Abstract Background As the world exits the COVID-19 pandemic era, it is very important to assess population-level and individual seroprotectivity against multiple circulating and emerging SARS-CoV-2 strains, especially in vulnerable populations. Current serological measurements for neutralizing antibodies (NAbs) are either done using complex and time-intensive pseudovirus- or micro-neutralization assays or as a high throughput competitive ELISA assay. These assays, by design, cannot be multiplexed to measure NAb titers against multiple strains and suffer from a limited dynamic range. The Q-NAb IgG Tests, which rely on blocking the non-neutralizing antibodies (NNAbs) using a novel fusion protein, were developed and validated to quantitatively measure NAb titer against the SARS-CoV-2 strains used to develop the first two mRNA vaccines viz. ancestral and BA.4/5 strains. Methods The Q-NAb assay measures the NAb titer in a sample by first sequestering the NNAbs using the fusion protein (made by covalently coupling SARS-CoV-2 BA.4/5 RBD and h-ACE2 protein) thereby enriching the NAbs in the sample. Variant-specific NAbs are then detected using an indirect ELISA where the wells are coated with the variant RBD and yield a signal that is directly proportional to the concentration of NAb. Clinical samples from subjects that have either had a SARS-CoV-2 infection (N=27), taken the initial vaccine series (N=123) and/or the bivalent booster (N=151) and pre-pandemic samples (N=29) were commercially sourced. Q-NAb NAb titers were measured using the ancestral and BA.4/5 kits and were compared against corresponding MN50 values using both parametric and non-parametric models. Single-site precision, low-end sensitivity, dilutional linearity, and interference screening of Q-NAb IgG Kits were evaluated in accordance with appropriate CLSI guidelines. Traceability and total uncertainty of the Q-NAb IgG Kits (Ancestral and BA.4/5) to WHO International Standard 21/340 and 21/338 respectively was established using a calibration hierarchy adapted from ISO 17511:2020. Results Both the ancestral and BA.4/5 Q-NAb IgG Test Kits correlated with MN50 titers with a Spearman correlation coefficient of 0.90 and 0.89 respectively. The total uncertainty in traceability of calibration to WHO International standards 21/340 and 21/338 was found to be less than 10% for both tests. Single site precision across the assay range for BA.4/5 kit was found to be 6-10% with a limit of quantitation of 312 IU/mL. Assays exhibited dilutional linearity across their respective dynamic range and had no significant interference from common endogenous and exogenous substances. Conclusions We have successfully validated the novel Q-NAb IgG neutralization assay platform as individual assays for measuring neutralizing titers against ancestral and BA.4/5 SARS-CoV-2 strains. The assays have expanded dynamic range, excellent analytical performance and correlation to accepted gold-standard microneutralization assays. Both assays are calibrated and traceable to WHO international standards, thereby enabling the compilation and comparison of data from multiple clinical cohorts run across multiple laboratories. The design of the Q-NAb IgG Test kit makes it amenable to multiplexing (for example, on an MSD platform) to assess neutralization titers against multiple variants at the same time. Such a multiplexed platform can be used to assess vaccine efficacy and seroprotection against multiple variants including emerging variants.

Abstract Background Following vaccination against SARS-CoV-2 (SC2), the human immune system produces neutralizing antibodies (NAb) that block the virus from entering host cells. These NAb levels are highly predictive of protection against SC2 infection and progression to severe disease [1]. Unfortunately, individual humoral responses vary and NAb titers decay over time, making measurement of NAb important in timing vaccine boosters, especially for at-risk individuals. Microneutralization assays (MNA) can be used to measure vaccine effectiveness by quantifying virus-specific NAb titers. However, these assays have long turnarounds, require a BSL3 lab, and have high inter-lab variability [2]. The Q-NAb™ test was developed to quantitatively measure NAb levels and correlate with MNA [3]. Q-NAb is available as both an ELISA for central laboratories, and a multiplexed POC centrifugal disc assay that measures NAb, anti-Nucleocapsid protein (NP), and anti-Spike antibodies. Methods The heart of the Q-NAb test is a recombinant fusion protein (FP) that blocks anti-RBD non-neutralizing antibodies (NNAb). FP consists of human ACE2 fused to the Wuhan-RBD of the SC2 Spike. Designed to conceal the the NAb binding epitopes of RBD, FP serves as a specific depleting agent for NNAb. The pretreated sample is then incubated with immobilized Wuhan-RBD to obtain a NAb readout. Both Q-NAb formats were calibrated to WHO international standard 20/136 using a set of 7 calibrators and correlated to MNA results. NAb levels were determined using >200 clinical samples collected 2–12 weeks post monovalent or bivalent vaccine booster and compared across age groups (<60, 60–70, 70–80, 80+ years). Results Both Q-NAb Disc and ELISA show correlation to each other (n = 167, WLS, R2 = 0.85) and are traceable to international standards. Q-NAb also correlated with MN50: ELISA (n = 137, Spearman's ρ = 0.88) and Disc (n = 208, Spearman's ρ = 0.91). After receiving a bivalent booster, no difference in median NAb levels across age groups was observed (Kruskal-Wallis, P = 0.3). While 99.4% of samples had a positive anti-Spike, using a NAb cutoff of 1000 IU/mL derived from the literature [2, 4–5], we found the percentage of individuals below that cutoff increased with age (9.8% for <60 years, 20.4% for 60–70 years, 19.5% for 70–80 years, and 34.1% for 80+ years). In addition, 32% of samples had an anti-NP level indicating recent infection. Conclusion The Q-NAb test, available in both central lab and POC formats, measures NAb levels, correlates to MNA, and is traceable to international standards. Q-NAb was used to assess humoral responses following vaccine boosters in a clinical sample set. While virtually all subjects had anti-Spike levels, the percentage of individuals falling below a NAb cutoff of 1000 IU/mL ranged from 9.8% to 34.1%, depending on age. Additionally, about one-third of patients had anti-NP activity, indicating NAb levels resulted from a combination of vaccination and infection. This data shows Q-NAb is an easily accessible and more accurate tool to identify a subset of at-risk patients that anti-Spike antibody tests alone do not discriminate.

