Name: fish for pre-implantation genetic diagnosis
Uploaded: 2011-02-23T18:30:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about FISH for Pre-implantation Genetic Diagnosis at JoVE.com

1。从活检卵裂球的细胞核裂解和传播<ol><li>蔓延细胞（0.2％Tween20 0.01 M盐酸，pH值2.0）的细胞裂解液应提前做好准备，24小时，并储存在-20 ° C准备100毫升和过滤器20毫升到30 mL无菌普遍使用无菌20 ml注射器和注射器过滤器容器。分送1毫升分装成10-15 1.7 mL无菌离心管，关闭和标签管前冻结。建议是使用两个不同批次，新批和前一批，其中已通过测试，可用于新批次是不能令人满意的的。 </li><li>除霜裂解液在室温下活检前30分钟。出于实用的目的，工作温度将室温和手的温度之间。 </li><li>分数胺涂层幻灯片用金刚石笔和标签前的幻灯片与案件的数量，独特的幻灯片编号，和活检日期（如Genetix）的底面上的小圆圈（直径约5毫米）。使用一个单独的幻灯片的数字顺序为每个卵裂球，并与胚胎数量的标签。幻灯片应标有一个硬如4H铅笔，和“抹杀”与乳胶手套，以消除任何石墨粉尘。 </li><li>广场内圆一个小体积的裂解缓冲液。 </li><li>活检卵裂球转移到细胞裂解液。如果需要添加的圆圈内的细胞裂解液，直到开始裂解的细胞裂解完全和细胞质分散的缓冲区干燥之前。 </li><li>观察细胞核，以确保它仍然是圆内，并不会丢失，如果细胞不具有细胞核或有多个细胞核，活检另一个细胞。 </li><li>保留在室温下风干的幻灯片。 </li></ol> 在一个单个卵裂球核原位杂交2。 <ol><li>准备在无菌蒸馏水，乙醇（70，90和100％）。 </li><li>打开并设置热块（如Hybaid Omnislide或Vysis Hybrite）至75 ° C。 </li><li>除霜探头，旋涡，离心机。所用试剂应见证并查实为探针混合物，然后才作出的正确。移液器，作为制造商，以弥补在0.65 mL无菌的离心管和涡和使用离心机前探针混合物指定的卷。探针混合物的总体积应足以让每核2μL进行测试，并四舍五入到最近的10μL，允许一个安全边际。 </li><li>预洗使用磷酸盐缓冲液（PBS）（pH值7.0：0.14米3毫米的氯化钾，氯化钠，10毫米的Na 2 HPO 4 2毫米KH 2 PO 4）在coplin罐子的幻灯片，在室温下5分钟。 </li><li>无菌蒸馏水冲洗两次幻灯片。 </li><li>乙醇系列（70，90和100％）每2分钟，在室温和空气干燥脱水的幻灯片。确保幻灯片完全沉浸，如果任何石墨粉尘浮在表面，用干净的纸巾浸泡。 </li><li>相衬显微镜可视化记录的圆圈内的细胞核中的位置。 </li><li>与100％的乙醇，在室温和空气干燥2分钟的脱水。 </li><li>应用探针混合物2μL，并涵盖与一个9 × 9毫米盖玻片（18 × 18毫米第一盖玻片的四分之一）。 </li><li>在与橡皮泥（如牛胶，牛校对）盖玻片密封的边缘。 </li><li>在炎热的块Codenature幻灯片在75 ° C为5分钟，然后杂交的幻灯片，在湿盒过夜（16-20小时）在37 ° C探头完全由着丝粒探针（即性联系的情况下）的混合杂交后60分钟的一个令人满意的结果。 </li><li>水浴准备足够coplin罐和加热到71 ° C。 </li><li>准备一个标准的0.4倍生理盐水柠檬酸（SSC），严格的洗涤液（pH值7.0，在71 ° C，0.06 M氯化钠，6毫米的C 6 H 5娜3 O 7 .2 H 2 O），在水浴热。 </li><li>要求严格，每coplin JAR洗分送50毫升和检查，温度为71 ° C立即使用前用干净的温度计。 </li><li>小心地从每张幻灯片的橡皮泥，用清水冲洗干净盖玻片，在室温下使用4X SSC/0.05％Tween20（pH值7.0）。 </li><li>洗净的0.4倍SSC严格洗的幻灯片，在71℃5分钟。每coplin JAR清洗不超过6张幻灯片。 </li><li> 2分钟，在室温下清洗4X SSC/0.05％Tween20的幻灯片。 </li><li>如果探头组合包含间接标记的探针（S），排出多余液体的幻灯片，并适用于下一个20 × 20平方毫米的封口膜20μL荧光标记的抗体。 </li><li>在湿盒中孵育37 ° 15分钟的彗星。 </li><li>取出的封口膜，在室温洗一次4X SSC/0.05％Tween20为2分钟。 </li><li>在PBS Å 2分钟两次清洗吨室温和流失的幻灯片。 </li><li>将6μL（160纳克4&#39;,6 - diamidino - 2 -苯基吲哚盐酸盐1毫升Vectashield安装媒介，媒介实验室）的DAPI / Vectashield 22 × 22毫米无。 0盖玻片倒置在盖玻片的幻灯片。 </li><li> Blot和清除指甲油密封盖玻片边缘。 </li></ol> 3。适当的装备，分析采用荧光显微镜的探头适当的过滤器。 <ol><li>分数直接可视化的信号，利用荧光显微镜和单带通过滤器为每个荧光检测。每个核应取得两位分析师。一般原则应导致一个单一的信号，两个相隔很近的信号是不到一个域（信号宽度）除得分，然而，根据经验判断需要行使解释信号，不同规模，强度和分离。 </li><li>使用成像软件（例如，ISIS，MetaSystems，Altlussheim，德国; CytoVision，Genetix）捕捉到的图像确认的视觉诊断的核心，和图像归档的实验室质量保证计划的一部分。 </li></ol> 4。代表性的成果： 从体外培养的外周血淋巴细胞核中期和相间应进行检查，以确认所选择的探头易位染色体的特定信息，断点（subtelomere探针杂交只易位段和着丝粒探针（S）的中心段（S））和间期核中的信号应该是明亮和离散。评分从每个合伙人每个探头的信号，在100间期核的建议，以评估检测的效率。在这种情况下，取得了2个信号在95％-99％，每个探头的原子核。图1显示了从一个编制为5日和9号染色体短臂之间的相互易位的中期和间期核。 在胚胎卵裂球间期细胞核的信号应该是明亮和离散，并取得使用单独的带通滤波器中使用的颜色。图2显示了与一个正常的信号模式（2份），所有的测试位点为5和第9号染色体正常或平衡的染色体补充，和不平衡易位的产品是一致的异常信号模式的核心卵裂球核单体为5号染色体三体（3份）9号染色体易位片段易位片段（一个副本）。 <img src="/files/ftp_upload/2570/2570fig1.jpg" alt="图1" /> 图1一个中期的蔓延和间期细胞核从一个运营商之间的5号染色体的短臂和9与断点在5p14.3和9p24.1的相互易位培养外周血淋巴细胞的准备：46，XY，T（ 5 9）（p14.3; p24.1）ISH T（5 9）（5ptel48 - 9ptel30 + 9ptel30的，5ptel48 +，9cen）。 FISH探针被选定为易位段（5p14.3→5pter，红色Cytocell subtelomere5ptel48 TexasRed; 9p24.1→9pter，绿色Cytocell subtelomere 9ptel30 FITC）和9号染色体的中心段（的9p24.1→9qter，蓝色雅培CEP 9阿尔法卫星SpectrumAqua）。 <img src="/files/ftp_upload/2570/2570fig2.jpg" alt="图2" /> 从3天的胚胎图2。相间卵裂球核信号捕捉每个荧光和合并，形成一个复合图像使用不同的过滤器。 （a）两个每个指示每个位点，这是与正常或平衡易位染色体的补充一致的两个副本的探针信号。 （二）一个红色信号，绿色信号，表明一个副本的5号染色体易位片段，三份9号染色体的易位段的9号染色体中，中心部分的两个副本，这是与相邻的两个蓝色信号-1 9p24.1→9pter 5p14.3→5pter和三体的不平衡与单体产品造成易位的隔离。

