Name: interphase fluorescence in situ hybridization of bone marrow smears of multiple myeloma
Uploaded: 2022-04-15T14:00:00
Description: Watch this Scientific Journal Video about Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma at JoVE.com

该项目由WNLO 2018WNLOKF023创新基金支持。

本研究根据《赫尔辛基宣言》的原则进行，并经武汉大学中南医院伦理委员会批准（第2019065号）。标本采集自武汉大学中南医院血液科的一名MM患者。
1. BM涂片的准备
<ol><li>将第一个0.2 mL BM溶液放在干净的一次性载玻片上。</li>
	<li>通过快速去除BM，用锋利的尾巴将BM涂抹到均匀的厚度。然后，自然干燥。</li>
	<li>在室温下用1 mL Wright-Giemsa溶液覆盖BM薄膜约10秒。</...

<ol>
	<li>Sonneveld, 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=Treatment+of+multiple+myeloma+with+high-risk+cytogenetics:+a+consensus+of+the+International+Myeloma+Working+Group.">Treatment of multiple myeloma with high-risk cytogenetics: a consensus of the International Myeloma Working Group.</a> Blood. 127, 2955-2962 (2016).</li><li>Radbruch, A., 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=Competence+and+competition:+the+challenge+of+becoming+a+long-lived+plasma+cell.">Competence and competition: the challenge of becoming a long-lived plasma cell.</a> Nature Reviews Immunology. 6, 741-750 (2006).</li><li>Lai, J. 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=Improved+cytogenetics+in+multiple+myeloma:+a+study+of+151+patients+including+117+patients+at+diagnosis.">Improved cytogenetics in multiple myeloma: a study of 151 patients including 117 patients at diagnosis.</a> Blood. 85 (9), 2490-2497 (1995).</li><li>Hallek, M., Bergsagel, P. L., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+myeloma:+increasing+evidence+for+a+multistep+transformation+process.">Multiple myeloma: increasing evidence for a multistep transformation process.</a> Blood. 91, 3-21 (1998).</li><li>Munsh, N. C., 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=Consensus+recommendations+for+risk+stratification+in+multiple+myeloma:+report+of+the+International+Myeloma+Workshop+Consensus+Panel+2.">Consensus recommendations for risk stratification in multiple myeloma: report of the International Myeloma Workshop Consensus Panel 2.</a> Blood. 117, 4696-4700 (2011).</li><li>Gole, 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=cIg-FISH+On+Patients+with+Multiple+Myeloma+-+A+Modified+and+Simple+Technique+Easily+Incorporated+Into+Routine+Clinical+Service.">cIg-FISH On Patients with Multiple Myeloma - A Modified and Simple Technique Easily Incorporated Into Routine Clinical Service.</a> Blood. 120, 4792-4792 (2012).</li><li>Lee, S. H., Erber, W., Porwit, A., Tomonaga, M., Peterson, L. I. C. S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematology,+ICSH+guidelines+for+the+standardization+of+bone+marrow+specimens+and+reports.">Hematology, ICSH guidelines for the standardization of bone marrow specimens and reports.</a> International Journal of Laboratory Hematology. 30, 349-364 (2008).</li><li>&#352;tifter, S., 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=Combined+evaluation+of+bone+marrow+aspirate+and+biopsy+is+superior+in+the+prognosis+of+multiple+myeloma.">Combined evaluation of bone marrow aspirate and biopsy is superior in the prognosis of multiple myeloma.</a> Diagnostic Pathology. 5, 30 (2010).</li><li>Huegel, A., Coyle, L., McNeil, R., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+interphase+fluorescence+in+situ+hybridization+on+direct+hematological+bone+marrow+smears.">Evaluation of interphase fluorescence in situ hybridization on direct hematological bone marrow smears.</a> Pathology. 27, 86-90 (1995).</li><li>Jacobsson, B., Bernell, P., Arvidsson, I., Hast, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+morphology,+esterase+cytochemistry,+and+interphase+cytogenetics+of+peripheral+blood+and+bone+marrow+smears.">Classical morphology, esterase cytochemistry, and interphase cytogenetics of peripheral blood and bone marrow smears.</a> The Journal of Histochemistry and Cytochemistry. 44, 1303-1309 (1996).</li><li>Dunning, K., Safo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultimate+Wright-Giemsa+stain:+60+years+in+the+making.">The ultimate Wright-Giemsa stain: 60 years in the making.</a> Biotechnic &amp; Histochemistry. 86, 69-75 (2011).</li><li>Woronzoff-Dashkoff, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wright-giemsa+stain.+Secrets+revealed.">The wright-giemsa stain. Secrets revealed.</a> Clinics in Laboratory Medicine. 22, 15-23 (2002).</li><li>McPherson, R. A., Pincus, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Henry's+Clinical+diagnosis+and+Management+by+Laboratory+Methods+E-book.">Henry's Clinical diagnosis and Management by Laboratory Methods E-book.</a> Elsevier Health Sciences. , (2017).</li><li>Ross, F. M., 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=Report+from+the+European+Myeloma+Network+on+interphase+FISH+in+multiple+myeloma+and+related+disorders.">Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders.</a> Haematologica. 97, 1272-1277 (2012).</li></ol>

