我们想感谢 Gráinne 的编辑协助。

外周血和血清被收集和储存的健康捐赠者和患者谁给予书面知情同意参与该项研究。该研究议定书是根据《1964赫尔辛基宣言》规定的道德标准, 由地方道德委员会批准的。
1. 星展集团的血液贮存
<ol><li>
采血样本收集
	<ol>
<li>收集3毫升的外周血样本在3毫升管与5.4 毫克乙二胺乙酸 (EDTA)。 注: 在8小时内尽快将外周血储存在 DBS 内。</li>
		<li>在保护卡的右下角写上病人代码号。</li>
		<li>小心地用5毫升的吸管并用重悬血液。</li>
	</ol>
</li>
	<li>
星展的血液斑点
	<ol>
<li>吸管和转移一点血液 (50 µL) 到保护卡 (材料表) 与50–200µL 提示。</li>
		<li>重复步骤1.2.1。有一共有3–5的血点。扔掉旧的小费, 并采取一个新的每一个血液整除。</li>
		<li>让星展集团在室温下过夜, 在一个开放的不吸水表面, 避免阳光直射的水平位置。把保护卡的盖子打开, 方便血液干燥。 注: 在干燥过程中, 空气或粉尘中的任何微粒都不会影响下游的应用。</li>
		<li>将卡在室温下存放在带有干燥剂的塑料袋中, 直到使用。</li>
	</ol>
</li>
</ol>2. dna 提取试剂盒 (用于干血斑点切片) 从 DBS 开始
<ol><li>
样品准备
	<ol>
<li>使用卡上的3个干血点。从卡上切下每个干血点, 并用镊子将它们与卡分开。将分离的干血点切成小块 (直径约1毫米)。</li>
		<li>将小块放入1.5 毫升离心管中。</li>
	</ol>
</li>
	<li>
样品消化
	<ol>
<li>将试剂盒 (材料表) 中提供的180µL 裂解缓冲液1添加到管中。添加20µL 蛋白酶 K。不要将裂解缓冲剂1与蛋白酶 k 混合, 然后再用血斑点卡吹打到1.5 毫升管。混合涡流。</li>
		<li>把管子放在 thermoincubator 中, 在56摄氏度处孵化60分钟。如果 thermoincubator 没有配备摇床, 则每10–15至少十年代将样品涡流, 注意不要让样品的温度降低。简单地离心管子从盖子去除下落。</li>
	</ol>
</li>
	<li>
洗涤步骤
	<ol>
<li>在十年代加入200µL 溶解缓冲 2 (材料表) 和漩涡。</li>
		<li>将管子放在 thermomixer 或加热的轨道孵化器中, 在70摄氏度处孵化10分钟。如果 thermoincubator 没有配备振动筛, 则至少在十年代将样品涡流一次, 注意不要让样品的温度降低。</li>
		<li>重复步骤2.3.1。将400µL 的裂解物从1.5 毫升管转移到洗脱柱 (材料表)。离心柱在 6000 x g 1 分钟。</li>
		<li>将洗脱柱放入新的2毫升收集管中, 丢弃旧的。</li>
		<li>添加500µL 洗涤缓冲 1 (材料表) 和离心机在 6000 x g 为1分钟。</li>
		<li>将该列放入一个新的2毫升收集管并丢弃旧的。</li>
		<li>添加500µL 洗涤缓冲 2 (材料表) 和离心机在 6000 x g 为1分钟。</li>
		<li>将该列放入一个新的2毫升收集管并丢弃旧的。</li>
		<li>离心机在 2万 x g 3 分钟干燥膜。</li>
	</ol>
</li>
	<li>
洗脱步骤
	<ol>
<li>将该柱放入新的1.5 毫升离心管中, 并丢弃收集管。</li>
		<li>将50µL 蒸馏水放到膜的中心。室温下孵育10分钟。</li>
		<li>离心管在 2万 x g 1 分钟收集洗脱液并再次放置在膜的中心。</li>
		<li>离心管在 2万 x g 1 分钟丢弃膜。</li>
		<li>使用分光光度计测量 DNA 浓度。</li>
		<li>储存的 DNA 在-20 摄氏度, 直到使用。 注: 从星展中提取的 DNA 可以用来进行几种分析, 如 SNP 评估的实时 PCR 或 DNA 测序。</li>
	</ol>
</li>
</ol>3. 用于 DSS 的血清贮存
<ol><li>
采血样本收集
	<ol>
<li>收集7毫升的外周血样本在7毫升管没有 EDTA。让血样在室温下凝固30分钟。</li>
		<li>在保护卡的右下角写上病人代码号。</li>
		<li>在室温下离心 980 x g的血液样本15分钟。 注: 在8小时内尽快将血清作为 DSS 贮存。</li>
		<li>小心地从试管中取出上清, 将其转移到新的15毫升管上。小心地用5毫升的吸管并用重悬血液。</li>
	</ol>
</li>
	<li>
DSS 中的血清斑点
	<ol>
<li>用吸管将一点血清 (50 µL) 转移到保护卡上。重复这一步, 有一个总的3–5血点。 注意: 要非常小心地将50µL 的血清注入每个部位。血清不应超出卡的虚线。</li>
		<li>让 DSS 在室温下过夜。把保护卡的盖子打开, 方便血液干燥。</li>
		<li>将卡在室温下存放在带有干燥剂的塑料袋中, 直到使用。如果存储时间超过14天, 请将卡存储在-20 摄氏度。</li>
	</ol>
</li>
</ol>4. 从 DSS 开始的 ELISA 检测
<ol><li>
洗脱蛋白来自 DSS。
	<ol>
<li>如有必要, 解冻每个示例的 DSS。将 DSS 切成小块 (直径约1毫米)。把这些碎片放入一个1.5 毫升管与400µL PBS。</li>
		<li>在4摄氏度一夜之间摇动样品。 注: 在低强度下摇动。在震动时, PBS 不应将管子的盖子弄湿。</li>
	</ol>
</li>
	<li>根据 ELISA 试剂盒的指示, 在适当的稀释后, 使用蛋白洗脱液作为血清标本进行分泌蛋白/生长因子分析。 注: 本程序适用于 DBP 检测9。对于其他 ELISA 化验, 可能需要一个优化步骤。</li>
</ol>

