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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

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

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

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

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

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

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

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

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

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

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

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...