应从同意的健康志愿者处采集血液，并按照当地法规进行处理。该方案通过透光聚集法（LTA）测量激动剂诱导的富血小板血浆（PRP）中的血小板聚集。ISTH 提供标准化方案，包括 2013 年 PRP 制备方法和 LTA9。 根据当地伦理委员会的规定，根据ISTH的建议，将全血收集到4%柠檬酸钠中。排除在过去 2 周内服用过任何非甾体抗炎药的供体。
1.检测程序
<ol><li>取等分试样的储备CRP-XL，并在测定（Tyrodes10）缓冲液（表1）中将其稀释至将导致最大聚集的起始浓度（参见步骤1.3下面的注释）。 注：在文献中，1 μg/mL 的浓度将诱导最大聚集，但一些实验室需要更高的浓度才能达到相同的效果 - 相应地调整稀释度系列</li>
	<li>用标准品进行相应的稀释系列（参见步骤 1.3 和 图 1 下面的注释），使浓度与测试试剂相同。</li>
	<li>再次在测定缓冲液中为测试品和标准品生成 6 至 8 点稀释系列，使聚集响应从 100% 降至 0%。 表2显示了8点稀释系列浓度曲线的示例。 注：一些实验室以总共 10% 的测定体积添加激动剂，例如，将 50 μL 激动剂添加到 450 μL 血小板中，以便它们在测定中将 10 倍浓度稀释至最终 1 倍浓度，而其他实验室则添加更小（更浓缩）体积的激动剂，例如，将 5 μL 激动剂添加到 495 μL 血小板中 - 只需确保稀释系列适合测定，同时牢记体积置换如何影响血小板浓度 - 再次保持一致。在该示例中，将 30 μL 10x 激动剂加入到 270 μL 富血小板血浆 （PRP） 中。</li>
	<li>一旦血小板静置并准备好使用，将它们分装到LTA比色皿中，加入搅拌棒，并让它们在保持孔中达到37°C。</li>
	<li>达到温度后，将比色皿放入聚集计并开始记录迹线。加入激动剂（搅拌）并记录数据。</li>
	<li>对测试和标准品的每种浓度重复步骤1.4，直到收集到绘制曲线所需的数据（示例曲线如图 2所示）。</li>
	<li>计算测试试剂相对于标准品的效力;同样，使用任何指标 - 最大聚合或曲线下面积 - 只要存在一致性并且分析模型适当拟合数据即可。</li>
</ol>2. 检查浓度曲线
<ol><li>首先，检查浓度响应曲线是否平行。有许多软件包可以做到这一点，但这里描述了 Graphpad Prism 的过程（参见 图 3）。<ol><li>输入数据，使log（浓度）位于X轴上，响应（%最大聚集/ AUC）位于Y轴上（图3A）</li>
		<li>转换CRP-XL浓度（图3B）</li>
		<li>从分析函数中选择非线性回归，并使用剂量反应刺激/抑制方程。</li>
		<li>选择对数（激动剂）与响应（可变斜率）- 使用3向或4向拟合，具体取决于哪种拟合最适合数据。</li>
		<li>使用 “比较 ”选项卡确定曲线是否平行（ 如图 3C 所示），然后单击按钮比较数据集，以确保斜率（山坡）和渐近线（曲线 的顶部 和 底部 ）相等。在本例中，测试的 山坡为 2.279，标准坡度为 2.025，比率为 1.125。</li>
		<li>如果斜率是平行的（例如，斜率比在0.8-1.25之间的验收标准）并且渐近线是等效的，则使用步骤2.1.3和2.1.4继续进行效力（EC50）计算。在此处显示的示例中，渐近线已受到约束，因为步骤 2.1.5 中的评估确认渐近线是等效的。</li>
		<li>将标准的 EC50 值除以测试值以确定相对效力。在此处显示的示例中，test/standard （0.07259/0.1498） = 0.4846，即“test”的效力是标准的一半。</li>
		<li>调整质量/体积浓度以考虑效力差异，即，如果之前以 1 μg/mL 的浓度使用标准品，则新材料的使用浓度为 1/0.4846 = 2.063 μg/mL），以确保在实验中使用相当量的活性材料</li>
	</ol></li>
</ol>

