通过测量免疫反应，预测自身免疫性疾病活动性的变化
"我们需要的分析不仅要能充分节省有限的样本量，还要让我们能够快速有效地检测出许多标志物。事实证明，Luminex 和 Simple Plex 分析是符合此要求的理想之选，让我们可以获得其他平台无法提供的准确度和重现性。"
- Dr. Melissa Munroe, 俄克拉荷马医学研究基金会 (OMRF) 研究员
系统性红斑狼疮 (SLE) 是一种自身免疫性疾病，可导致人体免疫系统攻击自身组织和细胞，引起广泛炎症且可能会损害受累器官。尽管治疗方法不断改善，但许多患者经常经历疾病活动性稳定一段时间后出现病情恶化和急性发作。急性发作的严重程度从轻度或中度到需要住院治疗的重要医学事件不等1。
Melissa Munroe 博士（医学博士）是俄克拉荷马医学研究基金会 (OMRF) 的一名研究员（教授）。OMRF 是一个非营利性生物研究机构，专注于基础研究、转化研究和临床研究工作，以确定潜在的致病机制，并在多个核心领域（包括癌症、自身免疫性疾病、衰老和心血管疾病）制定更优的疾病管理策略。Munroe 在 OMRF 的早期工作使她能够运用专业知识识别新的生物标志物，并使用高等数学建模预测狼疮疾病活动性的变化，并因此担任 Progentec Diagnostics, Inc. 的首席科研学者。
Profiling Immune Mediators in Lupus Patients
The increased risk of adverse outcomes related to lupus flare, coupled with the observation that immune system alterations precede clinical changes in disease activity, has led to a growing interest in finding ways to better assess immune mediators and predict episodes of heightened activity. The ability to predict impending flare would allow clinicians to make preemptive adjustments in the use of steroid-sparing, immune-modulating treatments to help prevent clinical worsening. For those patients with predicted stable disease, there is a potential opportunity to draw down immune-modulating treatments that carry significant side effects and contribute to organ damage.
While recent studies support the idea that immune mediator profiles are altered prior to changes in disease activity, a deeper analysis of key biomarkers is needed to better understand the mechanisms underpinning immune pathway dysregulation2. In a recent study of lupus patients with increased disease activity, Munroe and her team found that changes in the balance of inflammatory and regulatory mediators are detectable several weeks prior to clinical flare and are highly predictive of impending flare occurrence.
In their initial studies,(3,4) researchers examined a panel of 52 soluble mediators and found significant alterations in at least 34 of the 52 mediators, with increased levels of innate and adaptive cytokines, as well as IFN-associated chemokines and soluble TNF superfamily members. Conversely, circulating levels of regulatory mediators, including IL-10 and active TGF-β, were lower in SLE patients at risk of impending clinical disease flare.
“Our findings confirmed the idea that pro-inflammatory mediators are notably elevated, while regulatory mediators are notably decreased, prior to disease flare,” Munroe said. “The challenge is that the number and type of immune pathway alterations have been shown to vary widely in association with disease flare—both between lupus patients and even within the same patients over time.”
Improving Autoimmune Disease Flare-up Prediction with Accurate, Reproducible Results
Although traditional biomarkers, such as anti-dsDNA and complement, have been used to try and predict lupus flare, these biomarkers are affected in only a subset of SLE patients and are not necessarily the earliest immune system components altered prior to imminent clinical disease flare. Analysis involving a broader panel of immune pathway informed soluble mediators is needed to better understand their association with disease activity and imminent flare.
To gain deeper insight into these associations, Munroe and her team initially examined a broad panel of analytes across innate, adaptive, and effector immune mediators in a retrospective study of SLE patients prior to imminent disease flare compared to a comparable period of stable disease activity.(3,4) As part of their initial biomarker analysis, researchers relied on BLyS Quantikine™ ELISAs (considered the gold standard in lupus research) and Luminex® assay resources from Bio-Techne to screen a panel of 50 different biomarkers.
These studies confirmed the alteration of immune mediators prior to imminent disease flare and yielded a number of potential biomarkers for additional evaluation in prospectively collected samples.