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H., Tian, G. L., Huang, Q. W., Wang, Y. M., Xu, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+glutathione+and+glutathione+disulfide+in+the+blood+of+glucose-6-phosphate+dehydrogenase-deficient+newborns.">Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.</a> BMC Pediatr. 20 (17), 172 (2017).</li><li>Lukacs, Z., Barr, M., Hamilton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practice+in+the+measurement+and+interpretation+of+lysosomal+acid+lipase+in+dried+blood+spots+using+the+inhibitor+Lalistat+2.">Best practice in the measurement and interpretation of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2.</a> Clin Chim Acta. 471, 201-205 (2017).</li><li>Skogstrand, P. T., Bent, N. 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The question of the vitamin D pathway.</a> Oncotarget. 8 (8), 13142-13156 (2017).</li><li>Gupta, B. P., Jayasuryan, N., Jameel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+hepatitis+B+virus+from+dried+blood+spots+by+polymerase+chain+reaction+amplification.">Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification.</a> J Clin Microbiol. 30 (8), 1913-1916 (1992).</li><li>Colson, K. E., Potter, A., Conde-Glez, C., Hernandez, B., R&#237;osZertuche, D., Z&#250;&#241;iga-Brenes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+commercial+ELISA+for+the+detection+of+measles-specific+immunoglobulin+G+(IgG)+in+dried+blood+spots+collected+from+children+living+in+low-resource+settings.">Use of a commercial ELISA for the detection of measles-specific immunoglobulin G (IgG) in dried blood spots collected from children living in low-resource settings.</a> J Med Virol. 87 (9), 1491-1499 (2015).</li><li>Drabe, C. H., Blauenfeldt, T., Ruhwald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA-based+assay+for+IP-10+detection+from+filter+paper+samples.">ELISA-based assay for IP-10 detection from filter paper samples.</a> Methods Mol Biol. 1172, 27-37 (2014).</li><li>St Julien, K. 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作者没有披露的利益冲突。