标准化对血小板激动剂胶原蛋白相关肽的反应

<ol>
	<li>Faya, 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=Potency+assignment+of+biotherapeutic+reference+standards.">Potency assignment of biotherapeutic reference standards.</a> Journal of Pharmaceutical and Biomedical Analysis. 191, 113577 (2020).</li><li>Morton, L. F., 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=Integrin+alpha+2+beta+1-independent+activation+of+platelets+by+simple+collagen-like+peptides:+collagen+tertiary+(triple-helical)+and+quaternary+(polymeric)+structures+are+sufficient+alone+for+alpha+2+beta+1-independent+platelet+reactivity.">Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.</a> The Biochemical Journal. 306 (2), 337-344 (1995).</li><li>Pugh, N., 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=Synergism+between+platelet+collagen+receptors+defined+using+receptor-specific+collagen-mimetic+peptide+substrata+in+flowing+blood.">Synergism between platelet collagen receptors defined using receptor-specific collagen-mimetic peptide substrata in flowing blood.</a> Blood. 115 (24), 5069-5079 (2010).</li><li>Farndale, R. W., 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=Cell-collagen+interactions:+the+use+of+peptide+Toolkits+to+investigate+collagen-receptor+interactions.">Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions.</a> Biochemical Society Transactions. 36 (Pt 2), 241-250 (2008).</li><li>Dunster, 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=Multiparameter+phenotyping+of+platelet+reactivity+for+stratification+of+human+cohorts.">Multiparameter phenotyping of platelet reactivity for stratification of human cohorts.</a> Blood Advances. 5 (20), 4017-4030 (2021).</li><li>Hubbard, A. R., 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=Establishment+of+the+WHO+2nd+International+Standard+Factor+V,+plasma+(16/374):+communication+from+the+SSC+of+the+ISTH.">Establishment of the WHO 2nd International Standard Factor V, plasma (16/374): communication from the SSC of the ISTH.</a> Journal of Thrombosis and Haemostasis. 17 (4), 695-697 (2019).</li><li>Sergaki, 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=Developing+whole+cell+standards+for+the+microbiome+field.">Developing whole cell standards for the microbiome field.</a> Microbiome. 10 (1), 123 (2022).</li><li>Alessi, M. 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=Multi-center+evaluation+of+light+transmission+platelet+aggregation+reagents:+Communication+from+the+ISTH+SSC+Subcommittee+on+Platelet+Physiology.">Multi-center evaluation of light transmission platelet aggregation reagents: Communication from the ISTH SSC Subcommittee on Platelet Physiology.</a> Journal of Thrombosis and Haemostasis. (23), (2023).</li><li>Cattaneo, 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=Recommendations+for+the+standardization+of+light+transmission+aggregometry:+A+consensus+of+the+working+party+from+the+platelet+physiology+subcommittee+of+SSC/ISTH.">Recommendations for the standardization of light transmission aggregometry: A consensus of the working party from the platelet physiology subcommittee of SSC/ISTH.</a> Journal of Thrombosis and Haemostasis. , (2013).</li><li>Taylor, 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=Discovery+of+novel+GPVI+receptor+antagonists+by+structure-based+repurposing.">Discovery of novel GPVI receptor antagonists by structure-based repurposing.</a> PLoS One. 9 (6), e101209 (2014).</li><li>Coxon, C. H., Longstaff, C., Burns, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+the+science+of+measurement+to+biology:+Why+bother.">Applying the science of measurement to biology: Why bother.</a> PLoS Biology. 17 (6), e3000338 (2019).</li></ol>

