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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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><tbody><tr><td>4% sodium citrate</td><td>Sigma-Aldrich</td><td>S1804</td><td>Anticoagulant dissolved in water&lt;sup&gt;9&lt;/sup&gt;</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 peptide&lt;sup&gt;2,4&lt;/sup&gt; 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>&amp;nbsp;P5405</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&amp;nbsp;&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M4880</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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 days&lt;sup&gt;8&lt;/sup&gt;.</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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

970 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 

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

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 ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Biology

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

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

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Chapters in this video

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

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

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Microfluidics in Assessing Platelet Function

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets