Dried blood spot improves global access to aquaporin-4-IgG testing for neuromyelitis optica

Abstract

Objective: This study aimed to evaluate the diagnostic accuracy of dried blood spot (DBS) compared with conventional serum Aquaporin-4-IgG (AQP4-IgG) testing.

Methods: Prospective multicenter diagnostic study was conducted between April 2018 and October 2023 across medical centers in the United States, Uganda, and the Republic of Guinea. Neuromyelitis optica spectrum disorder (NMOSD) patients and controls collected blood on filter paper cards along with concurrent serum samples. These samples underwent analysis using flow cytometric live-cell-based assays (CBA) and enzyme-linked immunosorbent assay (ELISA) to determine AQP4 serostatus. The accuracy of AQP4-IgG detection between DBS and serum (gold standard) was compared.

Results: Among 150 participants (47 cases, 103 controls), there was a strong correlation between DBS and serum samples (Spearman's correlation coefficient of 0.82). The AUC was 0.97 (95% CI: 0.92-0.99). AQP4-IgG detection through DBS showed 87.0% sensitivity (95% CI: 0.74-0.95) and 100% specificity (95% CI: 0.96-1.00) using CBA, and 65.2% sensitivity (95% CI: 0.43-0.84) and 95.2% specificity (95% CI: 0.76-0.99) using ELISA. Serum ELISA demonstrated 69.6% sensitivity (95% CI: 0.47-0.87) and 98.4% specificity (95% CI: 0.91-0.99). The stability of DBS in detecting AQP4-IgG persisted over 24 months for most cases.

Interpretation: The DBS represents a viable alternative for detecting AQP4-IgG in resource-limited settings to diagnose NMOSD, offering high sensitivity and specificity comparable to serum testing. Moreover, DBS has low shipping costs, is easy to administer, and is suitable for point-of-care testing.

Figure 1

Workflow of the study: patients suspected of NMOSD (remote participants) in the United States received Whatman® 903 Protein Saver Cards by mail. They used these cards for blood spot collection and then returned their samples using pre‐paid business reply envelopes through regular post (non‐temperature controlled). DBS and concurrent serum samples from Uganda were collected on the same day, the serum samples were stored at −80°C, while the DBS samples were stored at room temperature (RT) until testing. Both DBS samples and simultaneous serum samples were mailed to the United States using a FedEx Express Pak. In the Republic of Guinea, a clinician collected DBS samples, stored them at RT, and mailed them using regular post upon return to the United States. All these collected samples were sent to the Mayo Clinic Neuroimmunology Laboratory for analysis. The DBS samples were extracted and subjected to AQP4‐IgG testing using a live‐cell‐based flow cytometer with FACS and ELISA, alongside the evaluation of serum samples. In the future, there is potential for developing a kit that employs an immunodot assay for detecting AQP4‐IgG. Notably, Patient 1 and Patient 2 demonstrated positive results, while Patient 3 tested negative. Abbreviations: NMOSD, neuromyelitis optica spectrum disorder; DBS, dried blood spot; FACS, fluorescence‐activated cell sorting; ELISA, enzyme‐linked immunosorbent assay; AF647, Alexafluor 647; AcGFP, Aequorea coerulescens green fluorescent protein; GFP, green fluorescent protein.

Figure 2

Whatman® 903 Protein Saver Cards.

Figure 3

Scatter plot demonstrates a correlation of 0.82 between AQP4‐IBI from DBS and serum, displayed in both full scale (A) and a zoomed‐in scale based on the subset where both DBS and serum IBI values are ≤2, revealing a correlation of 0.17 (B). Additionally, it includes subgroup analysis focusing on patients from the United States and Uganda. In the plot, NMOSD cases are indicated in red, while negative controls are represented in black. Reference lines at 2 indicate the positive cutoff results. The overall performance of DBS in AQP4‐IgG detection, indicated by the AUC of the ROC curve, was 0.97 (95% CI: 0.92–0.99) (C). Abbreviations: ROC curve, receiver operating characteristic curve; AQP4‐IBI, aquaporin‐4‐IgG binding index; DBS, dried blood spot; NMOSD, neuromyelitis optica spectrum disorder.

Figure 4

Stability over time of DBS in the detection of AQP4‐IgG was assessed in seropositive cases by evaluating AQP4‐IBI values approximately at 6, 12, and 24 months utilizing the same DBS. This revealed the persistence of a positive status throughout the 24‐month period for most cases, except for one patient with an initially low IBI (initial IBI of 2.69), who transitioned from a positive to a negative status. Abbreviations: DBS, dried blood spot; IBI, IgG binding index.