Blood should be collected from consenting healthy volunteers and processed in accordance with local rules. This protocol measures agonist-induced platelet aggregation in platelet-rich plasma (PRP) by light transmission aggregometry (LTA). The ISTH provides standardized protocols, including a 2013 method for PRP preparation and LTA9.&#160; Collect whole blood into 4% sodium citrate as per ISTH recommendations in accordance with local ethics committee rules. Exclude donors that have taken any NSAID within the past 2 weeks.
1. Assay procedure
<ol>
	<li>Take an aliquot of stock CRP-XL and dilute it in assay (Tyrodes10) buffer (Table 1) to a starting concentration that will cause maximum aggregation (see the NOTE below step 1.3). 
	NOTE: In the literature, a concentration of 1 &#956;g/mL will induce maximum aggregation, but some labs need higher concentrations to reach the same effect - adjust the dilution series accordingly</li>
	<li>Make a corresponding dilution series with the standard (see the NOTE below step 1.3 and Figure 1) such that the concentrations are the same as the test reagent.</li>
	<li>Produce a 6- to 8-point dilution series for the test and the standard, again in assay buffer, that takes the aggregation response from 100% to 0% aggregation. An example showing an 8-point dilution series concentration curve is shown in Table 2. 
	NOTE: Some labs add agonist at a total of 10% assay volume, e.g., 50 &#956;L of agonist to 450 &#956;L of platelets, such that they dilute a 10x concentration to a final 1x concentration in the assay, while others add smaller (more concentrated) volumes of agonist, e.g., 5 &#956;L of agonist to 495 &#956;L of platelets - just make sure that the dilution series is appropriate for the assay bearing in mind how the volume displacement affects platelet concentration - again, be consistent. In the example, 30 &#956;L of 10x agonist is added to 270 &#956;L of platelet rich plasma (PRP).</li>
	<li>Once the platelets are rested and ready to use, aliquot them into LTA cuvettes, add a stir bar, and allow them to reach 37 &#176;C in the holding wells.</li>
	<li>Once at temperature, place the cuvette(s) into the aggregometer and begin recording the trace(s). Add the agonist (with stirring) and record the data.</li>
	<li>Repeat step 1.4 for each concentration of the test and standard until the data needed to plot the curves has been collected (example curves shown in Figure 2).</li>
	<li>Calculate the potency of the test reagent relative to that of the standard; again, use any metric - maximum aggregation or area under the curve - as long as there is consistency and the analysis model appropriately fits the data.</li>
</ol>
2. Checking the concentration curves
<ol>
	<li>First, check that the concentration response curves are parallel. There are many software packages that do this, but here the process is described for Graphpad Prism (see Figure 3).
	<ol>
		<li>Enter the data so that log(concentration) is on the X axis and response (% maximum aggregation/AUC) is on the Y axis (Figure 3A)</li>
		<li>Transform CRP-XL concentrations (Figure 3B)</li>
		<li>Select Non-Linear Regression from the Analysis functions and use the equation for dose-response stimulation/inhibition.</li>
		<li>Select log (agonist) vs. response (variable slope) - use 3- or 4-way fit depending on which is best for the data.</li>
		<li>Determine whether the curves are parallel by using the Compare tab (as shown in Figure 3C) and click on the button to compare the datasets to ensure that the slope (Hill slope) and asymptotes (the Top and Bottom of the curves) are equivalent. In this example, the Hill slope for the test is 2.279, and that of the standard is 2.025 giving a ratio of 1.125.</li>
		<li>If the slopes are parallel (for example, an acceptance criterion of slope ratio between 0.8-1.25) and the asymptotes are equivalent, proceed with potency (EC50) calculation using steps 2.1.3 and 2.1.4. In the example shown here, the asymptotes have been constrained as the evaluation in step 2.1.5 confirmed that the asymptotes are equivalent.</li>
		<li>Divide the EC50 value of the standard by that of the test to determine the relative potency. In the example shown here, test/standard (0.07259/0.1498) = 0.4846, i.e., the &#39;test&#39; is half as potent as the standard.</li>
		<li>Adjust mass/volume concentrations to account for the difference in potency, i.e., if the standard was used previously at 1 &#181;g/mL, the new material would be used at 1/0.4846 = 2.063 &#181;g/mL) to ensure comparable amounts of active material are being used in experiments</li>
	</ol>
	</li>
</ol>

Standardizing the Responses to Platelet Agonist Collagen-Related Peptide

<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>

The authors have nothing to declare.

As shown here, the process of standardizing the materials is very straightforward. Although it requires a little bit of time to evaluate new batches of material and/or to check that the current batch is stable and not losing activity over time, the reward is that experiments are reproducible time and time again and comparable over years rather than just the lifetime of a batch of reagent. It also means that other researchers can accurately recreate assay conditions and that the community is harmonized at the global level. Critical steps in the protocol include whole blood collection and PRP preparation9, as well as precision when making the dilution series. It is also important to ensure the test and standard curves are parallel before proceeding with the EC50 comparison. If the lines are not parallel, or the asymptotes are not equivalent, it could indicate that the test and standard material are different to some degree.
Everyone that works in the biological sciences knows that biological assays do not always perform consistently, so one needs to be mindful of this when evaluating the data. In the example shown here, even though we are using the same donors to measure the potency of two very similar, if not identical, materials, the hill slopes and asymptotes are not identical. Yet we accept a degree of variability and conclude that the curves are, in this case, parallel, and therefore&#160;suitable for comparing and adjusting potency. Biological molecules are large and complex, and post-translational modifications during manufacture can influence their potency in bioassays. Standardizing biomolecules and bioassays cannot be done using just mass or volume and must be done by quantifying and comparing biological activity in a bioassay11. One must use his/her training and experience to evaluate the data in the context of the assay.
Limitations of the technique are that while the data may indicate an issue with the reagent(s), it cannot tell you what the&#160;issue is. Further work will be needed to determine why the materials are not comparable. In an ideal world, any material critical for&#160;experiment(s) and subsequent scientific conclusions would be verified by mass spectroscopy, but this is not always an option. However, if the data are not comparable further investigation is still warranted in some capacity. Another limitation of this approach is the time and resources needed to do it. Ideally, the test and standard should be compared in 3-6 donors to provide some confidence in assigning biological activity to the material, but we feel this is justified by supporting reproducibility and reliability. Standardizing biological material is not needed every time experiments are run, but at a minimum, it should be done for every new batch of agonists, preferably more frequently, depending on stability and use. Indeed, should a standard be made available, this is something that international standardization organizations can provide clarity on.
While the example shown here is for CRP-XL in the context of measuring platelet aggregation, the protocol of comparing the biological activity of biomolecules in bioassays is widely applicable across the sector.

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

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.

<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

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.

Figure 1&#160;is a diagrammatic representation of a dilution series and meant to highlight that a dilution series is needed for both the standard and the test. Figure 2A shows aggregation traces for the standard, while the test data is shown in Figure 2B. At 0.075 &#181;g/mL, there is a clear response to the standard, while for the test, the corresponding concentration (0.075 &#181;g/mL) is flat, i.e., no response. These data are used to plot the subsequent graphs to give the EC50. In the example shown here, 100% maximum aggregation was used to plot the concentration-response curve and determine EC50, as shown in Figure 3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65410/65410fig01.jpg" /> 
Figure 1: Graphical description of the two dilutions series of the test (left) and the standard (right). Starting with the top concentration (suggested concentration: 10 &#181;g/mL CRP-XL), sequential 1 in 2 dilutions are made in parallel over 8 points (&#62;3 log units). <a href="https://www.jove.com/files/ftp_upload/65410/65410fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65410/65410fig02.jpg" /> 
Figure 2: Aggregation traces for the standard (left) and test dilution series (right). As platelet aggregation proceeds (x-axis), the amount of light passing through the sample and reaching the sensor increases (y-axis). (A) Standard. (B) Test. <a href="https://www.jove.com/files/ftp_upload/65410/65410fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65410/65410fig03.jpg" /> 
Figure 3: Data entry and analysis. (A) Data is entered, (B) transformed, and (C,D) analyzed for parallelism (ask the software to tell if the Hill slopes and asymptotes are comparable [C]). (E) If the curves are parallel, determine the potency (EC50) using non-linear regression curve fitting. (F) Plotted transformed data. Please refer to the method for more detail. <a href="https://www.jove.com/files/ftp_upload/65410/65410fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Tyrodes buffer9</td>
		</tr>
		<tr>
			<td>Constituents</td>
			<td>Concentration</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>134 mM&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>0.34 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.9 mM&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>12 mM&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>20 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 mM</td>
			<td>pH 7.3</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of Tyrodes buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">8 point dilution series</td>
		</tr>
		<tr>
			<td>Dilution 1</td>
			<td>10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 2</td>
			<td>5&#160;&#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 3</td>
			<td>2.5&#160;&#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 4</td>
			<td>1.25&#160;&#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 5</td>
			<td>0.625&#160;&#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 6</td>
			<td>0.3125&#160;&#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 7</td>
			<td>0.15625&#160;&#181;g/mL</td>
		</tr>
		<tr>
			<td>Dilution 8</td>
			<td>&#160;0.073125&#160;&#181;g/mL</td>
		</tr>
	</tbody>
</table>
Table 2: An example showing an 8-point dilution series.

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

Author Spotlight: Biological Standardization to Ensure Reproducibility and Harmonization in Research 

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.

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

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

reproducibility-harmonization-research-using-biological-standards

Research

JoVE Journal

Medicine

934 Views.  Medicines and Healthcare products Regulatory Agency. 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.

Video: Author Spotlight: Biological Standardization to Ensure Reproducibility and Harmonization in Research 

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.
Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (&ge; 2.0 x1011 platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.
Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.

This article describes the process used by &#214;rebro University Hospital to produce double dose buffy coat platelet concentrates prepared from whole blood donations and treated with the INTERCEPT Blood System for pathogen inactivation. The in vitro quality of the final platelet units are evaluated over 7 days of storage.

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds &#8211; PP2, rottlerin, and curcumin &#8211; in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.