作者没有什么可声明的。

如图所示，材料标准化的过程非常简单。虽然评估新批次的材料和/或检查当前批次的材料是否稳定且不会随着时间的推移而失去活性，但回报是实验可以一次又一次地重复，并且可以在数年内进行比较，而不仅仅是一批试剂的寿命。这也意味着其他研究人员可以准确地重建检测条件，并且社区在全球范围内得到协调。方案中的关键步骤包括全血采集和PRP制备9，以及进行稀释系列时的精确度。在进行 EC50 比较之前，确保测试曲线和标准曲线平行也很重要。如果线不平行，或者渐近线不等效，则可能表明测试材料和标准材料在某种程度上不同。
每个从事生物科学工作的人都知道，生物检测并不总是始终如一地执行，因此在评估数据时需要注意这一点。在这里显示的示例中，即使我们使用相同的供体来测量两种非常相似（如果不是完全相同）材料的效力，山坡和渐近线也不完全相同。然而，我们接受一定程度的可变性，并得出结论，在这种情况下，曲线是平行的，因此适合比较和调整效力。生物分子大而复杂，制造过程中的翻译后修饰会影响其在生物测定中的效力。生物分子和生物测定的标准化不能仅使用质量或体积来完成，必须通过量化和比较生物测定中的生物 活性 来完成11.必须利用他/她的培训和经验来评估分析环境中的数据。
该技术的局限性在于，虽然数据可能表明试剂存在问题，但它无法告诉您问题是什么。需要进一步的工作来确定为什么这些材料没有可比性。在理想情况下，任何对实验和随后的科学结论至关重要的材料都将通过质谱进行验证，但这并不总是一种选择。但是，如果数据不具有可比性，则仍有必要以某种身份进行进一步调查。这种方法的另一个局限性是这样做所需的时间和资源。理想情况下，应在 3-6 个供体中比较测试和标准品，以提供为材料分配生物活性的一定信心，但我们认为这可以通过支持可重复性和可靠性来证明是合理的。不需要在每次进行实验时都对生物材料进行标准化，但至少应该对每批新的激动剂进行标准化，最好更频繁地进行，具体取决于稳定性和用途。事实上，如果有一个标准可用，国际标准化组织可以对此提供澄清。
虽然这里显示的示例是在测量血小板聚集的背景下针对 CRP-XL，但在生物测定中比较生物分子生物活性的方案在整个行业中广泛适用。

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65410">Reproducibility and Harmonization in Research using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide</a>. The Authors section was updated from:
Carmen H. Coxon1 
Peter Rigsby1 
1National Institute for Biological Standards and Control
to:
Carmen H. Coxon1 
Peter Rigsby1 
1Medicines and Healthcare products Regulatory Agency

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65410">Reproducibility and Harmonization in Research using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide</a>. The Authors section was updated.

Erratum: Reproducibility and Harmonization in Research using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide

我们中的许多人在实验中使用生物激动剂和拮抗剂，最常见的是在生物测定中，该测定量化了它们在特定条件下对细胞功能的影响。这里描述的方法适用于血小板激动剂胶原相关肽交联 （CRP-XL），这是一种激活血小板的糖蛋白 VI 激动剂，其活性可以在各种测定法（光透射聚集法、显微镜、流式细胞术、Ca2+ 释放等）中测量，但激动剂标准化方案适用于任何生物激动剂/拮抗剂和/或生物测定法。物理科学精通在日常工作实践中使用标准，在商业领域开发生物治疗药物的公司了解使用参考标准的价值1，但许多生物科学家，特别是在学术研究界，它们仍然没有得到充分利用，缺乏使用会对科学产出的质量产生负面影响。
CRP-XL 是一种特异性 GPVI 特异性激动剂，自 1995 年被描述以来，血小板领域已经使用了几十年2，帮助社区自 3,4 以来将 GPVI 的作用与其他胶原结合血小板蛋白的作用区分开来。这种激动剂可以从各种商业来源采购或内部生产。肽单体可以从供应商处购买，然后在内部进行交联，也可以由供应商支付额外费用。通过降低交付时所需的纯度（例如，70% 对 95%），可以进一步节省成本。关于CRP-XL的储存方式也没有达成共识，有些选择低温储存，有些选择冷藏，有些选择将材料冻干直到使用，有些则将其储存在溶液（水或缓冲液，取决于偏好）中。因此，实验室储备的质量和组成是非标准化或协调的。由于该激动剂以规定的浓度（质量/体积）而不是活性单位使用，因此在一个实验室中，CRP-XL的效力（例如，1μg/mL）将与另一个实验室不同。值得注意的是，该领域使用的其他血小板激动剂已经标准化（例如，凝血酶，其以活性单位而不是质量/体积提供和使用），因此从质量/体积到生物单位的运动对社区来说应该不会太具有挑战性。还应该注意的是，患者对血小板激动剂（包括 CRP-XL）的反应在对激动剂的敏感性以及反应能力（反应程度）方面是可变的5，因此在测定中使用一致量的激动剂活性更为重要。
如果血小板激动剂可以通过在规定的活性而不是重量/体积下使用它们来协调，则可以确保一个实验室的实验与其他实验室的实验直接比较，并且可以放心地准确再现。在现场就如何存储和标准化 CRP-XL 活动达成一致之前，必须对材料进行定期检查，以确保其在使用月/年内的一致性。
生物标准品的活性值分配必须通过协作的多中心研究（例如6,7）完成，并且需要专业知识。最近，由国际血栓形成和止血学会 （ISTH） 支持的一项多中心合作研究8 强调了建立血小板激动剂生物学标准的必要性;在国际CRP-XL标准可用之前，研究人员可以在当地对自己的材料进行标准化，以确保长期保持一致性，并且商业或内部采购的新材料与以前的制剂以及社区其他部分的制备具有相当的活性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td>Anticoagulant dissolved in water9</td>
		</tr>
		<tr>
			<td>4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>54457</td>
			<td></td>
		</tr>
		<tr>
			<td>CRP-XL</td>
			<td>Peptide Protein Research Ltd.</td>
			<td></td>
			<td>A crosslinked, triple-helical peptide2,4 with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline was obtained as a custom order from Peptide Protein Research Ltd.</td>
		</tr>
		<tr>
			<td>Cuvettes and stir bars</td>
			<td>Stago</td>
			<td>86921</td>
			<td>Consumables for aggregometer</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M4880</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Analysis software package</td>
		</tr>
		<tr>
			<td>Platelet rich plasma</td>
			<td></td>
			<td></td>
			<td>Isolated from whole blood from healthy volunteers free of non-steroidal anti-inflammatory drugs for a minimum of 10 days8.</td>
		</tr>
	</tbody>
</table>

