JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

滑液分析以识别骨关节炎

Published: October 20th, 2022

DOI:

10.3791/64351

1Rheumatology Unit, Department of Medicine-DIMED, University of Padova

透射、偏振和补偿光学显微镜下的滑液分析用于通过简单的步骤评估样品的炎症或非炎症性质。在骨关节炎中,检测钙晶体和识别更严重的骨关节炎亚群特别有用。

滑液 (SF) 分析对于诊断骨关节炎 (OA) 很重要。宏观和微观特征,包括总白细胞和分类白细胞 (WBC) 计数,有助于确定 SF 的非炎症性质,这是 OA 的标志。在OA患者中,SF样本中的WBC通常不超过每微升2000个细胞,并且炎症细胞(如中性粒细胞)的百分比非常低或不存在。钙晶体常见于从OA患者收集的SF。尽管它们在OA发病机制中的作用尚不清楚,但它们与轻度炎症过程和更严重的疾病进展有关。最近,钙晶体在OA的早期和晚期都有描述,表明它们可能在诊断OA的不同临床亚群和药物治疗中发挥重要作用。OA中SF分析的总体目标是双重的:确定SF的非炎症程度和突出钙晶体的存在。

骨关节炎 (OA) 是一种复杂的多因素慢性关节疾病,估计 15 岁及以上受试者的全球综合患病率为 16%,40 岁及以上受试者的汇总患病率为 23%。由于人口老龄化和肥胖和代谢综合征等危险因素的增加,OA的发病率预计将增加2。

与OA相关的主要问题包括在早期阶段诊断疾病的困难,以及目前可用的治疗方法仅限于疼痛管理和症状性慢效药物(SYSADOA),如糖胺聚糖。骨关节炎的诊断基于临床症状和影像学表现。然而,两项评估之间缺乏相关性可能导致OA诊断和治疗开始延迟数年2。OA的特征是退行性过程和低度炎症,可以通过滑液(SF)分析 轻松 研究。SF是一种富含透明质酸的粘性血浆透析液,可润滑关节空间,为软骨提供营养和氧气,并清除代谢废物。SF还充当减震器,从而在应力和应变期间保护接头3

SF分析是一种简单可靠的方法,在对有肌肉骨骼症状和关节积液的患者进行初始评估时必须始终进行3。美国风湿病学会、英国风湿病学会等的建议包括 SF 分析,包括风湿性疾病诊断测试,必须主要在评估急性单关节炎时进行4

Log in or to access full content. Learn more about your institution’s access to JoVE content here

本协议符合帕多瓦大学医院伦理委员会的指导方针。SF是在患者同意下从接受治疗性关节穿刺术的患者的膝关节中收集的,以便在他们首次到诊所就诊时或对关节炎发作做出反应。该手术的禁忌症是:凝血功能障碍,抗凝药物,皮肤病变,皮炎或关节上的蜂窝织炎。所有SF样品均已去识别化。

1.滑液准备

  1. 将肿胀关节15 关节穿刺术期间收集?.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

受OA影响的大关节经常肿胀并产生大量的SF,这些SF通过关节穿刺术 15。关节穿刺术后立即评估的SF的宏观特征主要包括数量,颜色,清晰度和粘度17。尽管它们的特异性低,但它们提供了关于炎症程度的初步数据。颜色取决于SF细胞和细胞外基质大分子的碎裂程度。从OA患者收集的SF为淡黄色,这种阴影通常与非炎症样本有关,而深黄色与炎性积液有?.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

在OA中,SF分析通过简单的步骤帮助确定疾病特征:白细胞总数和差异白细胞计数以及搜索晶体,包括CPP和BCP。此外,MSU 晶体的检测可能突出重要的合并症。

尽管成本低且执行简单,但由于分析师经验不足,测试的敏感性和结果的可靠性可能会受到影响 - 主要是因为它与晶体鉴定有关。分析师的培训和经验对于区分CPP和MSU晶体的形状和双折射以及将其形状与其双折射程度相匹.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

作者希望感谢Leonardo Punzi教授在滑液分析领域的宝贵指导以及帕多瓦大学医院的支持。

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
Alizarin red SMerckA5533For BCP crystal search
Burker chamberMerckBR718905For total white blood cell count
Cover glassesMerckC7931For microscopic examination 
EDTA tubesBD368861For SF collection 
Glass slides MerckS8902For crystal search
Lambda filter (compensator)Any Refer to microscope companyFor crystal identification 
Malassez-Potain pipetteArtiglass54830000For dilution of synovial fluid
Methylene blue solutionMerck3978For total white blood cell count
Polarized microscope Leica, Nikon, othersDepending on the model and companyFor complete synovial fluid analysis
Polarizing lensAny Refer to microscope companyFor crystal identification 
Testsimplet Waldeck14386Supravital staining for cell differentiation

