We thank the blood donors and the personnel at Canadian Blood Services Network Centre for Applied Development for collection and production of the platelet units used in this study. We acknowledge the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the UBC Centre for Blood Research. Funding for the publication was provided by LightIntegra Technology Inc., manufacturer of ThromboLUX.

The following protocol has been performed in compliance with all Canadian Blood Services (CBS) guidelines. Volunteers gave consent that their donations could be used to carry out these studies. Platelet concentrates were prepared according to CBS standard operating procedures. All performance testing described here was carried out at the Centre for Blood Research at the University of British Columbia, Vancouver, Canada with institutional Research Ethics Board approval.
1. Quality Control Check
<ol>
	<li>Perform a quality control check at least once per testing day to verify the proper operation of the DLS system. Measure the provided control beads (50 nm radius) following the manufacturer&#39;s instructions for use.</li>
	<li>Retrieve the control vial from cold storage and allow 15 min to warm to room temperature. The beads must equilibrate to room temperature prior to use.</li>
	<li>Turn the DLS system on prior to preparing the sample so the 15 min laser warm up period is completed by the time the first sample is ready for testing.</li>
	<li>Once the console has started up, follow the instructions on the touch screen to log in and select the System Test option to measure the Control sample.</li>
	<li>Vortex mix the vial for 10 s to ensure a representative sample.</li>
	<li>Fill a capillary with the control beads using a 100 &#181;L fixed volume pipette, pipette tip and capillary from the test kit.</li>
	<li>When the test is completed follow the instructions on the screen of the DLS system. 
	NOTE: The control bead test results are automatically compared to the information stored in the control bead barcode label and a PASS or FAIL notification as well as sample information are shown on the DLS system screen at the end of the test.</li>
</ol>
2. Obtaining a sample from a platelet concentrate
NOTE: These steps describe the process for non-invasive sampling from the platelet transfusion into the DLS testing capillary for routine platelet inventory management. An overview is shown in Figure 1. Required accessories: tube sealer, manual tube stripper, scissors, and splash shield.
<ol>
	<li>Remove the platelet concentrate from the untested inventory.</li>
	<li>Obtain the sample of the platelet product either from an unused sampling pouch (proceed to step 2.3) or a tubing segment (proceed to step 2.4 if empty or step 2.5 if not empty). 
	To disconnect the pouch tubing or tubing segment use a tube sealer. To operate the tube sealer, follow the manufacturer&#39;s instructions. Briefly, clamp the tubing to be sealed in a precise position between two heated jaws. In a handheld device, press the molten tubing together and cool under high pressure while pressing the lever (the duration is indicated by the green light) resulting in a permanent, leak-proof seal.</li>
	<li>Sampling from a pouch
	<ol>
		<li>Mix the content of the platelet bag well by gentle horizontal movement for 5 s (tipping from end to end five times).</li>
		<li>Open the clamp to the pouch. The pouch is evacuated and will fill by itself. It is not necessary to fill the pouch completely as only 100 &#181;L sample will be required for DLS testing.</li>
		<li>Disconnect the pouch from the bag by heat sealing the connecting tubing with a tube sealer. Continue with step 3.</li>
	</ol>
	</li>
	<li>Sampling from empty tubing
	<ol>
		<li>Verify that the platelet bag tubing has been stored empty and the tubing block is still in place. Visually inspect the tubing to verify that there is not a significant amount of platelet concentrate in the tubing. If the tubing is not empty continue with step 2.5.</li>
		<li>Close the tube stripper on the tubing as close to the tubing block as possible by compressing the handles to squeeze the tubing between the rollers.</li>
		<li>Hang the bag vertically and keep compressing the stripper handles.</li>
		<li>While keeping the stripper closed, release the tubing block.</li>
		<li>Slowly pull the stripper down the empty tubing allowing for the tubing to slowly fill behind the stripper. Continue until the section below the stripper has fully inflated or until the stripper is within 1 inch of the end of the tubing.</li>
		<li>Once the stopping point is reached with the stripper, use the tube sealer to heat seal the tubing 1 inch above the stripper.</li>
		<li>Release the handles of the manual tube stripper.</li>
		<li>Heat seal again 1 to 2 inches above the previous seal to create the testing segment.</li>
		<li>Cut off the testing segment from the platelet unit. This segment will be used for DLS testing.</li>
	</ol>
	</li>
	<li>Sampling from tubing that is not empty
	<ol>
		<li>Hang the bag vertically and release the tubing block if in place.</li>
		<li>Visually inspect the tubing to determine if there are any significant solid clumps. If solid clumps do exist, seal above them such that they cannot be stripped into the bag.</li>
		<li>Close the tube stripper on the tubing as close to the sealed end of the tubing as possible.</li>
		<li>Strip the contents of the tubing into the bag by compressing the handles to squeeze the tubing between the rollers and, while maintaining the clamping force, move the tube stripper along the tubing towards the bag.</li>
		<li>Remove the platelet bag from the hanging hook and gently mix the bag for 5 s by tipping it from end to end five times while keeping the stripper closed.</li>
		<li>Hang the bag vertically while keeping the stripper closed.</li>
		<li>Slowly pull the stripper down the empty tubing, allowing for the tubing to slowly fill behind the stripper. Continue until the section below the stripper has fully inflated or until the stripper is within 1 inch of the end of the tubing.</li>
		<li>Once the stopping point is reached with the stripper, use the tube sealer to heat seal the tubing 1 inch above the stripper.</li>
		<li>Release the stripper.</li>
		<li>Heat seal again 1 to 2 inches above the previous seal to create the testing segment.</li>
		<li>Cut off and discard the last portion of the tubing.</li>
		<li>Cut off the testing segment from the platelet unit. This segment will be used for DLS testing.</li>
	</ol>
	</li>
</ol>
3. Filling the sample into the DLS testing capillary
<ol>
	<li>Using a splash shield and clean, dry scissors, cut one end of the pouch tubing or testing segment. 
	NOTE: The capillary used for DLS testing should be filled immediately.</li>
	<li>Fill the capillary by using the sampling tool (assembled 100 &#181;L fixed volume pipette, pipette tip and capillary) to draw the sample directly from the opened pouch or tubing segment into the capillary.</li>
	<li>Seal the bottom of the filled capillary by gently pushing the filled capillary into the capillary tube sealant while applying a gentle twist and some pressure against the tray.</li>
	<li>Disconnect the 100 &#181;L fixed volume pipette and tip from the capillary, ensuring the capillary remains firmly embedded in the capillary tube sealant.</li>
	<li>Remove the capillary from the capillary tube sealant tray, wipe with an isopropyl alcohol pad and ensure that no air bubbles are trapped by gently flicking the bottom of the capillary.</li>
	<li>Place the capillary into the DLS system.</li>
</ol>
4. Performing the DLS test
<ol>
	<li>Select the MP test option to initiate a new DLS test for measuring the microparticle content of a platelet sample.</li>
	<li>Enter the sample information (Donation Identification Number (ISBT), Collection/Production Expiration Date, and Product Code) using the barcode scanner or manually.&#160;</li>
	<li>If no product code is available from which the DLS system can extract the fluid medium information, select the appropriate fluid medium manually (for most platelet concentrates select plasma but for samples containing Platelet Additive Solution (PAS), enter the percentage of residual plasma). 
	NOTE: A small deviation in the PAS percentage from the nominal 65% will cause a small error in the viscosity (automatically calculated by the DLS system) which minimally affects the calculated MP radius but not %MP.&#160;</li>
	<li>Place the capillary into the DLS system when instructed and start the test.</li>
	<li>When the test is completed, remove the sample from the capillary holder and dispose of the consumables appropriately according to facility guidelines. Remove the sample from the capillary holder and dispose of the consumables appropriately according to facility guidelines.</li>
	<li>Tag the platelet bag with the color corresponding to the result, for example orange for non-activated (homogeneous) with %MP equal or below 15% and pink for activated (heterogeneous) with %MP above 15%.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56893/56893fig1.jpg" /> 
Figure 1: Method overview. Overview of the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet concentrate, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles and using the reported microparticle content to identify activated platelets.&#160;<a href="//cloudfront.jove.com/files/ftp_upload/56893/56893fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

EMS is the founder of LightIntegra Technology Inc., a medical device company to develop the ThromboLUX System for microparticle and platelet quality testing. All other authors are employees of LightIntegra Technology. Production and Free Access to this article are sponsored by LightIntegra Technology Inc.