reproducibility and harmonization in research using biological standards: the example of platelet agonist collagen-related peptide

计量学 - 测量科学 - 是很少有生物科学家在培训中被教导的学科，这对他们不利;将简单的标准化流程应用于日常工作实践，可以提高数据的可信度以及随距离和时间变化的可重复性。
该方法演示了如何标准化止血研究和临床实践中广泛使用的核心实验室实验，特别是通过透光聚集法 （LTA） 测量对血小板胶原受体（糖蛋白 [GP]VI）激动剂胶原相关肽交联 （CRP-XL） 的反应。使用这种方法将确保实验室内的可重复性和实验室间的协调，无论激动剂库存或供应商如何。重要的是，这种方法适用于其他血小板激动剂，实际上也适用于许多其他生物分子和生物测定。
下面概述的过程包括制作“标准品”和“测试品”（您正在检查的材料）的 6-8 点稀释系列，并在选定的测定（在本例中为 LTA）中并排运行它们。CRP-XL 在质量/体积浓度下使用，但并非每种材料在给定浓度下都具有相同的生物活性，因此进行稀释系列以比较标准品和测试材料，并确定提供等效活性所需的浓度。稀释系列必须跨越 0-100% 的聚集。使用非线性回归绘制数据，并确定每个样品（标准品和测试品）的EC50 值。要分配活性，请将标准品的 EC50 值除以测试值，以确定其效力或多或少，并相应地调整浓度。这种方法将确保一次又一次地将相同的生物“活性”添加到测定中。

