Harris Annalie J, Toth Eszter, Treat Jennifer, Zerbe Christa S, Holland Steven M, Rosen Lindsey B
Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, Maryland, USA.
Open Forum Infect Dis. 2025 Aug 8;12(8):ofaf478. doi: 10.1093/ofid/ofaf478. eCollection 2025 Aug.
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.
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.
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.
Dried blood and plasma spots are simple, inexpensive, durable, and allow functional anti-IFNγ autoantibodies to be easily recovered.
高滴度、中和性抗细胞因子自身抗体（ACAA）针对干扰素γ（IFNγ）是成人免疫缺陷的一个新出现的病因，检测可指导临床管理。目前，ACAA的检测是在血液或血浆上进行，这对时间敏感，获取成本相对较高，且必须从偏远地区用专门容器运送。我们采用了一种更简便的方法，将血液或血浆收集到纸卡上制成干血斑（DBS），这种方法简单、便宜，且不需要医疗机构或特殊邮寄。
通过静脉穿刺或手指采血获得的全血被吸印到滤纸上制成DBS，然后从中洗脱样品。对4组样本进行分析：有或无中和活性的抗IFNγ自身抗体患者、患有其他ACAA的疾病对照以及健康对照（共50例）。然后使用基于颗粒的方法对洗脱液和配对的血浆样本进行抗体筛查。通过流式细胞术测定中和活性，该方法检测健康对照单核细胞中细胞因子刺激的直接下游效应。
在17例先前诊断为抗IFNγ自身抗体的患者的DBS洗脱液中检测到自身抗体。以健康对照单核细胞中IFNγ诱导的STAT-1磷酸化作为读数，从DBS洗脱液中回收的具有中和性抗IFNγ自身抗体的自身抗体仍保持完全中和活性。保存为DBS的抗IFNγ自身抗体在4°C、室温或40°C下储存一周后，对于下游功能测试仍保持稳定。
干血斑和干血浆斑简单、便宜、耐用，且能轻松回收具有功能的抗IFNγ自身抗体。