This protocol describes a dynamic light scattering method for microparticle screening optimized for the high particle concentrations found in biological samples such as platelet concentrates. The method of DLS is inherently standardized to accurately measure size. The relative concentration of microparticles can be converted into an absolute concentration if the platelet concentration is known and the platelet peak area is used as the reference peak19. As platelet concentrations are usually obtained with hematology analyzers or flow cytometers these methods can be considered companion technologies to DLS.
The functionality of the DLS system is insured by running control beads regularly. Distilled water can be measured to verify that background noise is minimal. Commercially available platelet standards can be analyzed as MP-negative controls and, after addition of 125 nm radius beads, as MP-positive controls. Within the biological range of MP concentrations of interest, the procedures are practical and quick to perform as part of the blood bank routine.
As opposed to flow cytometry, this method is not based on comparing the scattering intensities of particles but rather the speed of their Brownian motion. Thus, exosomes can also be detected despite their small size and are reported separately from MP.
Designed as a screening tool, the limitations of this method are related to its inability to differentiate between different types of microparticles. There is potential room for improvement if additional isolation steps are used; samples could be tested before and after the specific removal of microparticles through antibody coupled magnetic bead capture. In addition, it cannot be assumed that all detected microparticles are cell derived because chylomicrons formed in hyperlipidemia47,48 and small bacteria or viruses6 will also be reported in the microparticle range. However, other safeguards exist within the blood supply to avoid highly lipemic or contaminated platelets to enter the hospital blood bank inventory.
The choice of anticoagulant in the sample affects the extent of platelet activation and therefore the MP content49. For comparisons of different products this factor needs to be considered. Furthermore, exchange of plasma with MP free suspension media such as PAS will affect the MP content and the threshold for determining heterogeneity-if only about one third of the original MP content is left within the residual plasma in the concentrate an accordingly lower MP content threshold will indicate the same level of platelet activation as in 100% plasma. Percent MP is the MP content relative to platelets. It was previously reported that the average platelet count of PAS product was lower so that the average %MP was still 9.5%19. The %MP threshold for PAS platelets licensed in the US is currently set to 10%.
While the primary source of MP in platelet concentrates is the donor, processes that cause stress to platelets will increase the MP level depending on the susceptibility of the platelets to stress-if platelets are already highly activated, minor stressors such as extended shelf life, pathogen inactivation, washing, irradiation or long-distance transport could lead to significant increase in MP content. None of these stressors have been shown to significantly affect homogeneous, non-activated platelets19. In addition, attention should be paid to the potential for changes in sample composition within the capillary if not tested immediately after preparation (completion of Step 3 of this protocol).
The focus of this protocol is on determining the composition of particles present in platelet transfusions and to use microparticles as biomarkers of platelet activation. The platelet transfusions are tagged as either non-activated (orange) or activated (pink) based on a microparticle percentage threshold of 15%. The threshold of 15% MP for platelets in 100% plasma was empirically determined as the average 66th percentile from multiple sites.
The objective of platelet inventory management based on routine microparticle screening with DLS is to improve patient care and drive cost efficiencies by preventing non-immune platelet refractoriness. The implementation of the DLS system for screening of platelet bags will enable the user to direct non-activated platelets to patient populations most at risk for developing platelet refractoriness.

The interest in microparticles has revolved mostly around their involvement in cell to-cell communication and biological processes.1,2,3 More recently, microparticles have also attracted interest as potential early diagnostic markers of autoimmune and cardiovascular diseases.4,5 Microparticles, also known as extracellular vesicles or exosomes, have been widely investigated by flow cytometry. Unfortunately, despite efforts to standardize conventional flow cytometry protocols there is no consensus on the optimal protocol to use.6,7,8,9,10
Although conventional flow cytometry can characterize specific MP subpopulations,11 it has several reported limitations.11,12,13,14 Some of these limitations have been addressed by using higher power lasers and detectors in a so-called &#34;small particles option&#34;,15,16 as well as detection at 15&#176;-25&#176; forward scatter angle and modified sheath pressure.17,18 Nevertheless, a quick and easy-to-use screening method that can be combined with these sophisticated methods is still needed to utilize microparticles as early diagnostic markers. Elevated levels of microparticles are detectable in about one third of normal blood donors19 and might indicate subclinical conditions. Consequently, high microparticle levels in donated blood products might be incompatible with vulnerable recipients.20
When providing platelet transfusions, there is a risk of non-immune refractoriness - a condition wherein a patient&#39;s body rejects consecutive transfusions and does not allow a significant portion of platelets to circulate.21,22 Non-immune refractoriness can result in wasted transfusions, health complications for patients, and extended hospital stays.23 Platelet transfusions containing high numbers of microparticles can be a contributing factor for the development of non-immune refractoriness in vulnerable hematology-oncology patients.20 It is possible to manage the platelet inventory according to the composition of platelet transfusions by screening for microparticles thereby reducing the risk for vulnerable patients. However, most microparticle tests require isolation of microparticles from platelets5,12 or are otherwise very labor intensive24,25 and can therefore not be implemented routinely in hospital blood banks.
The technique described here uses dynamic light scattering (DLS) - also known as photon correlation spectroscopy or quasi-elastic light scattering. For decades, dynamic light scattering has been extensively used in the pharmaceutical industry to characterize liposomal drug formulations or emulsions where particle sizes are in the sub-micron range.26,27 However, tools developed for these applications are not optimized to screen blood products. A new DLS system was developed to overcome technical limitations and make dynamic light scattering useful for screening of platelet transfusions.28
Dynamic light scattering measurements are performed by illuminating the suspended particles with laser light and analyzing the time variation of the scattered light intensity which is a consequence of particles moving in suspension. Further, this method uses the inverse relationship between particle speed and size - small particles move fast and large particles move slowly - to provide information on the size distributions and relative concentrations of the sample components. Using DLS, the mean microparticle content and mean radius of the microparticle component can be quantified. The content of microparticles in the blood product is given as %MP based on the area in the measured histogram for particle radii from 50 - 550 nm. While particles with radii below 50 nm are detected by DLS and reported by the DLS system, they are not included in %MP. Rather than isolating microparticles from platelets, the heterogeneity of platelet transfusions is determined based on the relative content of microparticles and platelets in a sample. In fact, the ratio of the platelet and microparticle peaks can be used to calculate the absolute MP concentration when the platelet count is known.19
The DLS system provides health care professionals with qualitative and quantitative information about microparticles found in specimens derived from human blood or blood products. The main advantage of the described DLS technique over alternative techniques such as flow cytometry, electron microscopy,29 nanoparticle tracking analysis30 or tunable resistive pulse sensing31 is the sample preparation: aliquots of platelet concentrates can be directly measured without the need to isolate MP from platelets, sample dilution or other modifications.9,17 In addition, DLS is an absolute sizing method and does not suffer from the lack of calibration beads with appropriate refractive index.14,32
A correlation between platelet activation and MP content has previously been shown by microscopy33,20 and can be inferred from the increase in MP content in pathological conditions15,34,35,36 and under in vitro conditions known to activate platelets.19,37,38 However, further studies are needed to fully understand the relationship of DLS-measured microparticle content and platelet activation. Based on our current knowledge that activated platelets contain high numbers of microparticles, they are best used for therapeutic treatment of actively bleeding patients39, while hematology-oncology patients benefit from non-activated platelets with no or low levels of microparticles20. It has recently been reported that in vitro responsiveness of donor platelets did not significantly impact the recovery and survival of these platelets in single transfusions to stable, mostly non-bleeding hematology-oncology patients40. From this finding, it might be concluded that platelet activation identified by high MP content also plays no significant role in the prophylactic treatment of patients. However, due to the narrow selection of patients, this study did not address the impact of donor factors for complex patients who are febrile (excluded from the study), not stable and receive many more than just one platelet transfusion. The question how the complexity of these patient cases can be reduced - which holds the promise to reduce refractoriness - remains unanswered.
Microparticles are early markers of inflammation41,42,43,44 and platelet activation45 and are therefore detected in many normal donors.19 Consequently, activated platelets and microparticles are present in the platelet donations. It is reasonable to hypothesize that patients with fever, i.e., a fully activated innate immune system, cannot tolerate the additional challenge of a transfusion of activated platelets. However, studies are needed to prove this hypothesis. Microparticle screening can alleviate the current uncertainty about the content of platelet transfusions and reduce the complexity of patient treatment.
The ratio of activated to non-activated platelets in a hospital blood bank depends primarily on the donor population and to a much lesser degree on transport, irradiation, pathogen inactivation and other processes that might increase platelet activation in concentrates.19 Data from major hospital blood banks in the United States showed that the average composition of the platelet inventory is 49% activated and 51% non-activated (range for activated platelets: 38 - 62%, personal communications). If blood product providers or hospital blood banks want to know how many activated and non-activated platelet concentrates they produce or receive, and want to manage their inventory based on platelet activation as indicated by microparticle content, this protocol might be appropriate for them.
Based on platelet composition a hospital blood bank will be able to direct non-activated, homogeneous platelets for prophylactic use and activated, heterogeneous platelets for therapeutic use. Platelet screening allows hospitals to maximize usage of available inventory which improves patient care and decreases cost. This protocol is intended for laboratory personnel who are familiar with basic handling and manipulation of blood products.
Presented here is a screening method for microparticles in platelet transfusions that can be routinely applied to manage hospital blood bank inventory where selecting product based on microparticle content is desirable. The objective of this protocol is to outline the implementation and evaluation of DLS for screening of donated platelets. The described protocol addresses the common questions of non-invasive access to samples, integration of the testing into the blood bank work flow, and performance characteristics.