  1. Cui, A., et al. Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies. EClinicalMedicine. 29, 100587 (2020).
  2. Hunter, D. J., Bierma-Zeinstra, S. Osteoarthritis. Lancet. 393 (10182), 1745-1759 (2019).
  3. Mandell, B. F. . Synovial Fluid Analysis and the Evaluation of Patients with Arthritis. , (2022).
  4. Schmerling, R. H. Guidelines for the initial evaluation of the adult patient with acute musculoskeletal symptoms. Arthritis & Rheumatism. 39 (1), 1-8 (1996).
  5. Coakley, G., et al. RCGP and BSAC guidelines for management of the hot swollen joint in adults. Rheumatology. 45 (8), 1039-1041 (2006).
  6. Frallonardo, P., et al. Basic calcium phosphate and pyrophosphate crystals in early and late osteoarthritis: relationship with clinical indices and inflammation. Clinical Rheumatology. 37 (10), 2847-2853 (2018).
  7. Nalbant, S., et al. Synovial fluid features and their relations to osteoarthritis severity: new findings from sequential studies. Osteoarthritis Cartilage. 11 (1), 50-54 (2003).
  8. Fuerst, M., et al. Calcification of articular cartilage in human osteoarthritis. Arthritis & Rheumatism. 60 (9), 2694-2703 (2009).
  9. Campillo-Gimenez, L., et al. Inflammatory potential of four different phases of calcium pyrophosphate relies on NF-κB activation and MAPK pathways. Frontiers in Immunology. 9, 2248 (2018).
  10. Pazár, B. Basic calcium phosphate crystals induce monocyte/macrophage IL-1β secretion through the NLRP3 inflammasome in vitro. The Journal of Immunology. 186 (4), 2495-2502 (2011).
  11. Martín, M. J. A., et al. Automated cell count in body fluids: a review. Advances in Laboratory Medicine. 2, 149-161 (2021).
  12. Seghezzi, , et al. Optimization of cellular analysis of synovial fluids by optical microscopy and automated count using the Sysmex XN body fluid mode. Clinica Chimica Acta. 462, 41-48 (2016).
  13. Reginato, A. J., Maldonado, I., Reginato, A. M., Falasca, G. F., O'Connor, C. R. Supravital staining of synovial fluid with Testsimplets. Diagnostic Cytopathology. 8 (2), 147-152 (1992).
  14. Lumbreras, B., et al. Analysis for crystals in synovial fluid: training of the analysts results in high consistency. Annals of the Rheumatic Diseases. 64 (4), 612-615 (2005).
  15. Tieng, A., Franchin, G. Knee arthrocentesis in adults. Journal of Visualized Experiments. , e63135 (2022).
  16. Punzi, L., Oliviero, F. Arthrocentesis and synovial fluid analysis in clinical practice: value of sonography in difficult cases. Annals of the New York Academy of Sciences. 1154 (1), 152-158 (2009).
  17. Schumacher, H. R., Reginato, A. J. . Atlas of Synovial Fluid Analysis and Crystal Identification. , (1991).
  18. Mopin, A., Driss, V., Brinster, C. A. Detailed protocol for characterizing the murine C1498 cell line and its associated leukemia mouse model. Journal of Visualized Experiments. (116), e54270 (2016).
  19. Oliviero, F., Punzi, L. Basics of Polarized Light Microscopy. Synovial Fluid Analysis and the Evaluation of Patients with Arthritis. , 79-90 (2022).
  20. Paul, H., Reginato, A. J., Schumacher, H. R. Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis & Rheumatism. 26 (2), 191-200 (1983).
  21. Favero, M., et al. Synovial fluid fetuin-A levels in patients affected by osteoarthritis with or without evidence of calcium crystals. Rheumatology. 58 (4), 729-730 (2019).
  22. Frallonardo, P., et al. Detection of calcium crystals in knee osteoarthritis synovial fluid: a comparison between polarized light and scanning electron microscopy. Journal of Clinical Rheumatology. 22 (7), 369-371 (2016).
  23. Peffers, M. J., Smagul, A., Anderson, J. R. Proteomic analysis of synovial fluid: current and potential uses to improve clinical outcomes. Expert Review of Proteomics. 16 (4), 287-302 (2019).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved