV-2 antibodies, the focus is usually either on only one antigen at a time or multiple proteins from only one SARS-CoV-2 variant. These tests usually do not evaluate antibodies against viral proteins from different SARS-CoV-2 variants or from other coronaviruses. Here, we evaluated a multiplex serology test based on Luminex technology, where antibodies against multiple domains of SARS-CoV-2 wild type, SARS-CoV-2 mutants, and common coronavirus antibodies are detected simultaneously in a single assay. This Luminex-based multiplex serology assay can enhance our understanding of the immune response to SARS-CoV-2 infection and vaccination.

Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine Immunogenicity among Chimeric Antigen Receptor T Cell Therapy Recipients

Patients receiving chimeric antigen receptor T cell (CAR-T) therapy may have impaired humoral responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations owing to their underlying hematologic malignancy, prior lines of therapy, and CAR-T-associated hypogammaglobulinemia. Comprehensive data on vaccine immunogenicity in this patient population are limited. A single-center retrospective study of adults receiving CD19 or BCMA-directed CAR-T therapy for B cell non-Hodgkin lymphoma or multiple myeloma was conducted. Patients received at least 2 doses of SARS-CoV-2 vaccination with BNT162b2 or mRNA-1273 or 1 dose of Ad26.COV2.S and had SARS-CoV-2 anti-spike antibody (anti-S IgG) levels measured at least 1 month after the last vaccine dose. Patients were excluded if they received SARS-CoV-2 monoclonal antibody therapy or immunoglobulin within 3 months of the index anti-S titer. The seropositivity rate (assessed by an anti-S assay cutoff of ≥.8 U/mL in the Roche assay) and median anti-S IgG titers were analyzed. Fifty patients were included in the study. The median age was 65 years (interquartile range [IQR], 58 to 70 years), and the majority were male (68%). Thirty-two participants (64%) had a positive antibody response, with a median titer of 138.5 U/mL (IQR, 11.61 to 2541 U/mL). Receipt of ≥3 vaccines was associated with a significantly higher anti-S IgG level. Our study supports current guidelines for SARS-CoV-2 vaccination among recipients of CAR-T therapy and demonstrates that a 3-dose primary series followed by a fourth booster increases antibody levels. However, the relatively low magnitude of titers and low percentage of nonresponders demonstrates that further studies are needed to optimize vaccination timing and determine predictors of vaccine response in this population.

Influence of resuspension volume on dry sampling devices taken for human papillomavirus testing: implications for self-sampling.

Optimization of technical parameters that influence the performance of human papillomavirus (HPV) testing on self-taken samples is important. Here, the authors assessed the impact of resuspension volume on the detection of HPV using four validated HPV assays. Two self-sampling devices, FLOQSwabs® and Evalyn® Brushes, were inoculated with dilutions of HPV-16-positive cell line, then resuspended in various volumes of ThinPrep. The influence of vortexing during resuspension was also assessed. At target concentrations around the assay cutoff, larger volumes led to decreased HPV detection. Interestingly, the effect(s) of vortexing differed by the self-sampling device. Resuspension in 5ml or less may maximize the detection of HPV sequences. Using a proxy of clinical material, the current observations underline the importance of optimizing preanalytical laboratory processes to support high-quality HPV testing of self-samples.