本议定书研究当实验室没有必要的工作人员或基础设施来正确处理血液样本时, 在滤纸上储存血液和血清的潜力。特别是, 在标准管或手指刺中收集的全血或血清可以用这种方法储存, 并且没有必要在血液收集后立即冷冻-20 摄氏度或-80 摄氏度的样品。星展/DSS 可以保持室温14天, 不改变血液/血清完整性。储存的一个关键问题是水分的存在, 影响生物材料的性能超过温度。使用干燥剂的塑料袋可以避?...

研究癌症生物标志物的主要目的是确定新的生物学参数, 用于诊断、预测患者预后, 以及确定患者是否会对特定治疗作出反应。这个研究领域是发现创新癌症治疗的基础, 并在量身定做的治疗中发挥关键作用。
在生物标志物鉴定和验证的每一个步骤中执行的程序必须是可靠和可重现的。翻译研究成功的基石是正确储存血液和血清等生物样本。这是获得高质量生物材料的第一步, 可用于进行分子生物学实验或蛋白质化验。
多中心的研究往往需要招募足够的病人, 以获得可靠的数据。并非所有的研究所都能将样品储存在-80 摄氏度, 或者将样品送到其他国际中心的干冰中。使用滤纸收集血液是一种简单的方法来储存血液和血清, 不需要立即冻结样本1,2。一滴血或血清可以被发现在纸上, 留到干燥过夜, 然后储存14天的室温1,2。这使得研究者们有时间把样品送到其他实验室。利用干血点 (DBS) 和干血清点 (DSS) 可以简化发达国家和发展中国家研究所之间的合作。
由于它的易用性, DBS 取样被广泛应用于几种血清学或遗传下游分析的化验。例如, 在过去, 星展集团经常被用于发展中国家的艾滋病毒筛查1、2、3、4、5。这种存储方法的另一个优点是可以从手指刺中收集血液样本, 从而使其用于新生儿筛查试验6、7、8。方便的样品处理和运输是 DBS 的进一步优势, 特别是在没有实验室设备的偏远地区收集的样品。在以前的出版物中, 我们使用 DBS 和 DSS 测试维生素 d 和维生素 d 结合蛋白 (DBP) 在一系列的白种人和非洲患者9。我们的非洲同事无法获得干冰。为了比较生物标记物对两个族群之间维生素 D 通路的差异, 我们进一步改进了在标准条件下和滤纸上储存的匹配样品的使用方法。在优化了星展/DSS 程序后, 我们能够分析两个患者血清中的 DBP 和维生素 D。我们还评估了一些单核苷酸多态性 (SNPs) 后, 从全血 DNA 提取的白种人和星展集团的非洲9。本议定书允许高质量的血液样本储存在室温下, 而不影响不同类型的下游分析, 从分子生物学到 ELISA 化验。建议用于管理多中心研究中的生物材料或没有标准贮存条件设施的中心。下面的协议代表了这些优化过程的顶点。