“Once we began to see some common data points across patient demographics, we knew we were going to need highly accurate and consistent analytical techniques that would help establish the framework we needed for more longitudinal comparisons over time,” Munroe said.
As part of their new partnership with Progentec Diagnostics, Inc. resulting from these initial studies, researchers selected a subset of these markers for further evaluation using Simple Plex™ assays running on the automated Ella™ platform5. With the goal of translating the initial findings to a clinically actionable lab assay, a primary concern for researchers was finding immunoassays that could provide a high level of data accuracy and reproducibility while minimizing the potential for variability across assay lots, users, and testing sites.
“One of the things that attracted us to Bio-Techne has been their diligence in maintaining high standards for data accuracy and reproducibility across their analytical platforms and assay offerings," Munroe said.
Precise Immune Mediator Analysis with Next-Generation Techniques
While individual markers are unlikely to be universally associated with impending flare development, recent research has shown that the overall balance between inflammatory and regulatory mediators, particularly when considered en masse, may correlate with future disease activity.
“One mediator that surprised us was osteopontin,” Munroe said. “It’s one of those markers we’re starting to see more frequently relative to disease activity, but we’ve had trouble finding an assay that can consistently detect it.”
That’s where the broad detection capabilities and superior sensitivity of the Simple Plex assays proved to be a major advantage.
“If you are trying to measure some of the type I interferons or interleukins that float around at a low level, you need an assay that is up to the task,” Munroe said. “We’ve been extremely happy with the precision and sensitivity of the Simple Plex assays and their ability to detect analytes at extremely low levels.”
Gaining an Edge in Autoimmune Disease Research with Analytical Speed and Efficiency
As is the case in many research projects, a major challenge in this effort was the limited quantity of human samples from patients and healthy volunteers, which required Munroe and her team to find ways to get more data points out of each assay run.
“We needed assays that were not only excellent at conserving our limited sample volumes but would allow us to test a lot of markers quickly and efficiently,” Munroe said. “The Luminex and Simple Plex assays proved to be the ideal choice in this case, giving us a level of accuracy and reproducibility that we’ve been unable to get with other platforms.”
The simple, load-and-go workflow of the Ella platform allowed researchers to substantially shorten the time between assay runs. With Ella, assay setup is quick and easy. Simply pipette diluted samples into the cartridge, start the software, and come back to fully analyzed results in 75 minutes.
“With the Simple Plex assays running on Ella, we’re able to test hundreds of samples without the need for standard control preparations that can add variability,” Munroe said. “The less we rely on manual steps, the more reproducible the results.”
Overall, Munroe found Ella’s simplicity and reproducibility to be the ideal fit for their demanding research needs. The speed in which fully analyzed data can be produced with Ella has allowed Munroe and her team to get more data in less time while ensuring long-term data consistency.
Equally important in this case was the ability to reliably correlate data across three assay platforms at each stage of analysis.
“Early on in our evaluation of Ella, we were able to see the data comparisons between the Quantikine ELISAs, the Luminex assays, and the Simple Plex assays,” Munroe said. “The ability to maintain this high level of data consistency and reproducibility between assays was truly impressive—and a critical element that our research team took note of right away.”
REFERENCES
- Division of Population Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, 2018
- M.E. Munroe, E.S. Vista, J.T. Merrill, J.M. Guthridge, V.C. Roberts, J.A. James, Pathways of impending disease flare in African American systemic lupus erythematosus patients, 2017.
- Munroe ME, Vista ES, Merrill JT, Guthridge JM, Roberts VC, James JA. Pathways of impending disease flare in African-American systemic lupus erythematosus patients. J Autoimmun. 2017;78:70-8.
- Munroe ME, Vista ES, Guthridge JM, Thompson LF, Merrill JT, James JA. Pro-inflammatory adaptive cytokines and shed tumor necrosis factor receptors are elevated preceding systemic lupus erythematosus disease flare. Arthritis & rheumatology. 2014;66(7):1888-99.
- Munroe ME, Blankenship D, DeFreese D, Purushothaman M, DeJager W, Macwana S, et al. A Flare Risk Index Informed by Select Immune Mediators in Systemic Lupus Erythematosus Arthritis & rheumatology. 2022; Revisions requested.