Many of us use biological agonists and antagonists in our experiments, most often in a biological assay that quantifies their effect on cell function under specific conditions. The method described here is for the platelet agonist collagen-related peptide, cross-linked (CRP-XL), a glycoprotein VI agonist that activates platelets and whose activity can be measured in a variety of assays (light transmission aggregometry, microscopy, flow cytometry, Ca2+ release, etc.), but the agonist standardization protocol is applicable to any biological agonist/antagonist and/or bioassay. The physical sciences are well versed in the use of standards in their day-to-day working practices, and companies developing biotherapeutic medicines in the commercial sector understand the value of using reference standards1, yet they remain underutilized by many biological scientists, especially in the academic research community, and their lack of use negatively impacts the quality of scientific output.
CRP-XL is a specific GPVI-specific agonist that the platelet field has used for decades since its description in 19952, helping the community to delineate the role of GPVI from that of other collagen-binding platelet proteins ever since3,4. This agonist can be procured from a variety of commercial sources or produced in-house. The peptide monomer can be purchased from a supplier and then cross-linked in-house or this can be&#160;done by the supplier for an extra fee. Further cost-saving can be made by reducing the required purity upon delivery (70% versus 95%, for example). There is also no consensus on how CRP-XL should be stored, with some opting for cryostorage and others refrigeration, some keeping the material lyophilized until use, and others storing it in solution (water or buffer, depending on preference). Therefore, the quality and composition of laboratory stocks are non-standardized or harmonized. Because this agonist is used at a defined concentration (mass/volume) rather than units of activity, the potency of CRP-XL in one lab, e.g., at 1 &#956;g/mL, will not be the same as another. It is worth noting that other platelet agonists used in the field are already standardized (e.g., thrombin, which is supplied and used in units of activity rather than mass/volume), so the movement away from mass/volume to units of biological should not be too challenging for the community. It should also be noted that patient responses to platelet agonists, including CRP-XL, are variable in terms of their sensitivity to agonists as well as their capacity to respond (extent of response)5, so it is even more important to use consistent amounts of agonist activity in the assays.
If platelet agonists can be harmonized by using them at a defined activity rather than weight/volume, it can be ensured that an experiment in one lab is directly comparable to that in other labs and can be accurately reproduced with confidence. Until such time as the field reaches an agreement on how to store and standardize CRP-XL activity, it is essential to perform regular checks on the material to ensure its consistency over the months/years in which it is being used.
Activity value assignment for biological standards must be done through collaborative, multi-center studies (for example6,7) and require specialist expertise. The need for established biological standards for platelet agonists has been recently highlighted by a collaborative multi-center study8 supported by the International Society for Thrombosis and Haemostasis (ISTH); until an international CRP-XL standard is available, researchers can standardize their own material locally to ensure consistency over time, and that new material sourced commercially or in-house is of comparable activity to previous preparations, as well as that of the rest of the community.

作者聚焦：确保研究中的可重复性和协调性的生物标准化 

使用生物学标准品进行研究的可重复性和协调性：以血小板激动剂胶原相关肽为例

Researchers at the MHRA South Mimms Laboratories have been developing manufacturing biological standards for over 60 years, and these reference reagents are used to ensure the quality, consistency, and safety of biological medicines and diagnostics. NIB standards from the MHRA support translation of cutting edge therapeutic products, as well as the control of established medicines. At the MHRA South Mimms Laboratories, we use state-of-the-art technologies to produce materials that support the development and regulation of novel therapeutic innovations.Biological standards remain underutilized by academics in the biological sciences due to a lack of knowledge exchange. This negatively impacts the quality of research and contributes to the reproducibility crisis. Biological standardization ensures experiments are reproducible regardless of where or when they are performed.A lack of proper knowledge in basic metrological principles and training compromises scientific rigor and negatively impacts reproducibility. Although our protocol focuses on standardizing platelet function measurement, the practice can be applied to any biological system. Applying biological standardization to research practices will not only standardize biological experiments, but also help to address inconsistencies and the lack of harmonization that undermines research.

图 1 是稀释系列的示意图，旨在强调标准品和测试都需要稀释系列。 图2A 显示了标准的聚合迹线，而测试数据如图 2B所示。在 0.075 μg/mL 时，对标准品有明确的响应，而对于测试，相应的浓度 （0.075 μg/mL） 是平坦的，即没有响应。这些数据用于绘制后续图形，以给出 EC50。在此所示的示例中，使用100%最大聚集来绘制浓度-响应曲线并确定EC50， 如图3所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65410/65410fig01.jpg"> 图 1：测试（左）和标准品（右）的两种稀释系列的图形说明。 从最高浓度（建议浓度：10 μg/mL CRP-XL）开始，在 8 个点（>3 个对数单位）上平行进行连续 1/2 稀释。 <a href="https://www.jove.com/files/ftp_upload/65410/65410fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65410/65410fig02.jpg"> 图 2：标准品（左）和测试稀释系列（右）的聚集迹线。 随着血小板聚集的进行（x 轴），通过样品并到达传感器的光量增加（y 轴）。（a） 标准。（B） 测试. <a href="https://www.jove.com/files/ftp_upload/65410/65410fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65410/65410fig03.jpg"> 图 3：数据输入和分析。 （A） 输入数据，（B） 转换，（C，D） 分析并行性（要求软件判断希尔坡度和渐近线是否具有可比性 [C]）。（E） 如果曲线平行，则使用非线性回归曲线拟合确定效力 （EC50）。（F） 绘制的转换数据。有关更多详细信息，请参阅方法。<a href="https://www.jove.com/files/ftp_upload/65410/65410fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="3">Tyrodes 缓冲液9</td>
		</tr><tr><td>成分</td>
			<td>浓度</td>
			<td>评论</td>
		</tr><tr><td>氯化钠</td>
			<td>134 毫米</td>
			<td></td>
		</tr><tr><td>钠2HPO4</td>
			<td>0.34毫米</td>
			<td></td>
		</tr><tr><td>氯化钾</td>
			<td>2.9 毫米</td>
			<td></td>
		</tr><tr><td>氢氧化钠3</td>
			<td>12 毫米</td>
			<td></td>
		</tr><tr><td>4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）</td>
			<td>20毫米</td>
			<td></td>
		</tr><tr><td>葡萄糖</td>
			<td>5 毫米</td>
			<td></td>
		</tr><tr><td>氯化镁2</td>
			<td>1 毫米</td>
			<td>pH 值 7.3</td>
		</tr></tbody></table>表1：Tyrodes缓冲液的组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2">8点稀释系列</td>
		</tr><tr><td>稀释度 1</td>
			<td>10微克/毫升</td>
		</tr><tr><td>稀释度 2</td>
			<td>5微克/毫升</td>
		</tr><tr><td>稀释度 3</td>
			<td>2.5微克/毫升</td>
		</tr><tr><td>稀释度 4</td>
			<td>1.25微克/毫升</td>
		</tr><tr><td>稀释度 5</td>
			<td>0.625微克/毫升</td>
		</tr><tr><td>稀释度 6</td>
			<td>0.3125微克/毫升</td>
		</tr><tr><td>稀释度 7</td>
			<td>0.15625微克/毫升</td>
		</tr><tr><td>稀释度 8</td>
			<td>0.073125微克/毫升</td>
		</tr></tbody></table>表 2：显示 8 点稀释系列的示例。

Watch this Scientific Journal Video about Biological Standardization to Ensure Reproducibility and Harmonization in Research at JoVE.com

在这里，我们提出了一种使用光透射聚集法标准化血小板激动剂胶原相关肽交联 （CRP-XL） 的方法。虽然该方案针对的是血小板功能，但实验过程可以应用于大多数生物分子和生物测定，以确保科学的严谨性和可重复性。

Metrology - the science of measure - is a subject few biological scientists are taught about in their training to their detriment; the application of ...

60 多年来，MHRA South Mimms 实验室的研究人员一直在开发制造生物标准品，这些参比试剂用于确保生物药物和诊断的质量、一致性和安全性。MHRA的NIB标准支持尖端治疗产品的转化，以及对现有药物的控制。在MHRA South Mimms实验室，我们使用最先进的技术来生产支持新型治疗创新开发和监管的材料。由于缺乏知识交流，生物科学领域的学者仍然未充分利用生物标准。这会对研究质量产生负面影响，并导致可重复性危机。生物标准化确保无论在何时何地进行实验，实验都是可重复的。缺乏对基本计量原理和培训的适当知识会影响科学的严谨性，并对可重复性产生负面影响。虽然我们的方案侧重于标准化血小板功能测量，但这种做法可以应用于任何生物系统。将生物标准化应用于研究实践不仅将使生物实验标准化，还有助于解决破坏研究的不一致和缺乏协调的问题。

reproducibility-harmonization-research-using-biological-standards

Research

JoVE Journal

Medicine

Reproducibility and Harmonization in Research Using Biological Standards: The Example of Platelet Agonist Collagen-Related Peptide

639 次观看.  Medicines and Healthcare products Regulatory Agency. 60 多年来，MHRA South Mimms 实验室的研究人员一直在开发制造生物标准品，这些参比试剂用于确保生物药物和诊断的质量、一致性和安全性。MHRA的NIB标准支持尖端治疗产品的转化，以及对现有药物的控制。在MHRA South Mimms实验室，我们使用最先进的技术来生产支持新型治疗创新开发和监管的材料。由于缺乏知识交流，生物科学领域的学者仍然未充分利用生物标准。这会对研...

