Clinical sensitivity and specificity of a high-throughput microfluidic nano-immunoassay combined with capillary blood microsampling for the identification of anti-SARS-CoV-2 Spike IgG serostatus

Abstract

Objectives: We evaluate the diagnostic performance of dried blood microsampling combined with a high-throughput microfluidic nano-immunoassay (NIA) for the identification of anti-SARS-CoV-2 Spike IgG seropositivity.

Methods: We conducted a serological study among 192 individuals with documented prior SARS-CoV-2 infection and 44 SARS-CoV-2 negative individuals. Participants with prior SARS-CoV-2 infection had a long interval of 11 months since their qRT-PCR positive test. Serum was obtained after venipuncture and tested with an automated electrochemiluminescence anti-SARS-CoV-2 S total Ig reference assay, a commercial ELISA anti-S1 IgG assay, and the index test NIA. In addition, 109 participants from the positive cohort and 44 participants from the negative cohort participated in capillary blood collection using three microsampling devices: Mitra, repurposed glucose test strips, and HemaXis. Samples were dried, shipped by regular mail, extracted, and measured with NIA.

Results: Using serum samples, we achieve a clinical sensitivity of 98·33% and specificity of 97·62% on NIA, affirming the high performance of NIA in participants 11 months post infection. Combining microsampling with NIA, we obtain a clinical sensitivity of 95·05% using Mitra, 61·11% using glucose test strips, 83·16% using HemaXis, and 91·49% for HemaXis after automated extraction, without any drop in specificity.

Discussion: High sensitivity and specificity was demonstrated when testing micro-volume capillary dried blood samples using NIA, which is expected to facilitate its use in large-scale studies using home-based sampling or samples collected in the field.

Fig 1. Study participants.

Serum validation) 249 participants were included in this study and were invited for a venipuncture. Serum obtained was used for testing on the reference standard Roche Elecsys anti-SARS-CoV-2 S total Ig assay, Euroimmun anti-S1 IgG assay, and on the microfluidic nano-immunoassay (NIA) index test. DBS microsampling) On the same day, the participants were invited to perform a capillary blood collection using three different microsampling devices: Mitra, repurposed glucose test strips, and HemaXis. The samples were shipped by regular mail in a dried state and extracted upon reception. For filter cards collected with the HemaXis device, a subset of samples was extracted by flowthrough desorption using an automated instrument (DBS-A, Gerstel AG).

Fig 2. Sample validation with Roche Elecsys, Euroimmun ELISA, and NIA.

A) Roche Elecsys anti-SARS- CoV-2 S total Ig assay. 5 SARS-CoV-2 negative samples (purple color) with signal above the positivity threshold (>0.8) were excluded from the study. B) Euroimmun ELISA anti-S1 IgG assay. The thresholds for positivity (>1.1) and negativity (<0.8) classification as provided by the manufacturer are indicated. C) ROC curve with calculated AUC for Euroimmun ELISA. D) NIA measuring inactivated serum samples at 1:8 dilution. E) ROC curve with calculated AUC for NIA. F) Correlation between two different NIA chips (N = 198). Samples with saturating signals (>55’000 RFU) were removed from the plot and correlation calculation.

Fig 3. Performance of microfluidic nano-immmunoassay (NIA) test using dried blood microsamples.

Signal intensities with positivity threshold (dotted line), ROC curve, and chip-to-chip correlation for the microfluidic nano-immunoassay testing of dried blood samples collected on Mitra (A-C), (glucose) test strips (D-F), or HemaXis microsampling devices (G-I), and after automated extraction by flow-through desorption of dried blood spots (DBS-A) (J-L).

Fig 4. NIA serum and microsampling comparison.

Comparison of serum at 1:8 dilution and samples obtained by microsampling tested on NIA. Samples from SARS-CoV-2 negative and SARS-CoV-2 positive participants are labeled red and blue, respectively. Comparison of serum samples with A) Mitra, B) (glucose) test strips, C) HemaXis, and D) HemaXis with automated extraction (DBS-A). Serum samples with signal above 59000 RFU were removed from the plot and linear regression. Comparison of Mitra with E) glucose test strips, F) HemaXis, and G) HemaXis with automated extraction (DBS-A). In panels B) and F), samples with negative signals were excluded. The samples from SARS-CoV-2 negative participants were not included in the linear regression.

Fig 5. Sensitivity and specificity comparison.

Sensitivity and specificity with 95% confidence interval are shown. Euroimmun ELISA and NIA index test were performed on serum obtained by venipuncture. Mitra, (glucose) test strips, and HemaXis correspond to the device used for capillary blood collection after fingerprick, and DBS-A to automated extraction of samples obtained on HemaXis. Capillary blood samples were dried, shipped, and extracted before testing on the NIA assay. A dotted line at 95% sensitivity or specificity is shown for visual aid.