883. Stool Toxin Concentrations Are Higher in Children with Baseline Severe Disease, Severe Outcomes, and Recurrence

Abstract Background In adults with C. difficile infection (CDI), higher baseline stool concentrations of toxins A and B are associated with severe baseline disease, CDI-attributable severe outcomes, and recurrence. We evaluated whether stool toxin concentration predicts these presentations in children with CDI. Methods We performed a prospective cohort study from 2016-2019. Participants were inpatients ≤17 years old at two pediatric hospitals with diarrhea and positive C. difficile testing who received therapy. Patients were followed for 40 days after baseline stool sample for severe outcomes (intensive care unit admission, colectomy, or death, categorized as CDI primarily attributable, CDI contributed, or CDI not contributing) and recurrence (resolution followed by new diarrhea and re-initiation of therapy). Baseline stool toxin A &amp; B concentrations were measured using ultrasensitive single molecule array assay (cutoff for positive result = 20 pg/mL). Median baseline toxin concentrations were compared between groups using Wilcoxon tests. Results We enrolled 206 patients [median age 8.9 years (IQR, 4.7–13.2)]. Children with severe baseline disease by IDSA-SHEA criteria (n = 39) had higher median stool toxin A+B concentration than those without severe disease (n = 131) (2,912.6 vs. 500.5 pg/mL, P=0.05). Of the cohort, 40 (19%) had a severe outcome (4 primarily attributed to CDI, 19 with contribution from CDI, and 17 unrelated to CDI). Median toxin A+B concentration was non-significantly higher in children with a primarily-attributed severe outcome versus those without severe outcome (19,473 vs. 429.1 pg/mL, P=0.317) (Figure 1). Of 197 children with eligible data, recurrence occurred in 18 (9.1%); baseline toxin A+B concentration was significantly higher in patients with versus without recurrence (3,946.7 vs. 283.3 pg/mL, P=0.026) (Figure 2). Conclusion Higher stool toxin concentrations are present in children with baseline severe CDI, a CDI-attributable severe outcome, or recurrence compared with children without these presentations. Quantification of stool toxin concentration may be helpful in identifying severe CDI and predicting CDI outcomes, which could help guide decisions about clinical management. Disclosures Larry K. Kociolek, MD, MSCI, Merck: Grant/Research Support Timothy J. Savage, MD, MPH, MSc, UCB: Contract to Brigham and Women's Hospital Alice Banz, PhD, biomerieux: Simoa assays were performed by bioMerieux, and A.B. is an employee of bioMerieux Kevin W. Garey, PharmD, MS, Acurx: Grant/Research Support|cidara: Advisor/Consultant|cidara: Grant/Research Support|Paratek: Grant/Research Support|Seres Health: Grant/Research Support|Summit: Grant/Research Support Ciaran P. Kelly, n/a, Artugen: Advisor/Consultant|Facile Therapeutics: Advisor/Consultant|Ferring Pharma: Advisor/Consultant|Finch: Advisor/Consultant|Finch: Advisor/Consultant|First Light Biosciences: Advisor/Consultant|First Light Biosciences: Ownership Interest|Milky Way Biosciences: Advisor/Consultant|Milky Way Biosciences: Grant/Research Support|Pfizer: Advisor/Consultant|Seres Therapeutics: Advisor/Consultant|Summit Therapeutics: Advisor/Consultant.