<table >
	<tbody>
		<tr >
			<td >ThromboLUX-M</td>
			<td >LightIntegra Technology Inc.&#160;</td>
			<td >LPN120</td>
			<td >DLS System</td>
		</tr>
		<tr >
			<td >ThromboSight Software</td>
			<td >LightIntegra Technology Inc.&#160;</td>
			<td >LPN124</td>
			<td >DLS analysis software</td>
		</tr>
		<tr >
			<td >Capillaries</td>
			<td >LightIntegra Technology Inc. (included in test kit, vendor Optinova MLE)</td>
			<td >LPN065</td>
			<td >Custom extruded non-activating, non-birefringent plastic tube, inspected and irradiated</td>
		</tr>
		<tr >
			<td >MiniPet&#160;</td>
			<td >LightIntegra Technology Inc. (included in test kit, vendor TriContinent Scientific)</td>
			<td >LPN082</td>
			<td >100 &#181;L fixed volume pipette for sampling&#160;&#160;</td>
		</tr>
		<tr >
			<td >MicroFlex 1-200 &#181;L Pipette tips</td>
			<td >LightIntegra Technology Inc. (included in test kit, vendor ESBE Scientific)</td>
			<td >LPN080</td>
			<td >Pipette tips for sampling</td>
		</tr>
		<tr >
			<td >Critoseal&#160;</td>
			<td >LightIntegra Technology Inc. (included in test kit, vendor Fisher Scientific)</td>
			<td >LPN075</td>
			<td >Capillary Tube Sealant</td>
		</tr>
		<tr >
			<td >Dukal Alcohol Pad or equivalent</td>
			<td >LightIntegra Technology Inc. (included in test kit, vendor Dukal Corp.)</td>
			<td >LPN085</td>
			<td >Isopropyl pad (saturated with 70% isopropyl alcohol)</td>
		</tr>
		<tr >
			<td >Control beads, 50 nm radius</td>
			<td >LightIntegra Technology Inc. (included in test kit, vendor PolySciences Inc.)</td>
			<td >LPN128</td>
			<td >Control beads</td>
		</tr>
		<tr >
			<td >Microbead NIST Traceable Particle Size Standard, 3 &#181;m</td>
			<td >PolySciences Inc.</td>
			<td >64060</td>
			<td >Standard microbeads: 1.5 &#181;m radius Polystyrene beads; range 1.43-1.58 &#181;m; 1% solids suspensions in de-ionized water</td>
		</tr>
		<tr >
			<td >Nanobead NIST Traceable Particle Size Standard, 250 nm</td>
			<td >PolySciences Inc.</td>
			<td >64014-15</td>
			<td >Standard nanobeads: 125 nm radius Polystyrene beads; range 120 - 130 nm; 1% solids suspensions in de-ionized water</td>
		</tr>
		<tr >
			<td >Genesis BPS RapidSeal II or equivalent</td>
			<td >Genesis BPS</td>
			<td >428-SE640</td>
			<td >Tube sealer</td>
		</tr>
		<tr >
			<td >Fresenius Kabi Tube Stripper or equivalent</td>
			<td >Fresenius Kabi</td>
			<td >6R4452</td>
			<td >Manual tube stripper</td>
		</tr>
		<tr >
			<td >Biohazard Shield - Cell Counter or equivalent</td>
			<td >CardinalHealth</td>
			<td >S1389-75</td>
			<td >Splash Shield</td>
		</tr>
		<tr >
			<td >Corning LSE Vortex Mixer or equivalent</td>
			<td >Corning Incorporated</td>
			<td >6775</td>
			<td >Vortex mixer</td>
		</tr>
		<tr >
			<td >FACSCanto II Flow cytometer&#160; with FACSDiva software version 6.1.3 or equivalent</td>
			<td >BD Biosciences</td>
			<td >338960</td>
			<td >Flow cytometer</td>
		</tr>
	</tbody>
</table>

