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 a dried blood spot (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 a DBS from which samples were eluted. Samples from four 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 for IFNγ-induced STAT-1 phosphorylation in healthy control monocytes. 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. Conclusion Dried blood and plasma spots are simple, inexpensive, durable, and allow functional anti-IFNγ autoantibodies to be easily recovered.