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应用荧光原位杂交（FISH），以一个单一的胚胎细胞（卵裂球）提出了特殊的挑战，无论是在实用性和信号模式的解释。幻灯片上的预先设定的区域内传播，以促进其本地化以下的鱼;活检细胞需要格外小心，需要采取的确保，细胞裂解，细胞质已经分散，和细胞核是可见的和完整的;，诊断从这个单细胞，严格评分和解释准则应适用的结果而定。然而，对于有经验的，鱼是一个强大的技术在临床实践中植入前遗传学诊断（PGD）。 PGD​​的鱼的原则，具体目标不同的荧光染料或半抗原的DNA标记的探针，可用于检测的特异位点的拷贝数，并以此来检测染色体减数分裂染色体重排的隔离（1）相关的不平衡，包括罗伯逊易位，相互易位，倒置，和复杂的重排（2）的。鱼也可以用来选择与X染色体连锁的疾病的女性在家庭中的胚胎，其中有没有特定的基因突变试验（3，4）。更有争议的是，鱼也被用于筛选零星的染色体非整倍体，以尝试和改进辅助繁殖（5，6）的效率;然而，预测值这个使用FSIH测试是可能到是低得无法接受在大多数人的的手，这是不推荐用于临床常规使用（7）。本文集中在技术方面和鱼应用于临床诊断的单细胞的局限性。 传播和操纵单个卵裂球的方法，包括甲醇/醋酸（5，8），吐温/盐酸（9），吐温/ HCl和甲醇/乙酸（10）的组合。变化包括低渗处理细胞固定后蔓延和/或胃蛋白酶和多聚甲醛处理。该方法应适当验证实验室（14）。吐温/盐酸的方法是在这篇文章中详细描述。吐温/盐酸的方法是技术上简单，在不同实验室的高度重复性。这种方法可以用来准备的性别鉴定和染色体重排与可接受的诊断准确率（7）鱼诊断的单核。 探针混合物可以结合直接标记和间接来自不同制造商的标记，探针和探头。已知多态性染色体区域的探头（11，12），或与其他染色体（13）显着交叉杂交的的应该是可以避免的，虽然可以使用，如果显示为PGD的具体和适当的生殖伙伴都事先测试。歧视探头可用荧光/半抗原和战略包括以下内容：德克萨斯红（TR），异硫氰酸荧光素（FITC），SpectrumGreen（Vysis），SpectrumOrange（Vysis），SpectrumAqua，TR - 亲和素检测生物素标记探针，FITC标记的抗生物素蛋白，或CY - 5链霉亲和素（可视化使用FarRed的过滤器），红色和绿色的探头的组合，以产生一种黄色信号，杂交的第二轮和第三个颜色由SpectrumOrange使用一个TexasRed和SpectrumGold过滤器）连续捕捉创建。 性别决定（14）一个探针组，其中包含三个探针，X和Y染色体和一个常染色体着丝粒区域的具体建议;常染色体探针是用来建立套数，从而区分三体综合征的X（2N，47 XXX）和三倍体（3N，69，XXX），四体X（48，XXXX）和四倍体（4N，92，XXXX）之间。一个典型的探头设置，此设置适用于含有α-卫星的X，Y，18雅培AneuVysion混合，这种探头设置已被证明有一个非常低的多态性利率，并因此治疗前的生殖伙伴工作不需要（14）。 任何特定重排的探头组合：理想情况下含有至少有足够的探测器检测到所有的染色体不平衡重排的预期产品。如果这是不可能的，探头混合未被发现的不平衡产品已被评定为在一个公认的妊娠非可行的，并可能有非常低发病率是可以接受的的（14）的。生殖伙伴培养淋巴细胞中期进行测试。应至少有10个中期利差特异性探针，基因多态性和交叉杂交研究，染色体重排载体，以确保探针杂交有望重排的不同环节。此外，这些准备工作至少有100间期核应取得评估信号的特殊性，亮度和离散（14）。 COMMErcial PGS的探头套（如雅培MultiVysion PB或PGT），针对最经常发现，在受孕的产品非整倍体的染色体，包括每个染色体的单探头针对性。通常细胞核内可能有一个与第二个杂交探针检测染色体13，15，16，18，21，22，XY（14）提供一个额外的染色体。本测试使用的蔓延，这里描述的FISH技术的预测值可能低得无法接受，在大多数人的手中，这是不推荐用于临床常规使用（7 15）。 如适用于单核细胞的比较基因组杂交（CGH）技术能够测试每一个染色体的拷贝数（威尔顿等2001），并利用单核苷酸多态性（SNP）的阵列和定量分析 （ Wells等2008）或“karyomapping”基因分型（Handyside 等人，2009年），是很有前途的替代技术来检测染色体非整倍体。但是，分辨率的限制和段失衡的准确性仍然不明朗，这是有可能，鱼类将继续涉及小段的染色体重排的首选技术。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hydrochloric Acid, 2.5L</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>101254H</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride, 5Kg</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>443827W</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride, 250g</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>26764.232</td>
<td>&nbsp;</td>
<tr>
<td>di-Sodium Hydrogen Orthophosphate Anhydrous, 500g</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>102494C</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Dihydrogen Orthophosphate, 500g</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>10203 4B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide Pellets, 500g</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28245.265</td>
<td>&nbsp;</td>
<tr>
<td>Tween20, 500mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>663684B</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol, 2.5L</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>8.18760.2500</td>
<td>Duty Paid</td>
</tr>
<tr>
<td>Tri-Sodium Citrate, 5Kg</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>27833.363</td>
<td>&nbsp;</td>
<tr>
<td>Cow Gum, 250mL</td>
<td>&nbsp;</td>
<td>Cow Proofings Ltd.</td>
<td>No longer available</td>
<td>Old stocks can still be found in art shops</td>
</tr>
<tr>
<td>DAPI/Antifade ES, 0.125&mu;g/mL, 500ul</td>
<td>&nbsp;</td>
<td>Cytocell</td>
<td>DES500L</td>
<td>150&mu;l vial rec&#x2019;d with each Cytocell TEL Probe</td>
</tr>
<tr>
<td>Nail Varnish, 12mL</td>
<td>&nbsp;</td>
<td>Boots</td>
<td>10088316001</td>
<td>&nbsp;</td>
<tr>
<td>Amine-coated Slides, 25</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>K2615</td>
<td>&nbsp;</td>
<tr>
<td>DAPI, 0.1mL</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-9542</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield, 10mL</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H1000</td>
<td>&nbsp;</td>
<tr>
<td>Texas-Red-avidin, 0.001mL</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>A-2016</td>
<td>&nbsp;</td>
<tr>
<td>FITC-avidin, 0.001mL</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>A-2011</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 Streptavidin, 0.001mL</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>PA45001</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge Tubes, 1.75mL, box of 500</td>
<td>&nbsp;</td>
<td>Thistle Scientific</td>
