Detection of Anti-IFNγ Autoantibodies From Dried Blood Spots.

Background: High titer, neutralizing anti-cytokine autoantibodies (ACAA) to interferon gamma (IFNγ) are an emerging cause of adult-onset immunodeficiency, and detection can direct clinical management. Currently, the detection of ACAAs is performed on blood or plasma, which is time-sensitive, relatively expensive to obtain, and must be shipped from remote locations in specialized containers. We have adapted an easier method of collecting blood or plasma onto a paper card as dried blood spots (DBS), which is simple, inexpensive, and does not require a medical facility or special mailing. Methods: Whole blood, either from venipuncture or finger stick, was blotted onto filter paper to yield DBS from which samples were eluted. Samples from 4 groups were analyzed: patients with anti-IFNγ autoantibodies, either with or without neutralizing activity, disease controls with other ACAAs, and healthy controls (n= 50 total). Eluates and paired plasma samples were then screened for antibodies using a particle-based approach. Neutralizing activity was determined by flow cytometry, which examined immediate downstream effects of cytokine stimulation in healthy control monocytes. Results: Autoantibodies were demonstrated in DBS eluate from 17 previously diagnosed patients with anti-IFNγ autoantibodies. The autoantibodies recovered from DBS eluate with neutralizing anti-IFNγ autoantibodies remained fully neutralizing using IFNγ-induced STAT-1 phosphorylation in healthy control monocytes as a readout. Anti-IFNγ autoantibodies preserved as DBS were stable for downstream functional testing when stored at either 4°C, room temperature, or 40°C for a week. Conclusions: Dried blood and plasma spots are simple, inexpensive, durable, and allow functional anti-IFNγ autoantibodies to be easily recovered.