FISH在MM的遗传风险分层中的应用至关重要。FISH报告的关键部分不是所有有核细胞，而是通过用抗CD138磁珠分选或用细胞质免疫球蛋白轻链κ或λ标记特异性纯化或鉴定的克隆PC。PC分选或cIg-FISH后的相间FISH在MM的诊断中具有显着意义，因为BM中PC的比例相对较低。然而，这些方法存在一些缺点，包括工艺复杂，样品要求高，实验成本高。50台PC可以通过显微镜级游标卡尺快速定位。BM 涂片比抗凝剂 BM 标...

多发性骨髓瘤（MM）是一种恶性浆细胞（PC）疾病，具有很强的生物学异质性和临床疗效的个体差异，生存期从数月到数十年不等。细胞遗传学特征是MM的重要预后指标。基于遗传特征的风险分层系统和个体化治疗已成为MM1临床研究中备受关注的话题。在骨髓（BM） 荧光原位 杂交（FISH）面板中测试的PC的像差包括del 13q14（RB1），del 17p13（TP53），t（4;14）（IGH / FGFR3），t（11;14）（IGH / MYEOV），t（14;16）（IGH / MAF），t（14：20）（IGH / MAFB），1q21（CKS1B）增益/扩增和1p（CDKN2C）缺失。
在 MM 患者的初始评估中应包括标准的中期细胞遗传学。虽然传统的G带核型分析具有全染色体分析的优势，但该方法的低产量导致许多假阴性结果2。传统上，PC被认为在很大程度上无法分裂，因为它们是B淋巴细胞分化的终末期产物。这使得很难获得分割图像。通过长期培养（6 天）改进 MM 的细胞遗传学分析以及通过细胞因子刺激培养物可能是识别新诊断 MM 患者细胞遗传学异常的有希望的方法3。然而，即使将培养时间延长至6天，30%-50%的MM患者也准确报告了细胞遗传学异常4。此外，MM的特征在于复杂的细胞遗传学畸变，反映了其预后异质性。然而，传统染色体条带技术的分辨率较低，这很容易导致MM染色体异常的检测失误。
相间FISH，优选在CD138阳性磁珠基PC分选或用细胞质免疫球蛋白轻链κ/λ标记后，在MM5，6的分析中是必不可少的。强烈建议选择CD138阳性样品以优化肿瘤细胞的产量。然而，细胞遗传学畸变的准确定量需要至少 4 mL 抗凝 BM 进行 PC 分选。此外，CD138免疫磁珠分选与FISH或细胞质κ / λ轻链免疫球蛋白与FISH测试（cIg-FISH）的组合增加了许多实验成本并且非常耗时。
通常，PC比率首先通过染色的BM涂片或BM活检部分的形态学检查来评估7，8。最高质量的BM样本（第一个骨髓抽吸样本）用于形态学测试，而那些送去FISH或其他检测的标本通常是与外周血稀释比例高的二级抽吸物样本。
早在20世纪90年代，多项研究表明，BM涂片可以直接用于间质FISH检查，这已被证明是一种可靠且可重复的方法9。一项基于细胞形态学和酯酶细胞化学结合外周血FISH和BM涂片的优雅研究证实了其对阐明粒细胞和淋巴细胞白血病患者染色体异常的巨大临床意义10。
本文为提高MM患者相间FISH检测的成功率提供了一种新颖的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>automatic FISH machine</td>
			<td>Leica&#160; Corporation</td>
			<td>S500-24</td>
			<td>FINAL Assy Thermobrite 240V</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abbott Molecular Inc.</td>
			<td>06J49-001</td>
			<td>DAPI Counterstain</td>
		</tr>
		<tr>
			<td>FISH Analysis Software</td>
			<td>IMSTAR&#160; Corporation</td>
			<td>IMSTAR</td>
			<td>FISH Analysis Software</td>
		</tr>
		<tr>
			<td>FISH Probe</td>
			<td>Abbott Molecular Inc.</td>
			<td>05N56-020</td>
			<td>Vysis Locus Specifc Identifer TP53 / CEP 17 FISH Probe Kit</td>
		</tr>
		<tr>
			<td>Fixed volume pipette</td>
			<td>Eppendorf China Ltd.</td>
			<td>M33768H</td>
			<td>10&#160; microliter</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Olympus Corporation</td>
			<td>BX53</td>
			<td>Forward Fluorescence Microscope</td>
		</tr>
		<tr>
			<td>Karyotype Analysis Software</td>
			<td>IMSTAR&#160; Corporation</td>
			<td>IMSTAR</td>
			<td>Karyotype Analysis Software</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus&#160; Corporation</td>
			<td>BX41</td>
			<td>Forward Light Microscope</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abbott Molecular Inc.</td>
			<td>07J05-001</td>
			<td>NP-40</td>
		</tr>
		<tr>
			<td>Plastic staining dyeing rack</td>
			<td>Guangzhou Kaixiu Trading Co., Ltd.</td>
			<td>RSJ-501</td>
			<td>&#160;24 slides</td>
		</tr>
		<tr>
			<td>Plastic staining dyeing tank</td>
			<td>Guangzhou Kaixiu Trading Co., Ltd.</td>
			<td>RSJ-516</td>
			<td>&#160;24 slides</td>
		</tr>
		<tr>
			<td>Rubber Cement</td>
			<td>Marabu&#160;GmbH &#38; Co. KG</td>
			<td>FixoGum&#160;</td>
			<td>Rubber Cement</td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>Abbott Molecular Inc.</td>
			<td>02J10-032</td>
			<td>20&#215;SSC</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Shanghai Boxun Medical Bio-Instrument Co., Ltd.</td>
			<td>DK-8D</td>
			<td>Multiple Temperature Water bath</td>
		</tr>
	</tbody>
</table>

interphase fluorescence in situ hybridization of bone marrow smears of multiple myeloma

荧光 原位 杂交（FISH）检测是多发性骨髓瘤（MM）遗传风险分层中不可或缺的方法，MM是最常见的血液系统恶性肿瘤之一。MM的识别特征是骨髓中积聚的恶性浆细胞。MM的FISH报告主要关注纯化或鉴定的克隆浆细胞，而不是所有有核细胞，通过用抗CD138磁珠分选或用细胞质免疫球蛋白轻链κ或λ标记。骨髓间期核通常从新鲜的骨髓细胞中获得。然而，浆细胞标本的令人满意的富集需要大量的新鲜肝素抗凝骨髓，这在骨髓提取困难或骨髓干抽取的情况下无法获得。在本文中，我们建立了一种新颖的方法，以提高对染色或未染色的骨髓涂片进行FISH检测的成功率。骨髓涂片比抗凝骨髓标本更容易获得。

在新诊断的MM患者的初始形态学评估中，发现BM涂片中15%的PC具有比正常PC更大，更暗的细胞核以及更多的细胞质（图2A）。通过免疫表型技术检测到的第一管肝素抗凝BM仅显示2.3%的单克隆异常PC.CD138免疫磁珠与FISH或cIg-FISH技术联合分选对于获得准确的FISH结果至关重要。但是，如果吸气BM是干式水龙头，则吸气BM是有限的。使用BM涂片来测试相间FISH。通过荧光显微镜载物台游标?...

Watch this Scientific Journal Video about Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma at JoVE.com

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

在这里，我们提出了一种方案，用于提高相间荧光 原位 杂交检测对多发性骨髓瘤患者骨髓涂片的成功率。

多发性骨髓瘤骨髓涂片的相间荧光 原位 杂交

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

将称为FISH的荧光原位杂交应用于多发性骨髓瘤的遗传风险分层至关重要。FISH报告的关键部分不是所有有核细胞，而是通过用抗CD138磁珠分选或用细胞质kappa或Lambda轻链免疫球蛋白标记称为cIg FISH的FISH测试特异性纯化或鉴定的克隆浆细胞。浆细胞分选或cIg FISH后的相间FISH在多发性骨髓瘤的诊断中具有显着意义，因为浆细胞在骨髓中的比例相对较低。然而，这些方法存在一些缺点，包括工艺复杂，样品要求高和实验成本高。浆细胞可以快速定位在骨髓涂片中。基于此，我们使用骨髓涂片FISH来检测多发性骨髓瘤细胞。本研究根据《赫尔辛基宣言》的原则进行，并经武汉大学中南医院伦理委员会批准。标本采集自中国武汉大学中南医院血液科的一名多发性骨髓瘤患者。将前0.2毫升骨髓溶液放在干净的一次性载玻片上，通过快速取出骨髓然后将其自然干燥，将骨髓涂片铺成均匀的厚度和锋利的尾巴。在室温下用一毫升Wright-Giemsa溶液覆盖骨髓膜约10秒。向载玻片中加入一毫升磷酸盐缓冲液，需要小心防止染料溶液干燥或从载玻片中流出。轻轻混合染料溶液，并在室温下保持15分钟。用清水冲洗载玻片，并在室温下在空气中干燥。使用正向光显微镜检测恶性浆细胞，浆细胞应分散良好。现在我们在现场看到的是相当分散的浆细胞。这是一种典型的双核核浆细胞。用固定溶液覆盖杂交区域10分钟，以变色并固定浆细胞。用去离子水冲洗载玻片，并在室温下在空气中干燥。在水浴中将含有两倍SSC缓冲液的罐子预热至56摄氏度。将制备的骨髓涂片在预热的两次SSC缓冲液中洗涤10分钟，然后在室温下浸入去离子水中。在乙醇梯度70%85%和100%乙醇中对涂片进行脱水一分钟。将涂抹物在空气中干燥10分钟。首先将17号染色体探针混合物的着丝粒上的TP53添加到杂交区域，并用盖子覆盖，轻轻地按压细尖镊子以除去下面的气泡。用橡胶水泥密封盖玻片的所有侧面，以确保良好的密封性。在橡胶水泥凝固后，将载玻片置于自动FISH机上，在78摄氏度下进行变性五分钟，然后在37摄氏度下杂交过夜。从鱼机上拿起幻灯片。使用细尖镊子轻轻去除橡胶水泥。将载玻片浸入室温下两次SSC中约一到两分钟，以洗掉盖玻片。在水浴中以68摄氏度的预热缓冲液洗涤载玻片两分钟。在37摄氏度的水浴中用两次SSC洗涤载玻片一分钟。在黑暗中设置，在10微升DAPI下彻底干燥10分钟到杂交区域，盖上盖子。使用荧光显微镜上设置的合适滤光片查看杂交载玻片，使用单色蓝色荧光团滤光片观察细胞核的荧光强度。在DAPI过滤器集下拍摄细胞核图像。CEP17用绿色荧光标记。使用光谱绿色荧光团滤光片观察CEP17的位置。在绿色滤光片集下拍摄CEP17信号图像。TP53基因用橙色荧光标记。使用光谱橙色荧光团滤光片观察TP53，在集合下拍摄TP53信号图像，将这三幅图像合并为一个整体，并检查数值异常。在正常的相间细胞中，在具有蓝色荧光的细胞核内观察到两个橙色和两个绿色信号，表明一对正常的染色体17。结果显示了多发性骨髓瘤患者骨髓的形态和遗传学。图A显示，骨髓涂片中15%的浆细胞被发现具有比正常更大，更暗和核以及大量的细胞质。图B显示了由骨髓涂片中有核浆细胞代表的灰度图像。图C显示浆细胞一个相间细胞核中的四个橙色和四个绿色信号，表明17号染色体的四个拷贝定位于一个细胞核中。图D显示，该患者的异常染色体核电图证实一个细胞核中有两对17号染色体。骨髓涂片比抗凝剂骨髓标本更容易获得，因为获得抗凝骨髓需要复杂的程序来获得细胞核。因此，我们建立了一种令人信服的骨髓涂片FISH方法，用于检测多发性骨髓瘤细胞。

Research

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Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

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Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Femoral Bone Marrow Aspiration in Live Mice

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

白消安作为骨髓抑制剂在小鼠生成稳定的高水平骨髓嵌合体

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em&gt;原位</em&gt;杂交

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

多发性骨髓瘤骨髓涂片的相间荧光 原位 杂交

多发性骨髓瘤骨髓涂片的相间荧光原位杂交