<td>AX-MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge Tubes, 0.6mL, box of 1000</td>
<td>&nbsp;</td>
<td>Thistle Scientific</td>
<td>AX-MCT-060-C-CS</td>
<td>&nbsp;</td>
<tr>
<td>30ml Universal Containers, box of 400</td>
<td>&nbsp;</td>
<td>Sterilin</td>
<td>128B</td>
<td>Available from NHS Supply Chain, Cat No. KCP053</td>
</tr>
<tr>
<td>Hot Block</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Spirit Burner Spirit Burner Socket Spirit Burner Wick</td>
<td>&nbsp;</td>
<td>VWR VWR VWR</td>
<td>451-0107 451-3112 451-3111</td>
<td>&nbsp;</td>
<tr>
<td>Plugged Glass Pipettes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>PMK-300-052R</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Wild Heerbrugg</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissues, 100boxes</td>
<td>&nbsp;</td>
<td>NHS SUPPLY CHAIN</td>
<td>MJT058</td>
<td>&nbsp;</td>
<tr>
<td>Metal Culture Trays</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Melamine Trays</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>682-009</td>
<td>&nbsp;</td>
<tr>
<td>Oven</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouth Pipette Mouthpiece</td>
<td>&nbsp;</td>
<td>Scientific Laboratory Supplies</td>
<td>HAE3700 </td>
<td>&nbsp;</td>
<tr>
<td>Tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Filter, 0.2&mu;m pore size, 25mm diameter</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>FDM-340-010U</td>
<td>&nbsp;</td>
<tr>
<td>Diamond Pen</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>818-0021</td>
<td>&nbsp;</td>
<tr>
<td>Hot Block for Slides</td>
<td>&nbsp;</td>
<td>Thermo Hybaid</td>
<td>HBOSBB</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Chamber</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Roll</td>
<td>&nbsp;</td>
<td>NHS SUPPLY CHAIN</td>
<td>MJT004</td>
<td>&nbsp;</td>
<tr>
<td>Schieferdecker Jar</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-9313</td>
<td>&nbsp;</td>
<tr>
<td>Coplin Jar</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-9331</td>
<td>&nbsp;</td>
<tr>
<td>Forceps, Fine</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>232-2123</td>
<td>&nbsp;</td>
<tr>
<td>Forceps, Blunt</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>232-2113</td>
<td>&nbsp;</td>
<tr>
<td>1000mL Beaker</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>213-1642</td>
<td>&nbsp;</td>
<tr>
<td>Phase Contrast Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>E200</td>
<td>&nbsp;</td>
<tr>
<td>Fibre-free Blotting Cards, box of 100</td>
<td>&nbsp;</td>
<td>Fisher Scientific (Raymond Lamb)</td>
<td>SDE1</td>
<td>&nbsp;</td>
<tr>
<td>Blue-Frosted Microscope Slides, box of 100</td>
<td>&nbsp;</td>
<td>Scientific Laboratory Supplies</td>
<td>MIC3022</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips, 18x18mm, No. 1, box of 200</td>
<td>&nbsp;</td>
<td>Scientific Laboratory Supplies</td>
<td>MIC3110</td>
<td>&nbsp;</td>
<tr>
<td> Coverslips, 22x22mm, No. 0, box of 200</td>
<td>&nbsp;</td>
<td>Scientific Laboratory Supplies</td>
<td>MIC3104</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips, 22x50mm, No. 0, box of 100</td>
<td>&nbsp;</td>
<td>Scientific Laboratory Supplies</td>
<td>MIC3206</td>
<td>&nbsp;</td>
<tr>
<td>1mL Syringe, box of 100</td>
<td>&nbsp;</td>
<td>NHS Supply Chain</td>
<td>FWC045</td>
<td>&nbsp;</td>
<tr>
<td>1mL Pipette Tip, 10racks of 1000</td>
<td>&nbsp;</td>
<td>Thistle Scientific</td>
<td>AX-T-1000-C-L-R</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>LEEC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Waterbath</td>
<td>&nbsp;</td>
<td>Grant</td>
<td>W22</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol Thermometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Various sources available</td>
</tr>
<tr>
<td>pH Meter</td>
<td>&nbsp;</td>
<td>Jenway</td>
<td>3305</td>
<td>&nbsp;</td>
<tr>
<td>pH Electrode</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>662-1759</td>
<td>BNC Plug to fit Jenway pH meter 3305</td>
</tr>
<tr>
<td>Heated Stirrer</td>
<td>&nbsp;</td>
<td>Bibby</td>
<td>HC502</td>
<td>&nbsp;</td>
<tr>
<td>1mL Pasteur Pipettes, box of 500</td>
<td>&nbsp;</td>
<td>Scientific Laboratory Supplies</td>
<td>PIP4202</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm, 50mm x 75m</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>291-1214</td>
<td>&nbsp;</td>		</tbody>
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fish for pre-implantation genetic diagnosis

植入前遗传学诊断（PGD）是一个既定的替代产前诊断，涉及选择植入前胚胎，辅助生殖技术（ART）生成一个队列。这种选择可能是必要的，因为家族的单基因疾病（如囊性纤维化），或因为一个合作伙伴进行的染色体重排（例如一个双向的相互易位）。 PGD​​是谁曾以前影响的儿童，和/中的染色体重排，习惯性流产或不孕的情况下，或夫妇。卵母细胞吸气以下卵巢刺激浸泡在精液在体外 （试管婴儿），或由单个精子胞浆内注射（ICSI）的受精。通常是由一个单细胞受精后第3天的清除，植入前胚胎卵裂期活检，活检细胞测试，以确定胚胎的遗传地位。荧光原位杂交（FISH）技术活检细胞与特定目标的DNA探针固定核是首选的技术检测染色体不平衡染色体重排，并与X -连锁的疾病有选择家庭中的女性胚胎没有突变，具体的测试。鱼也被用于屏幕胚胎自发的染色体非整倍体（又称PGS或PGD - AS），以尝试和改进辅助生育的效率;然而，这次试验的预测值，使用的传播和FISH技术在这里描述可能低得无法接受，在大多数人的手中，这是不推荐用于临床常规使用。我们描述了单细胞的鱼，卵裂球核的传播技术，原位杂交和信号打分以胚胎植入前诊断，在临床上应用选择合适的探头。