1939. COVID-19 vaccine immunogenicity among CD19 receptor T-cell (CAR-T) therapy

Abstract Background Patients receiving CAR-T therapy may have impaired humoral responses to SARS-CoV-2 vaccinations due to their high net state of immunosuppression associated with the underlying disease, prior lines of therapy and CAR-T treatment associated hypogammaglobinemia. Comprehensive data on vaccine immunogenicity in this patient population are currently lacking. Methods A single-center retrospective study of adults receiving CD19 CAR-T therapy for non-Hodgkin’s lymphoma was conducted between 3/27/2018 – 8/31/2021. Patients received at least two doses of COVID-19 vaccinations with BNT162b2 (Pfizer, BioNTech), mRNA-1273 (Moderna), or 1 dose of Ad26.COV2.S (Janssen) and had SARS-CoV-2 anti-spike (S) levels measured at least one month after the last vaccine dose. We excluded patients who received COVID-19 monoclonal antibody therapy or immunoglobulin within 3 months of the index anti-S titer. Patients were followed from the time of the first COVID-19 vaccines through their index anti-S antibody result. Patients were censored on the first day of any additional antineoplastic therapy after disease relapse. Our primary endpoint was the percentage of patients who develop a positive anti-S response (assessed by anti-S assay cutoff of &amp;gt;0.8 U/mL, Roche assay). Results Twenty-five patients met eligibility. Median age was 65 years (range 41 – 78), and majority of patients were male (72%). The number of patients with a positive antibody response was 12 (48%). Median number of vaccines received was 3. 18 patients (72%) received Pfizer vaccines, 4 patients (16%) received Moderna, 2 patients (8%) received Moderna and Pfizer, and 1 patient (4%) received Janssen and Pfizer. Median anti-S titers among patients with a positive response was 111 U/mL (range 2.44 – 12500). Two patients (8%) had COVID-19, both with negative anti-S responses. Conclusion Our analysis shows that only 48% of patients who received CAR-T therapy developed a positive antibody response after at least two COVID-19 vaccine doses, with a low median titer among responders. This patient population is at higher risk for developing severe COVID-19 disease and likely remains vulnerable even after vaccination. Alternative approaches are needed to prevent COVID-19 and mitigate disease severity in patients undergoing CAR-T. Disclosures Nicolas C. Issa, MD, AiCuris: Grant/Research Support|Merck: Grant/Research Support Sarah P. Hammond, MD, F2G: Advisor/Consultant|F2G: Grant/Research Support|GSK: Grant/Research Support|Scynexis: Grant/Research Support Caron Jacobson, MD, MMSc, Abintus Bio: Advisor/Consultant|Bluebird Bio: Advisor/Consultant|BMS/Celgene: Advisor/Consultant|Daiichi-Sankyo: Advisor/Consultant|Epizyme: Advisor/Consultant|ImmPACT Bio: Advisor/Consultant|Instill Bio: Advisor/Consultant|Ipsen: Advisor/Consultant|Kite/Gilead: Advisor/Consultant|Kite/Gilead: Grant/Research Support|Lonza: Advisor/Consultant|Novartis: Advisor/Consultant|Pfizer: Grant/Research Support.

Utilization of the Abbott SARS-CoV-2 IgG II Quant Assay To Identify High-Titer Anti-SARS-CoV-2 Neutralizing Plasma against Wild-Type and Variant SARS-CoV-2 Viruses.

ABSTRACTThere is evidence that COVID-19 convalescent plasma may improve outcomes of patients with impaired immune systems; however, more clinical trials are required. Although we have previously used a 50% plaque reduction/neutralization titer (PRNT50) assay to qualify convalescent plasma for clinical trials and virus-like particle (VLP) assays to validate PRNT50 methodologies, these approaches are time-consuming and expensive. Here, we characterized the ability of the Abbott severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG II Quant assay to identify high- and low-titer plasma for wild-type and variant (Alpha, Beta, Gamma, and Delta) SARS-CoV-2 characterized by both VLP assays and PRNT50. Plasma specimens previously tested in wild-type, Alpha, Beta, Gamma, and Delta VLP neutralization assays were selected based on availability. Selected specimens were evaluated by the Abbott SARS-CoV-2 IgG II Quant assay [Abbott anti-Spike (S); Abbott, Chicago, IL], and values in units per milliliter were converted to binding antibody units (BAU) per milliliter. Sixty-three specimens were available for analysis. Abbott SARS-CoV-2 IgG II Quant assay values in BAU per milliliter were significantly different between high- and low-titer specimens for wild-type (Mann-Whitney U = 42, P < 0.0001), Alpha (Mann-Whitney U = 38, P < 0.0001), Beta (Mann-Whitney U = 29, P < 0.0001), Gamma (Mann-Whitney U = 0, P < 0.0001), and Delta (Mann-Whitney U = 42, P < 0.0001). A conservative approach using the highest 95% confidence interval (CI) values from wild-type and variant of concern (VOC) SARS-CoV-2 experiments would identify a potential Abbott SARS-CoV-2 IgG II Quant assay cutoff of ≥7.1 × 103 BAU/mL.IMPORTANCE The United States Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for the use of COVID-19 convalescent plasma (CCP) to treat hospitalized patients with COVID-19 in August 2020. However, by 4 February 2021, the FDA had revised the convalescent plasma EUA. This revision limited the authorization for high-titer COVID-19 convalescent plasma and restricted patient groups to hospitalized patients with COVID-19 early in their disease course or hospitalized patients with impaired humoral immunity. Traditionally our group utilized 50% plaque reduction/neutralization titer (PRNT50) assays to qualify CCP in Canada. Since that time, the Abbott SARS-CoV-2 IgG II Quant assay (Abbott, Chicago IL) was developed for the qualitative and quantitative determination of IgG against the SARS-CoV-2. Here, we characterized the ability of the Abbott SARS-CoV-2 IgG II Quant assay to identify high- and low-titer plasma for wild-type and variant (Alpha, Beta, Gamma, and Delta) SARS-CoV-2.

Serology versus nucleic acid amplification to diagnose acute hepatitis E, United Kingdom, 2014–18

ObjectivesDiagnosing hepatitis E infection usually involves specific IgM testing, but sensitivity/specificity concerns mean many guidelines and practices include confirmatory tests. We studied whether additional information confirmatory tests provide justifies their use. MethodsWe examined 9131 records of anti-hepatitis E IgM assays, 7615 of IgG assays, and 1726 of RT-PCR assays from our regional laboratory, spanning October 2014–October 2018. We paired 495 IgM assay results with a RT-PCR result. We examined whether IgM results predicted PCR results, reviewed discrepant pairs, and investigated the correlation between IgG and PCR results in patients with strongly reactive IgM assays. ResultsAnti-hepatitis E IgM titres are bimodal. A high cut-off value optimises prediction of RNA detectability. 7/404 low-IgM samples had detectable RNA, 6 from immunosuppressed patients. 26/91 high-IgM samples did not have detectable RNA. In high-IgM samples, RNA detectability was not associated with IgG titre (one-tailed Mann-Whitney U test, p = 0.14). ConclusionsIn immunocompetent patients, tests beyond IgM seldom add clinically useful information. In patients with immunocompromise, IgM and RNA could contribute information. Additional tests’ extra costs/intervention delays cannot be justified. IgM assay cut-offs should reflect titres’ bimodal distribution, with values standardised using international units.

Testing for Lupus Anticoagulants.

Lupus anticoagulant (LA) is one of the three criteria antiphospholipid antibodies (aPLs) employed in classification, and by default diagnosis, of antiphospholipid syndrome (APS). Detection of LA is not via calibrated assays but is based on functional behavior of the antibodies in a medley of coagulation assays. A prolonged clotting time in a screening test is followed by demonstration of phospholipid dependence and inhibitory properties in confirmatory and mixing tests, respectively, which are modifications of the parent screening test. Complications arise because no single screening test is sensitive to every LA, and no test is specific for LA, because they are prone to interference by other causes of elevated clotting times. Several screening tests are available but the pairing of dilute Russell's viper venom time (dRVVT) with LA-sensitive activated partial thromboplastin time (aPTT) is widely used and recommended because it is proven to have good detection rates. Nonetheless, judicious use of other assays can improve diagnostic performance, such as dilute prothrombin time to find LA unreactive with dRVVT and aPTT, and the recently validated Taipan snake venom time with ecarin time confirmatory test that are unaffected by vitamin K antagonist and direct factor Xa inhibitor anticoagulation. Expert body guidelines and their updates have improved harmonization of laboratory practices, although some issues continue to attract debate, such as the place of mixing tests in the medley hierarchy, and areas of data manipulation such as assay cut-offs and ratio generation. This article reviews current practices and challenges in the laboratory detection of LA.