routine screening method for microparticles in platelet transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Average preparation time 
The platelet screening process using a DLS system is summarized in Figure 1. Platelets are tested with the DLS system at the time of receipt from the blood supplier. As detailed in Table 1 , the average preparation time for trained users is 2 min 23 s while the clean-up and post-test work times are 14 s and 46 s, respectively. In total, the average user takes 3 min and 23 s of hands-on time per test.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Activity&#160;</td>
			<td>Active time</td>
			<td>Walk-away testing time</td>
			<td>TOTAL</td>
		</tr>
		<tr>
			<td>Prepare DLS System</td>
			<td>14 s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assemble sampling tool</td>
			<td>28 s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Obtain segment</td>
			<td>52 s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fill capillary and start test</td>
			<td>49 s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>5 min</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean-up</td>
			<td>14 s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tag and inventory platelet bag</td>
			<td>46 s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Total time needed per sample</td>
			<td>3 min 23 s</td>
			<td>5 min</td>
			<td>8 min 23 s</td>
		</tr>
	</tbody>
</table>
Table 1: Active testing time breakdown. The test itself is a walk away test with an average duration of 5 min. The average user takes 3 min 23 s to prepare the DLS system for a test, obtain and test a sample following this protocol, and tag the platelet bag.
Precision 
The precision of the DLS system was assessed at three microparticle levels, 0-7%, 12-25% and 28-75%. The clinically relevant range of %MP is 3 - 75%. Two operators tested low, medium, and high control samples for 16 operating days on two DLS systems in parallel. Samples were tested in duplicate, but in random order on each testing day.
Table 2 summarizes the within-device precision of the DLS measurements for Percent Microparticles (%MP) relative to platelets.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Microparticle Content</td>
		</tr>
		<tr>
			<td></td>
			<td>Low</td>
			<td>Medium</td>
			<td>High</td>
		</tr>
		<tr>
			<td>Mean %MP (%)</td>
			<td>4.4</td>
			<td>19.5</td>
			<td>53.8</td>
		</tr>
		<tr>
			<td>Standard deviation (%)</td>
			<td>1.8</td>
			<td>2.6</td>
			<td>5.8</td>
		</tr>
		<tr>
			<td>CV (%)</td>
			<td>40.4</td>
			<td>13.2</td>
			<td>10.6</td>
		</tr>
	</tbody>
</table>
Table 2: Within-device precision of Percent Microparticles (%MP). At very low microparticle content small platelets may contribute to %MP resulting in increased variability for low microparticle content samples.
Table 3 shows the within-device precision of the DLS measurements for average microparticle radius between 50-550 nm.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Microparticle Content</td>
		</tr>
		<tr>
			<td></td>
			<td>Low</td>
			<td>Medium</td>
			<td>High</td>
		</tr>
		<tr>
			<td>Mean Radius (nm)</td>
			<td>331</td>
			<td>161</td>
			<td>188</td>
		</tr>
		<tr>
			<td>Standard deviation (mm)</td>
			<td>133</td>
			<td>41</td>
			<td>25</td>
		</tr>
		<tr>
			<td>CV (%)</td>
			<td>40.1</td>
			<td>25.2</td>
			<td>13.5</td>
		</tr>
	</tbody>
</table>
Table 3: Within-device precision of microparticle radius. At very low microparticle content small platelets may contribute to Percent Microparticles (%MP) resulting in increased variability for low microparticle content samples.
Table 4 shows the reproducibility of DLS measurements for Percent Microparticles (%MP).
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Microparticle Content</td>
		</tr>
		<tr>
			<td></td>
			<td>Low</td>
			<td>Medium</td>
			<td>High</td>
		</tr>
		<tr>
			<td>Mean %MP (%)</td>
			<td>4.4</td>
			<td>29.2</td>
			<td>53.6</td>
		</tr>
		<tr>
			<td>Reproducibility (%)</td>
			<td>1.5</td>
			<td>2.3</td>
			<td>5</td>
		</tr>
		<tr>
			<td>CV (%)</td>
			<td>35</td>
			<td>11.8</td>
			<td>9.4</td>
		</tr>
	</tbody>
</table>
Table 4: Reproducibility of Dynamic Light Scattering (DLS) measurements for Percent Microparticles (%MP).
Linearity 
Figure 2&#160;shows that DLS results are linear, i.e., fit a straight line with respect to the assigned values of the samples. Seven samples were prepared with differing microparticle content. Samples with high (MP7) and low (MP1) microparticle content and matching platelet concentration were prepared and mixed at different ratios to create intermediate samples (MPx). Samples were tested with flow cytometry as described previously19 and DLS and %MP results at each concentration were plotted as input and output. The coefficient of determination was found to be 0.985. Examples of DLS histograms for low and high microparticle content are shown in Figure 3A. The DLS results were confirmed by flow cytometry (Figure 3B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56893/56893fig2.jpg" /> 
Figure 2: Comparison of flow cytometry and DLS results.&#160;Linear relationship between %MP determined by Flow Cytometry (input) and DLS (output), the original DLS data from the two samples marked by the open symbol &#9675; are shown in&#160;Figure 3.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56893/56893fig2large.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/56893/56893fig3.jpg" /> 
Figure 3: Microparticle content to differentiate between activated and non-activated platelets. (A)&#160;DLS results of MP content in activated platelets (dashed line) was 57% compared to 4% in non-activated platelets (solid line). Tests were performed at a measurement temperature of 37 &#176;C, plasma viscosity setting of 1.06 x10-3&#160;Pa&#183;s, and total intensity settings between 200-600 kHz.&#160;(B)&#160;Flow cytometry results (obtained as described previously19) of the same samples as shown in (A); in the forward scatter histograms P1 and P2 represent the MP and platelet gates, respectively; for activated platelets (left) 76% of events fell into the MP gate compared to 6% for non-activated platelets (right). The linear regression line suggests a constant relative contribution of background noise to %MP by flow cytometry leading to the consistently higher results.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56893/56893fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Specificity/Interference 
The International Organization for Standardization (ISO) defines analytical specificity as the ability of a measurement procedure to detect or measure only the measurand while there are other quantities present in the sample. The analytical specificity of microparticle screening might be affected by red blood cells (RBC). DLS measures MP content relative to platelet content. RBC might interfere because the scattering contribution of RBC is included in the scattering contribution of platelets, reducing the relative contribution of MP. Regulatory limits for the allowable RBC content in blood products exist; a conservative conversion of the threshold recommended by AABB for allowable RBC concentration in platelet concentrates-2 mL of packed RBC in one unit of platelets-resulted in 8.0 x1010 cells/L (assumptions: RBC volume is 8.5 x10-14L, hematocrit of packed RBC is 68%, volume of platelet unit is 200 mL). The reported residual RBC concentrations in different products are well below this threshold46.
Three different donors donated platelets and red blood cells (RBC) on two different days, as eligible, for three independent experiments. The initial RBC content in the platelet concentrates (reference sample) was 0.05 - 0.15 x109 cells/L as determined with a hemocytometer. Five additional samples were created by spiking-in known quantities of RBC into aliquots of the platelet concentrate; target RBC levels in these samples were 1.0, 5.0, 10, 40 and 80 x109 cells/L.
The interference threshold of red blood cells was approximately 1.0 x1010 cells/L (Figure 4), which also correlated with the level at which the presence of red blood cells was visually evident (Figure 5). Above this level, %MP was underestimated which means that in visually red samples-which contain RBC rather than hemoglobin-the reported microparticle content will be too low.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56893/56893fig4.jpg" /> 
Figure 4: Linear relationship between %MP (DLS) and RBC Concentration (cells/L).&#160;Increasing RBC concentrations of 0.1 x109, 1.0 x109, 5.0 x109, 1.0 x1010, 4.0 x1010, and 8.0 x1010&#160;cells/L (from left to right) from 3 independent experiments (&#9675; experiment 1, &#9679; experiment 2, &#9633; experiment 3, linear regression lines are shown for each experiment). Above 1.0 x1010&#160;RBC/L leads to underestimation of %MP. Visual appearance of RBC containing samples is shown in&#160;Figure 5.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56893/56893fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56893/56893fig5.jpg" /> 