The ability to automate immunodiagnostics testing is critical for the efficiency of clinical laboratories. For automation of Infectious Disease testing, special design considerations must be made to ensure the integrity of the testing result while at the same time delivering productivity. The ADVIA Centaur was designed with the intent of performing a complete Infectious Disease panel including HBV, HCV, and HIV markers. Features such as disposable sample tips, sample and reagent delivery verification, and clog and clog detection have been incorporated in the design for assay robustness. Productivity features deliver best in class throughput performance for the Infectious Disease assays. Moreover, the underlying technology incorporating flexible assay protocols, universal solid phases and proprietary acridinium ester technology contribute to the assay design flexibility and analytical performance of the ADVIA Centaur immunoassay system.

Summary The study investigated the clinical usefulness of a new method to evaluate platelet activation and the variability of platelet response to anti-platelet therapy in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). Platelet activation was assessed in parallel by a new method for platelet density measurements (MPC, Mean Platelet Component Concentration), on the automated ADVIA 120 Hematology System and by the classic measurement of P-selectin (CD62P) expression, on a fluorescence flow cytometer. Patients received a loading dose of clopidogrel (300 mg; n = 29) or a bolus of abciximab (0.25 mg/kg; n = 15). Blood samples were collected before (baseline) and at different times after PTCA and antiplatelet drugs administration. Our data showed a close inverse correlation between the change in MPC and the CD62P fluorescence surface marker expression (r = 0.776, P<0.0001). Individual platelet activation determinations in patients receiving either clopidogrel or abciximab showed a variation in platelet activation as assayed by MPC and CD62P expression. Patients were characterized as having either high platelet activity upon admission and positive response to treatment or no detectable platelet activation before or after treatment. This study demonstrates the heterogeneity of platelet activation states in ACS patients undergoing coronary angioplasty. The present work also illustrates the potential use of the MPC parameter, generated on an automated hematology system, to define high risk patients and to monitor the variability of platelet response to anti-platelet therapies.

SummaryPlatelet activation is reported to correlate with acute coronary syndromes. A platelet analysis method on the ADVIA®120 Hematology System provides rapid analysis of platelet density, reported as mean platelet component (MPC) concentration, utilizes routine hematology specimens, requires no pre-treatment, and thirty seconds to generate results. Sub-populations of platelets separated by density gradients showed excellent correlation with the ADVIA 120 MPC parameter (r = 0.997). Platelet activation induced by thrombin treatment resulted in a shift of platelets into the lowest density fraction (d ≤ 1.068 g/mL) with a corresponding reduction in MPC from 24.7 to 20.6 g/dL, N = 4 subjects (p < 0.004). There was also excellent correlation between expression of CD62P measured by fluorescence flow cytometry and the ADVIA 120 Hematology System MPC values (r = 0.85). These results indicate that the ADVIA 120 MPC parameter may be a useful new test for assessing activated platelets and platelet density.

In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.

BACKGROUND Monitoring of platelet activation by the ADVIA 120 Hematology System requires an anticoagulant and protocol that ensures that platelets are sphered and their activation status is not altered artifactually in vitro. METHODS Blood from healthy controls was collected into tripotassium EDTA; citrate, theophylline, adenosine, and dipyridamole (CTAD); or a combination of both (E/C) and stored at ambient temperature or at 4 degrees C (E/C only) and then analyzed between 0 and 180 min later on the ADVIA 120. In addition, immunofluorescent flow cytometry was used to identify activated platelets and platelet-leukocyte aggregates. RESULTS In blood stored with all three anticoagulants, the platelet count changed little, but the mean platelet volume (MPV) at first decreased and then increased, whereas the mean platelet component (MPC; an indicator of activation) changed in a reciprocal manner. The changes in MPV and MPC, which reflect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature, irrespective of which anticoagulant was used, and between 60 and 180 min when blood anticoagulated with E/C was stored at 4 degrees C. In all anticoagulants, the percentages of platelets expressing CD62P and of leukocytes in platelet-leukocyte aggregates increased significantly (P <0.01) over 180 min at ambient temperature. Only minimal (<2%) increases occurred when blood with E/C was stored at 4 degrees C. CONCLUSIONS When determining platelet activation ex vivo on the ADVIA 120, blood should be collected into E/C, stored at 4 degrees C, and analyzed between 60 and 180 min later; these conditions ensure maximum platelet sphering without concurrent artifactual platelet activation.

Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood.