感谢重庆市科研院所绩效激励指导项目（jxyn-2021-1-15 和 jxyn-2021-2-6）的财政支持。

临床试验已获得重庆市中医院审批委员会批准（编号 2022-KY-31）。典型病例涉及一名诊断为肺孢子菌肺炎的患者，并获得了患者的知情同意。
1. ROSE 的设备及材料要求
<ol><li>设备：有关本协议中使用的设备，请参阅 材料表 文件。 注意：必须使用专用的细胞学显微镜和图形成像系统（图 1A）。</li>
	<li>材料准备（见 材料表）。<ol><li>准备无菌细胞培养载玻片（具有很强的细胞粘附性）、吸水纸、无粉乳胶手套、一次性 2.5-5 mL 注射器针头，并将全套 Diff Quik （DQ） 染色溶液放入带有密封盖的玻璃染色罐中，以便于处理（图 1）。</li>
	</ol></li>
</ol>2. M-ROSE 工作流程
<ol><li>细胞学载玻片的制备 注意：制备玻片的方法有很多，以下是几种常用的制备方法。<ol><li>滚动幻灯片 注意：在此过程中确保组织样本的损失最小。<ol><li>使用容量为 2.5-5 mL 的一次性注射器针头提取组织颗粒（图 2A）。</li>
			<li>在无菌细胞学载玻片上，从染色端的内侧到外侧三分之一部分（表现出强烈细胞附着的末端）铺展一个直径约 1 厘米且厚度适中的圆形区域。</li>
		</ol></li>
		<li>画笔幻灯片 注：该方法适用于使用常规细胞刷、抗污染细胞刷或超细细胞刷获得的标本，以及痰液和粘性体液等半液体标本。<ol><li>伸出刷头，将其涂抹在无菌细胞学载玻片的远端三分之一处（载玻片具有很强的细胞粘附特性），以获得约 2 cm x 1 cm 的矩形区域，厚度适中。</li>
		</ol></li>
		<li>喷雾载玻片<ol><li>将穿刺针放置在具有强大细胞粘附的无菌细胞学载玻片染色端的三分之一处。</li>
			<li>对穿刺针的尖端施加气压，从内侧向外插入，形成直径约 1 cm 的中等厚度的圆形区域。</li>
		</ol></li>
	</ol></li>
	<li>细胞学玻片染色 注：世界卫生组织 （WHO） 建议使用 Diff 染料5 对 ROSE 细胞学玻片进行快速染色。Diff 染料可以重复使用，但不能多次重复。如有沉淀物，使用后应过滤。染色太深可以用甲醇或酒精适当脱色，最好不再染色。如果染色太深或太浅，应调整染色时间或工作液浓度;pH值对染色有一定影响，玻片应清洁无酸碱污染。Diff A 溶液和 Diff B 溶液均具有挥发性，使用后应密封保存。<ol><li>将载玻片浸入 Diff A 溶液中 10-30 秒（图 2B），然后在 PBS 染料浴中冲洗以去除多余的 Diff A 溶液并轻轻甩掉任何剩余的缓冲液（图 2C）。</li>
		<li>接下来，将载玻片浸入 Diff B 溶液中 20-40 秒（图 2E）。</li>
		<li>最后，在水染色槽中清洁载玻片（图 2F），用吸水纸吸干并擦拭载玻片的残留液体（图 2D），然后完成染色。 注：M-ROSE 能够直接观察病原微生物，并有助于评估呼吸道标本质量，能够区分感染和非感染以及感染和污染，这对评估感染严重程度和预后具有指导意义。讨论部分将涵盖这方面，因为它不涉及任何实验程序。</li>
	</ol></li>
</ol>

使用 M-ROSE 加强肺部感染治疗

<ol>
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L., Herth, F. J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+interventional+pulmonology.">Progress in interventional pulmonology.</a> Respiration. 95 (5), 287-288 (2018).</li><li>Hsia, D., Musani, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventional+pulmonology.">Interventional pulmonology.</a> Med Clin North Am. 95 (6), 1095-1114 (2011).</li><li>Moore, A. J., Mercer, R. M., Musani, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+interventional+pulmonology.">Advances in interventional pulmonology.</a> Clin Chest Med. 39 (1), 271-280 (2018).</li><li>Majid, A., Fernandez-Bussy, S., Folch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventional+pulmonology+and+solitary+pulmonary+nodule.">Interventional pulmonology and solitary pulmonary nodule.</a> Arch Bronconeumol. 54 (10), 497-498 (2018).</li><li>Ali, M. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endobronchial+ultrasound-guided+transbronchial+needle+aspiration:+performance+of+biomedical+scientists+on+rapid+on-site+evaluation+and+preliminary+diagnosis.">Endobronchial ultrasound-guided transbronchial needle aspiration: performance of biomedical scientists on rapid on-site evaluation and preliminary diagnosis.</a> Cytopathology. 27 (5), 344-350 (2016).</li><li>Pearson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+on-site+evaluation+of+fine-needle+aspiration+by+non-cytopathologists:+A+systematic+review+and+meta-analysis+of+diagnostic+accuracy+studies+for+adequacy+assessment.">Rapid on-site evaluation of fine-needle aspiration by non-cytopathologists: A systematic review and meta-analysis of diagnostic accuracy studies for adequacy assessment.</a> Acta Cytol. 62 (4), 244-252 (2018).</li><li>Arimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiopsy+with+endobronchial+ultrasonography+using+a+guide+sheath+for+peripheral+pulmonary+lesions+and+DNA+analysis+by+next+generation+sequencing+and+rapid+on-site+evaluation.">Cryobiopsy with endobronchial ultrasonography using a guide sheath for peripheral pulmonary lesions and DNA analysis by next generation sequencing and rapid on-site evaluation.</a> Respir Investig. 57 (2), 150-156 (2019).</li><li>Gianella, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+rapid+on-site+cytologic+evaluation+during+endobronchial+ultrasound-guided+transbronchial+needle+aspiration+in+malignant+and+nonmalignant+disease.">Utility of rapid on-site cytologic evaluation during endobronchial ultrasound-guided transbronchial needle aspiration in malignant and nonmalignant disease.</a> Acta Cytol. 62 (5-6), 380-385 (2018).</li><li>Natella, V., Cozzolino, I., Sosa Fernandez, L. V., Vigliar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+nodes+fine+needle+cytology+in+the+diagnosis+of+infectious+diseases:+clinical+settings.">Lymph nodes fine needle cytology in the diagnosis of infectious diseases: clinical settings.</a> Infez Med. 20, 12-15 (2012).</li><li>Baughman, R. P., Spencer, R. E., Kleykamp, B. O., Rashkin, M. C., Douthit, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator+associated+pneumonia:+quality+of+nonbronchoscopic+bronchoalveolar+lavage+sample+affects+diagnostic+yield.">Ventilator associated pneumonia: quality of nonbronchoscopic bronchoalveolar lavage sample affects diagnostic yield.</a> Eur Respir J. 16 (6), 1152-1157 (2000).</li><li>Petrone, M. 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作者报告没有利益冲突。

介入肺病学是现代呼吸疾病的一个有价值的分支;特别是，它已广泛用于肺部疾病的诊断 8,9。近年来，由于肺部恶性肿瘤患病率的增加、下呼吸道发生的耐药病原体感染增加以及对莫名其妙和危重呼吸系统疾病的诊断要求要求，诊断介入肺病学一直在蓬勃发展 10,11,12.介入诊断效率取?...

快速现场评估技术 （ROSE） 是肺病手术领域采用的一种高效方法。它支持实时采样和诊断干预，便于即时细胞学分析1。这种创新方法包括将组织标本的一部分印记到载玻片上，同时保持其完整性。ROSE 的主要优势之一在于它能够通过专门的显微镜技术促进对临床信息的及时解释。这包括细胞形态分析、分类、定量、成分比例测定、排列评估、相关性分析、背景评估和异物鉴定。通过将所有这些数据与患者的临床信息相结合，ROSE 在评估采样充分性和指导实时介入程序和技术方面发挥着关键作用2。
作为 ROSE 技术的子公司，M-ROSE 技术主要侧重于获取目标病灶的微生物背景，而不是区分良性和恶性细胞 3,4。一方面，M-ROSE 能够对曲霉菌、隐球菌、肺孢子菌和念珠菌5 等病原体进行显微镜鉴定。另一方面，它在评估呼吸道标本质量、区分感染和非感染性疾病、区分感染和污染以及评估感染严重程度和预后方面具有重要的指导意义 6,7。例如，在呼吸道标本中，表现出相同形态的细菌与浸润的炎症细胞共存表明存在感染;相反，存在多种形态不同的细菌并伴有上皮细胞表明污染。全面分析临床信息和预测结果的能力使 M-ROSE 成为肺部疾病手术中的宝贵工具。
总之，ROSE 是一种重要的细胞学载体，可显着提高诊断肺部疾病的效率和准确性。其多方面功能通过实时采样和诊断干预技术确保及时干预和促进准确诊断，有助于改善患者预后。然而，重要的是要注意，这些进步依赖于获得合格的样品。在此，我们提出了一个标准化的 M-ROSE 方案，包括载玻片制备、染色技术和解释指南。该方案为临床医生建立准确的评估和治疗计划提供了宝贵的参考，同时有助于对目标标本的后续处理做出决策。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cytological microscope</td>
			<td>Olympus Corporation</td>
			<td>CX43</td>
			<td></td>
		</tr>
		<tr>
			<td>Diff Quik (DQ) staining solutions</td>
			<td>Besso Biotechnology Co. LTD</td>
			<td>G1541</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 2.5-5 mL syringe needles</td>
			<td>Shandong Zhu Pharmaceutical Group</td>
			<td>20183150304</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder-free latex gloves</td>
			<td>Henan Yadu Industrial Co., LTD</td>
			<td>20182140728</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell culture slides</td>
			<td>Jinan Preret industry and trade Co., LTD</td>
			<td>7101</td>
			<td></td>
		</tr>
	</tbody>
</table>

microbiological rapid on-site evaluation for pulmonary infectious diseases

对于表现不明原因的肺部感染患者，迅速开始经验性抗感染治疗至关重要。尽管影像学检查在临床实践中相对简单，但缺乏特异性通常需要额外的耗时检查，例如痰培养、支气管肺泡灌洗液培养或基因测序，以准确识别疾病的潜在病因。此外，经验性抗感染治疗的有限疗效可能导致抗生素滥用。在快速现场评估 （ROSE） 玻片上解释微生物背景的最新进展使临床医生能够通过支气管镜检查（例如，肺泡灌洗、粘膜刷洗、组织夹）及时获取样本，便于床边染色和解释，从而提供必要的微生物背景信息。因此，这为制定有针对性的抗感染治疗和个体化药物治疗计划奠定了基础。通过更好地了解哪些病原体实时引起感染，医生可以避免不必要的广谱抗生素导致抗生素耐药性。在呼吸内科或重症监护病房内建立快速、标准化的 M-ROSE 工作流程将极大地帮助医生为患者制定准确的治疗策略，具有重要的临床意义。

The technique of rapid on-site evaluation (ROSE) is a highly efficient method employed in the field of pulmonary disease procedures. It enables real-time sampling and diagnostic intervention, facilitating immediate cytological analysis1. This innovative approach involves imprinting a portion of the tissue specimen onto a slide while preserving its integrity. One of the key advantages of ROSE lies in its capacity to facilitate prompt interpretation of clinical information through specialized microscopy techniques. This encompasses the analysis of cell morphology, classification, quantification, determination of constituent ratios, assessment of arrangement, correlation analysis, evaluation of background, and identification of foreign objects. By integrating all this data with the patient's clinical information, ROSE plays a pivotal role in evaluating sampling adequacy and guiding real-time interventional procedures and techniques2.
As a subsidiary of ROSE technology, M-ROSE technology primarily focuses on acquiring the microbiological background of target lesions rather than discerning between benign and malignant cells3,4. On one hand, M-ROSE enables the microscopic identification of pathogens such as Aspergillus, Cryptococcus, Pneumocystis, and Candida5. On the other hand, it holds significant guiding implications in assessing respiratory specimen quality, distinguishing infectious from non-infectious diseases, discriminating infection from contamination, as well as evaluating infection severity and prognosis6,7. For instance, within a respiratory specimen, the coexistence of bacteria exhibiting identical morphology alongside infiltrating inflammatory cells indicates an infection; conversely, the presence of multiple morphologically diverse bacteria accompanied by epithelial cells suggests contamination. The capacity to comprehensively analyze clinical information and predict outcomes renders M-ROSE an invaluable tool in pulmonary disease procedures.
In conclusion, ROSE serves as a crucial cytological carrier that significantly enhances the efficiency and precision of diagnosing lung diseases. Its multifaceted capabilities contribute to improved patient outcomes by ensuring timely intervention and facilitating accurate diagnosis through real-time sampling and diagnostic intervention techniques. However, it is important to note that these advancements rely on obtaining qualified samples. Herein, we present a standardized M-ROSE protocol encompassing slide preparation, staining techniques, and interpretation guidelines. This protocol serves as an invaluable reference for clinicians to establish accurate evaluation and treatment plans while facilitating decision-making regarding subsequent handling of target specimens.