Watch this Scientific Journal Video about FISH for Pre-implantation Genetic Diagnosis at JoVE.com

FISH for Pre-implantation Genetic Diagnosis

本文介绍了单细胞的鱼，卵裂球核的传播技术，原位杂交和信号的得分，在临床上的应用植入前遗传学诊断（PGD）选择合适的探头。

鱼的胚胎植入前基因诊断

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

fish-for-pre-implantation-genetic-diagnosis

Research

JoVE Journal

Medicine

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

缺血性心力衰竭在临床前模型的转基因

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

科研

医学

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7, including several species of farmed cervids8-19, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves&#039; Muntjac Deer

Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.

里维斯的蒙特雅克鹿怀孕诊断的跨性别超声波

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described to assess MGT. Still, a standardized examination procedure is missing. Literature argues for weightbearing examination but the degree of knee flexion needed to eliminate the restraining effect of the M. gastrocnemius on ankle dorsiflexion (ADF) is unknown. This manuscript investigates the effect of knee flexion on ankle dorsiflexion and provides a detailed description of a standardized examination protocol. Examination on 20 healthy individuals revealed, that 20&#176; of knee flexion is sufficient to fully eliminate the influence of the M. gastrocnemius on ADF. This builds the prerequisite for a standardized examination for MGT. Non-weightbearing and weightbearing examination of ADF has to be conducted with the knee fully extended and at least 20&#176; flexed. Two investigators should conduct non-weightbearing testing with the subject in supine position. In order to obtain reliable results, the axis of the fibula should be marked. One examiner can conduct weightbearing examination with the subject in lunge stance. Isolated MGT is present if ADF is impaired with the knee fully extended and knee flexion results in a significant ADF increase. The herein presented standardized examination is the prerequisite for future studies aiming at establishing norm values.

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Isolated Musculus gastrocnemius tightness is a common cause for foot and ankle pathologies. Currently no standardized examination procedure exists. This manuscript demonstrates that 20 degree of knee flexion eliminates the restraining effect of the M. gastrocnemius on ankle dorsiflexion and presents a video description of a standardized examination protocol.

诊断<em&gt;家鼠腓肠肌</em&gt;密封性 - 关键因素临床考试

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

一种计算机辅助心血管分析诊断平台的研究

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure

Pneumothorax (PTX) represents accumulation of the air in the pleural space. A large or tension pneumothorax can collapse the lung and cause hemodynamic compromise, a life-threatening disorder. Traditionally, neonatal pneumothorax diagnosis has been based on clinical images, auscultation, transillumination, and chest X-ray findings. This approach may potentially lead to a delay in both diagnosis and treatment. The use of lung US in diagnosis of PTX together with US-guided thoracentesis results in earlier and more precise management. The recommendations presented in this publication are aimed at improving the application of lung US in guiding neonatal PTX diagnosis and management.

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

国际专家共识与新生儿肺功能超声诊断与超声引导性胸腔手术建议

Pneumothorax (PTX) represents accumulation of the air in the pleural space. A large or tension pneumothorax can collapse the lung and cause hemodynamic ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

人类布鲁塞拉性皮诺皮诺皮炎的诊断和手术治疗

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary vasomotor disorders (impaired vasodilatation and/or enhanced vasoconstriction/spasm) represent important functional causes for such a clinical presentation. Although impaired vasodilatation may be assessed with non-invasive techniques such as positron emission tomography or cardiac magnetic resonance imaging, there is currently no reliable non-invasive technique for the diagnosis of coronary spasm available. Thus, invasive diagnostic procedures (IDP) have been developed for the diagnosis of coronary vasomotor disorders including spasm testing as well as assessment of coronary vasodilatation. The identification of the underlying type of disorder (so called endotype) allows the initiation of targeted pharmacological treatments. Despite the fact that such an approach is recommended by the current European Society of Cardiology guidelines for the management of chronic coronary syndromes based on the CorMicA study, comparability of results as well as multicenter trials are currently hampered by major differences in institutional protocols for coronary functional testing. This article describes a comprehensive IDP protocol including intracoronary acetylcholine provocation testing for diagnosis of epicardial/microvascular spasm, followed by Doppler wire-based assessment of coronary flow reserve (CFR) and hyperemic microvascular resistance (HMR) in search of coronary vasodilatory impairment.

Coronary vasomotion disorders represent frequent functional causes of angina in patients with unobstructed coronaries. The underlying mechanism of angina (endotype) in these patients can be determined by a comprehensive invasive diagnostic procedure based on acetylcholine provocation testing followed by Doppler-derived assessment of the coronary flow reserve and microvascular resistance.

检测乙酰胆碱，然后使用腺苷进行冠状动脉血管舒缩疾病的侵入性诊断

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

测量心肌乳酸生成用于诊断冠状动脉微血管痉挛

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

嗜碱性粒细胞活化试验用于过敏诊断

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...