视频: 作者聚焦：确保研究中的可重复性和协调性的生物标准化 

Metrology - the science of measure - is a subject few biological scientists are taught about in their training to their detriment; the application of simple standardization processes to everyday working practices provides confidence in data and reproducibility over distance and time.
This method demonstrates how to standardize a core laboratory experiment used widely in hemostasis research and clinical practice, specifically, measuring responses to the platelet collagen receptor (glycoprotein [GP]VI) agonist collagen-related peptide, cross-linked (CRP-XL) by light transmission aggregometry (LTA). Using this approach will ensure intra-lab reproducibility and inter-lab harmonization, regardless of agonist stock or supplier. Importantly, this method is applicable to other platelet agonists and, indeed, many other biological molecules and bioassays.
The process outlined below involves making a 6-8 point dilution series of the &#39;standard&#39; and the &#39;test&#39; (the material you are checking) and running them side by side in a chosen assay (in this case, LTA). CRP-XL is used at mass/volume concentrations, but not every material gives the same biological activity at a given concentration, so a dilution series is made to compare the standard and test material and determine what concentration is needed to give equivalent activity. The dilution series must span 0-100% aggregation. Data is plotted using non-linear regression, and the EC50 value of each sample (standard and test) is determined. To assign activity, divide the EC50 value of the standard by that of the test to determine how much more or less potent it is and adjust the concentration accordingly. This approach will ensure that the same biological &#39;activity&#39; is added to the assay time and time again.

Here we present a method to standardize the platelet agonist collagen-related peptide cross-linked (CRP-XL) using light transmission aggregometry. While the protocol is targeted at platelet function, the experimental process can be applied to most biological molecules and bioassays to ensure scientific rigor and reproducibility.

科研

医学

Light Transmission Aggregometry for Standardizing Platelet Collagen Receptor Agonist

To begin the assay using the platelet receptor agonist, Collagen-Related Peptide Cross-Linked, or CRPXL, dilute an appropriate amount of the CRPXL stock in the assay buffer to a concentration suitable to induce maximum platelet aggregation. The resultant solution will be henceforth referred to as the standard. Next, starting from the same highest prepared concentrations, sequentially perform two-fold serial dilutions for both the standard and the test reagent.Prepare a six-to-eight point dilution series for both the test and the standard in the assay buffer that generates aggregation responses from 100%down to 0%When the platelet-rich plasma, or PRP, is ready to use, add an aliquot of PRP into the light transmission aggregometry cuvette. Allow the samples to warm to 37 degrees Celsius in the holding wells for a couple of minutes. Once appropriately warmed, place the cuvettes in the aggregometer to start recording the traces.With constant stirring, add the agonist, and record the data. Perform data collection for each prepared dilution of the standard and the test reagent. Use Graphpad Prism, or any suitable software package, to check that the concentration curves for the standard and test are parallel.Enter the data such that the x-axis contains the concentrations, and the y-axis contains the responses indicated by either the percentage of maximum aggregation or the area under the curve. Next, transform the concentration values to the corresponding logarithms, or log concentration. Select non-linear regression from the analysis functions, and use the equation for dose response stimulation.Select log agonist versus response variable slope, and use a three or four-way fit, depending on which is best for the data. Determine if the curves generated are parallel by clicking the compare tab and comparing the datasets. Ensure that the slope and asymptotes of the test, and the standard curves, are equivalent.If these conditions are met, proceed to calculate the potency, or EC50, using non-linear regression curve fitting. Calculate the relative potency by dividing the EC50 value of the standard by that of the test. Adjust the mass per volume concentration of the test reagent to account for the difference in potency with respect to the standard.The platelet aggregation traces for the standard and the test reagent revealed the difference between the two aggregation responses. At 0.075 micrograms per milliliter, there was a clear response to the standard, while, for the test, the corresponding concentration showed no response. The EC50 values of the standard and test reagent were calculated by plotting the concentration response curve.