New protocol for kinetic assay seeding ability recovery "KASAR" from formalin-fixed paraffin-embedded tissues.
New protocol for kinetic assay seeding ability recovery "KASAR" from formalin-fixed paraffin-embedded tissues.
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动力学测定能力恢复的新方案从福尔马林固定的石蜡包裹的组织中恢复“ kasar”。
DOI：
10.3389/fmolb.2023.1087982
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发表时间：
2023
影响因子：
5
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实时震动诱导转换（RT-QuIC）α-突触核蛋白（aSyn）蛋白动力学接种测定对于检测包括帕金森病（PD）在内的各种突触核蛋白病中的病理性聚集体非常有用。该生物标志物测定依赖于新鲜冷冻组织来有效地接种和扩增aSyn聚集蛋白。随着福尔马林固定石蜡包埋（FFPE）组织的大量储存，利用动力学测定的力量来释放存档FFPE生物标本的诊断潜力至关重要。然而，在测定中福尔马林固定组织的扩增显著减少所带来的主要挑战表明福尔马林固定阻止了单体与样品种子的相互作用，并抑制了随后的蛋白质聚集。为了克服这一挑战，我们开发了一种动力学测定接种能力恢复（KASAR）方案，以保持组织和接种蛋白的完整性。为此，我们在组织切片的标准脱蜡后，将脑组织悬浮在由500 mM tris-HCl（pH 7.5）和0.02% SDS组成的缓冲液中，实施了一系列加热步骤。最初，来自七个人脑样本的样本，包括来自诊断患有路易体痴呆症（DLB）的患者的四个样本和来自没有DLB的健康对照的三个样本，在三种不同但临床常见的样本储存条件下与新鲜冷冻样本进行比较：福尔马林固定，FFPE和FFPE切片切割5 μm厚。KASAR方案能够在所有储存条件下恢复所有阳性样品的接种活性。接下来，对诊断为PD、偶发性路易体病（ILBD）或健康对照的患者的28份下颌下腺（SMG）FFPE样本进行了检测，设盲时结果重复率为93%。仅用几毫克的样品，该方案在福尔马林固定的组织中恢复了与新鲜冷冻组织相同的接种质量。展望未来，蛋白质聚集体动力学测定结合KASAR方案可用于更全面地了解和诊断神经退行性疾病。总体而言，我们的KASAR方案解锁并恢复福尔马林固定的石蜡包埋组织的接种能力，用于在动力学测定中扩增生物标志物蛋白质聚集体。
The real-time quaking-induced conversion (RT-QuIC) alpha-synuclein (aSyn) protein kinetic seeding assay has been very useful for detecting pathological aggregates in various synucleinopathies including Parkinson’s disease (PD). This biomarker assay relies on fresh frozen tissue to effectively seed and amplify aSyn aggregating protein. With vast repositories of formalin-fixed paraffin-embedded (FFPE) tissues, it is paramount to harness the power of kinetic assays to unlock the diagnostic potential of archived FFPE biospecimens. However, the major challenge posed by significantly reduced amplification of formalin-fixed tissues in the assay suggests that formalin fixation deterred monomer interaction with the sample seed and depressed subsequent protein aggregation. To overcome this challenge, we developed a kinetic assay seeding ability recovery (KASAR) protocol to maintain the integrity of the tissue and seeding protein. For this, we implemented a series of heating steps with the brain tissue suspended in a buffer composed of 500 mM tris-HCl (pH 7.5) and 0.02% SDS after the standard deparaffinization of the tissue sections. Initially, samples from seven human brain samples, including four samples from patients diagnosed with dementia with Lewy bodies (DLB) and three samples from healthy controls without DLB, were compared to fresh frozen samples under three different, but clinically common sample storage conditions: formalin-fixed, FFPE, and FFPE slices cut 5 µm thick. The KASAR protocol was able to recover seeding activity for all positive samples in all storage conditions. Next, 28 FFPE samples from the submandibular gland (SMG) of patients diagnosed with PD, incidental Lewy body disease (ILBD), or healthy controls were tested with 93% of results replicating when blinded. With samples of only a few milligrams, this protocol recovered the same quality of seeding in formalin-fixed tissue as fresh frozen tissue. Moving forward, protein aggregate kinetic assays, in conjunction with the KASAR protocol, can be used to understand and diagnose neurodegenerative diseases more comprehensively. Overall, our KASAR protocol unlocks and restores the seeding ability of formalin-fixed paraffin-embedded tissues for the amplification of biomarker protein aggregates in kinetic assays.