作者聚焦：使用 M-ROSE 实时推进病原体检测和疾病评估

肺部感染性疾病的微生物学快速现场评估

Our intent is to introduce a real time rapid assessment method, M-ROSE to assist the physicians for formulating accurate treatment strategies for patient with pulmonary infections. Rapid cell staining technology and a high definition microscopic imaging system are currently used to advance research in the field. The number of clinical studies on M-ROSE is relatively limited.The biggest challenge is interpretation of M-ROSE results, which requires personnel, and trained personnel as it is difficult to visually identify pathogens and differentiate between infection and contamination. With a better understanding of pathogens causing infections in real time, physicians can avoid unnecessary broad spectrum antibiotics contributing to antibiotic resistance and as a treatment of pulmonary infections. M-ROSE is real time and highly efficient.M-ROSE enables the microscopic identification of pathogens such as aspergillus, cryptococcus, numerous and candida. It holds significant guiding implications in sizing respiratory basic quality, distinguishing infections from non-infectious disease, discriminating, infection from contamination, as well as evaluating infection, severity, and prognose.

在一个典型病例中，一名 63 岁的男性因咳嗽、发热和胸痛到医院就诊。患者既往诊断为肾病综合征，以蛋白尿和水肿为特征，长期接受泼尼松和他克莫司治疗。初步实验室检查结果显示，白细胞计数为 10.49 x 109/L，中性粒细胞计数为 8.87 x 109/L，全血 C 反应蛋白和 （1-3）-β-D-葡聚糖浓度为 155.81 mg/L 和 249.7 pg/mL。胸部计算机断层扫描 （CT） 扫描显示磨玻璃样混浊，位于弥漫性、双?...

Read this Scientific Article Protocol about Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases at JoVE.com

这里的方案展示了一种快速和标准化的微生物快速现场评估 （M-ROSE） 工作流程，包括三个步骤：载玻片制作、染色和解释。该协议将帮助医生快速做出临床决策。

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging ...

我们的目的是引入一种实时快速评估方法 M-ROSE，以帮助医生为肺部感染患者制定准确的治疗策略。快速细胞染色技术和高清显微成像系统目前用于推进该领域的研究。关于 M-ROSE 的临床研究数量相对有限。最大的挑战是 M-ROSE 结果的解释，这需要人员和训练有素的人员，因为很难目视识别病原体并区分感染和污染。通过更好地了解实时引起感染的病原体，医生可以避免使用不必要的广谱抗生素，这些抗生素会导致抗生素耐药性并用于治疗肺部感染。M-ROSE 是实时且高效的。M-ROSE 能够对病原体进行显微镜鉴定，例如曲霉菌、隐球菌、许多和念珠菌。它在确定呼吸基本质量、区分感染与非感染性疾病、区分感染与污染以及评估感染、严重程度和预后方面具有重要的指导意义。

microbiological-rapid-on-site-evaluation-for-pulmonary-infectious

Research

JoVE Journal

Medicine

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

254 次观看.  Chongqing Hospital of Traditional Chinese Medicine. 我们的目的是引入一种实时快速评估方法 M-ROSE，以帮助医生为肺部感染患者制定准确的治疗策略。快速细胞染色技术和高清显微成像系统目前用于推进该领域的研究。关于 M-ROSE 的临床研究数量相对有限。最大的挑战是 M-ROSE 结果的解释，这需要人员和训练有素的人员，因为很难目视识别病原体并区分感染和污染。通过更好地了解实时引起感染的病原体，医生可以避免?...

视频: 作者聚焦：使用 M-ROSE 实时推进病原体检测和疾病评估

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging acquisition is relatively straightforward in clinical practice, its lack of specificity often necessitates additional time-consuming tests such as sputum culture, bronchoalveolar-lavage fluid culture, or genetic sequencing to identify the underlying etiology of the disease accurately. Moreover, the limited efficacy of empirical anti-infective treatment may contribute to antibiotic misuse. Recent advancements in interpreting microbial background on rapid on-site evaluation (ROSE) slides have enabled clinicians to promptly obtain samples through bronchoscopy (e.g., alveolar lavage, mucosal brushing, tissue clamp), facilitating bedside staining and interpretation that provides essential microbial background information. Consequently, this establishes a foundation for developing targeted anti-infection treatment and individualized drug therapy plans. With a better understanding of which pathogens are causing infections in real-time, physicians can avoid unnecessary broad-spectrum antibiotics contributing to antibiotic resistance. Establishing a rapid and standardized M-ROSE workflow within respiratory medicine departments or intensive care units will greatly assist physicians in formulating accurate treatment strategies for patients, which holds significant clinical implications.

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.