186-OR: Novel Autoantibodies to Deamidated IA2 Extracellular Domain in Type 1 Diabetes

Background: Insulinoma antigen-2 (IA2) is a major self-antigen in T1D with post translational modification (PTM) of the extracellular domain (IA2ec) by deamidation eliciting T cell responses in T1D patients. Yet the autoantibodies (Abs) to these IA2ec PTM epitopes in T1D has not been studied. Methods: IA2ec protein containing all the four T-cell responsive deamidated Q&amp;gt;E epitopes (aa 198-216, 467-482, 523-536 and 545-562) was produced. Sera from 269 new onset T1D children (median age= 12.6yrs) and 3age-matched healthy children were tested for Abs to IA2ecQ&amp;gt;E and wide type (WT) IA2ec with radiobinding assay (RBA) . Results: The assay cut-offs were set at the 99th percentile for both IA2ecQ&amp;gt;E Ab and IA2ecWT Ab. In T1D children, positivity for IA2ecQ&amp;gt;E Ab was 60.6% (163/269) vs. IA2ecWT Ab 19.3% (52/269, P&amp;lt;0.0001) . In two subgroups of T1D patients who were positive (n=157) or negative (n=112) for current Abs to IA2 intra-cellular domain (IA2ic) , the positivity of IA2ecQ&amp;gt;E Ab were significantly higher than IA2ecWT Ab (Fig) , 58% (91/157) vs. 15.3% (24/157, P&amp;lt;0.0001) and 64.3% (72/112) vs. 25% (28/112, p&amp;lt;0.0001) . However, the positivity of IA2ec Ab did not differ between the patients with or without IA2ic Ab. Conclusion: IA2ecQ&amp;gt;E Ab was detected in a large proportion of T1D patients, independent of the prototypical IA2ic Ab. IA2ecQ&amp;gt;E Ab is potentially a new novel predictive and diagnostic biomarker for T1D. Disclosure X.Jia: None. D.Miao: None. C.Zhang: None. A.W.Michels: Employee; ImmunoMolecular Therapeutics, Stock/Shareholder; ImmunoMolecular Therapeutics. L.Yu: None. J.M.Wenzlau: None. Funding National Institutes of Health (R21- AI153665) National Institutes of Health (RDK032083) Diabetes Research Center (DRC) grant P30 DK116073

The development and validation of a high-capacity serological assay for celiac disease

BackgroundThe aim of the present study was to develop and clinically validate a high-throughput assay for serum IgA and IgG antibodies against transglutaminase-2 (TG2) and to determine appropriate assay cut-offs for large-scale population screening for celiac disease. MethodAn automated method was developed using dual label time-resolved fluorometry on the AutoDELFIA platform. Individuals (n = 1920) from the general population were screened. Subjects with serum anti-TG2 concentrations above a preliminary cut-off (>0.3 mg*/L anti-TG2 IgA or >0.5 mg*/L anti-TG2 IgG) were offered endoscopic examination and biopsy. A diagnosis of celiac disease was given if villous atrophy (Marsh grade 3) was found. ResultsThe assay had a limit of quantification of 0.25 mg*/L (anti-TG2 IgA) and 0.60 mg*/L (anti-TG2 IgG) with imprecision (CV) < 16% and <18% respectively. A total of 66 individuals were above the preliminary cut-off, and 56 underwent endoscopy. Of these, 26 were diagnosed with celiac disease. Sixty-eight percent of subjects with anti-TG2 IgA ≥ 0.7 mg*/L or anti-TG2 IgG ≥ 1.0 mg*/L had biopsy-proven celiac disease, and utilization of these higher cut-offs identified 96% of biopsy-positive patients. At the time of endoscopy, all individuals with anti-TG2 IgA > 2.0 mg*/L had celiac disease, and this cut-off identified 88% of newly diagnosed celiac patients. Eight percent (2/26) of the newly diagnosed patients had primarily anti-TG2 IgG. ConclusionsIn this study we developed and clinically validated a robust and automated assay suitable for celiac disease screening in the general population.