<table><tbody><tr><td>Whatman protein saver cards 903 Protein saver card, 100/pk&amp;lrm;</td><td>Sigma</td><td>Z761575</td><td>Useful to store samples at room temperature for downstream analyses</td></tr><tr><td>Falcon Serological Pipettes, 5 mL</td><td>Stem cell</td><td>#38003</td><td></td></tr><tr><td>50-200 &amp;micro;L tips</td><td>Star-Lab</td><td>S1120-8810</td><td></td></tr><tr><td>1.5 mL centrifuge tube</td><td>Eppendorf</td><td>4036-3204</td><td></td></tr><tr><td>QIAamp DNA microkit</td><td>Qiagen</td><td>56304</td><td>This is a DNA extraction kit designed to isolate small quantities of DNA&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Buffer ATL (included in QIAamp DNA microkit)</td><td>Qiagen</td><td></td><td>This is reported as Lysis Buffer 1 in the text</td></tr><tr><td>Buffer AL (included in QIAamp DNA microkit)</td><td>Qiagen</td><td></td><td>This is reported as Lysis Buffer 2 in the text</td></tr><tr><td>QIAamp mini elute column</td><td>Qiagen</td><td></td><td>This is reported as column in the text</td></tr><tr><td>AW1&amp;nbsp; (included in QIAamp DNA microkit)</td><td>Qiagen</td><td></td><td>This is reported as Wash Buffer 1 IN THE TEXT</td></tr><tr><td>AW2&amp;nbsp; (included in QIAamp DNA microkit)</td><td>Qiagen</td><td></td><td>This is reported as Wash Buffer 2 in the text</td></tr><tr><td>Human Vitamin D BP Quantikine ELISA Kit</td><td>R&amp;amp;D systems</td><td>DVDBP0</td><td></td></tr></tbody></table>

dried blood and serum spots as a useful tool for sample storage to evaluate cancer biomarkers

血液样本质量是确保准确的下游分析, 如实时 PCR 或 ELISA 的关键。正确贮存生物材料是实现可重现性和可靠结果的出发点。所有样品的处理方式应与采集血液的方法相同。根据要执行的分析, 整个血液和血清样本应储存在-20 摄氏度或-80 °c, 直到使用。血液/血清标本也应 aliquoted, 以避免多种冻融。另一个重要的问题是从一个实验室装运到另一间化验室的样品条件。如果干冰不可用或装运时间超过几天, 则需要其他方法。一种选择是使用滤纸收集血液。在这里, 我们提出了血液和血清标本收集的方法, 利用干血点 (DBS) 和干血清点 (DSS)。我们开发了从 DBS 中提取 DNA 的程序, 用于对某些单核苷酸多态性 (SNPs) 进行实时 PCR 的下游评价。我们还优化了从 DSS 蛋白质洗脱开始的 ELISA 检测。该方法可与其他酶联免疫测定法或评估蛋白质的程序一起使用。

我们利用星展银行和 DSS 的程序, 在室温下储存血液和血清, 而不影响生物材料的质量。图 1显示了一个没有血液和血液收集后的蛋白卡保护程序的例子。为了确认滤纸的贮存和洗脱血液的过程不影响样品质量, 我们对标准贮存方法进行了比较。我们提取的 DNA 来自3匹配样本的全血收集的2毫升管或存储为 DBS, 如报告的协议部分。采用实时 PCR 技术对五个...

Watch this Scientific Journal Video about Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers at JoVE.com

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

该协议描述了一种简单有效的方法, 用于存储外周血和血清/血浆, 用于下游分析, 如单核苷酸多态性 (SNP) 评价和 ELISA 检测。

干血和血清斑点作为样品贮存的有用工具, 用于评估肿瘤生物标志物

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

dried-blood-serum-spots-as-useful-tool-for-sample-storage-to-evaluate

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

10.4K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. 该协议描述了一种简单有效的方法, 用于存储外周血和血清/血浆, 用于下游分析, 如单核苷酸多态性 (SNP) 评价和 ELISA 检测。

视频: Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工程

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

癌症研究

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

干生生物标志物的血斑收藏最大限度地参与人口研究

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

医学

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

从临床样本中自动分离和收集癌症相关物质

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

在生物标志物分析临床试验场单次抽血时，准备外周血单核细胞颗粒和血浆

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...