Figure 5: Visual appearance of RBC containing samples.&#160;Redness of platelet samples containing RBC concentrations of 0.1 x109, 1.0 x109, 5.0 x109, 1.0 x1010, 4.0 x1010, and 8.0 x1010&#160;cells/L from left to right.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56893/56893fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Accuracy 
Accuracy is defined as the difference between a single measurement result and a true quantity value assigned to the sample. This measurement error includes a systematic component estimated by measurement bias and a random component estimated by a standard deviation. Thus, the accuracy of a measurement result is a combination of trueness and precision.
A bead standard was used to determine the accuracy of the DLS test. Accuracy was evaluated at two concentrations within the clinically relevant range of the assay of 3 - 75% MP. Reference bead mixtures were used to prepare samples of known concentrations because reference beads have a known size and concentration. Consequently, reference beads can be mixed to obtain samples with desired particle sizes and concentrations.
Standard polystyrene beads with a 125 nm radius were used to represent microparticles and beads with 1.5 &#181;m radius were used to represent platelets. The accuracy of the microparticle assay was assessed with bead mixtures of approximately 20% and 50% MP content. The accuracy of the measured particle radii was compared to the particle radii documented on the Certificates of Analysis for the reference beads as shown in Table 5.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">125 nm beads</td>
			<td colspan="3">1.5 &#181;m beads</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td>Certificate of Analysis</td>
			<td colspan="2"></td>
			<td>Certificate of Analysis</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td></td>
			<td>20% MP</td>
			<td>50% MP</td>
			<td></td>
			<td>20% MP</td>
			<td>50% MP</td>
		</tr>
		<tr>
			<td>Mean Radius</td>
			<td>122.0</td>
			<td>113.8</td>
			<td>124.4</td>
			<td>1.5</td>
			<td>1.6</td>
			<td>1.60</td>
		</tr>
		<tr>
			<td>Std Dev</td>
			<td>4.5</td>
			<td>2.83</td>
			<td>3.6</td>
			<td>0.035</td>
			<td>0.06</td>
			<td>0.07</td>
		</tr>
		<tr>
			<td>CV</td>
			<td>3.60%</td>
			<td>2.48%</td>
			<td>2.89%</td>
			<td>2.20%</td>
			<td>3.74%</td>
			<td>4.05%</td>
		</tr>
		<tr>
			<td>Accuracy</td>
			<td></td>
			<td>6.70%</td>
			<td>2.00%</td>
			<td></td>
			<td>6.50%</td>
			<td>6.30%</td>
		</tr>
	</tbody>
</table>
Table 5: Accuracy of Dynamic Light Scattering (DLS) measurements. Size comparison for DLS results against certificate of analysis for beads with 125 nm radius and 1.5 &#181;m radius in mixtures.

Watch this Scientific Journal Video about Routine Screening Method for Microparticles in Platelet Transfusions at JoVE.com

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

routine-screening-method-for-microparticles-in-platelet-transfusions

Research

JoVE Journal

Medicine

15.8K Views.  LightIntegra Technology Inc.. Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Video: Routine Screening Method for Microparticles in Platelet Transfusions

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 ...

Bioengineering

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 ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Immunology and Infection

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

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Neuroscience

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by&#160;human platelets,&#160;understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,&#160;identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Author Spotlight: Innovative Techniques for ROS Detection and Implications for Platelet Research

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet regulation.

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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.

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Biology

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

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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.

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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 ...