Name: preparation and pathogen inactivation of double dose buffy coat platelet products using the intercept blood system
Uploaded: 2012-12-07T11:00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System at JoVE.com

Funding for the publication is provided by Cerus Corp, manufacturer of the INTERCEPT Blood System.

1. Whole Blood Collection
<ol>
	<li>Collect whole blood from volunteer donors in 450 ml top/bottom collection sets according to local blood collection guidelines.</li>
	<li>The whole blood is stored for a minimum of 2 hr on a cooling plate before centrifugation and separation.</li>
</ol>
2. Buffy Coat Preparation
<ol>
	<li>Centrifuge the whole blood using a hard spin to separate the blood into three layers: red blood cells, buffy coat, and plasma. Centrifuge parameters used were 4,880 RCF for 11 min at 22&plusmn;2 &deg;C.</li>
	<li>Use an automated blood separator to express the plasma into the top satellite bag and the red blood cells (RBC) into the bottom satellite bag, leaving the buffy coat in the collection container. Target mean volume and hematocrit ranges for buffy coats are approximately 48 ml and 37%, respectively.</li>
	<li>The buffy-coats are stored overnight on a platelet agitator at 22&plusmn;2 &deg;C.</li>
</ol>
3. Buffy Coat Pooling
<ol>
	<li>Sterile connect 7 buffy coats and 300 ml of SSP+ platelet additive solution (PAS) in a train configuration with parallel lines to reduce the train length; the PAS should be at the top of the train. Clamp the line between the PAS and the first buffy coat.</li>
	<li>Open the sterile connections between the buffy coat units and allow the buffy coats to drain into the last container.</li>
	<li>Open the weld and the clamp between the PAS and the first buffy coat. Allow one-third of the additive solution to rinse through each of the buffy coat containers sequentially. Repeat 2 more times each time using one-half of the remaining PAS.</li>
	<li>The average buffy coat pool volume should be approximately 600 ml. If the target volume and hematocrit of the individual buffy coats are met, the plasma ratio will be 32 - 47% as required for the INTERCEPT pathogen inactivation treatment later in the process.</li>
	<li>Discard the empty SSP+ and buffy coat containers.</li>
	<li>For optimal platelet recovery, keep the pool on the agitator for 1 hr prior to centrifugation.</li>
	<li>Sterile connect a platelet storage container with integrated leukoreduction filter to the buffy coat pool.</li>
	<li>Perform a "soft spin" of the buffy coat pool to separate the red blood cells from the platelets in suspension (462 RCF for 9 min, 20 sec). Express the platelet suspension using an automated blood separator through the leukoreduction filter into the platelet storage container.</li>
	<li>The previously described processing requirements ensure that the platelet suspension meets the INTERCEPT processing specifications of 300 ml - 420 ml volume, 2.5-7.0 x 1011 platelet dose, and &le; 4 x 106/ml red cells.</li>
</ol>
4. INTERCEPT Treatment
<ol>
	<li>Perform the INTERCEPT treatment before the end of Day 1 after collection (day 0 is the day of collection).</li>
	<li>Unwrap the INTERCEPT Processing Set with Dual Storage Containers from the clear plastic pouch.</li>
	<li>Sterile connect the platelet suspension container to the tubing of the amotosalen container on the INTERCEPT processing set.</li>
	<li>Label the INTERCEPT processing set storage containers with the appropriate blood product identification following local requirements.</li>
	<li>Hang the platelets and break first the bottom cannula on the amotosalen container, allowing the amotosalen solution to flow into the illumination container. Break the top cannula on the amotosalen container allowing the platelets to flow through the amotosalen container into the illumination container.</li>
	<li>Gently mix the platelet and amotosalen mixture and express the air from the illumination container into the amotosalen container.</li>
	<li>Express a small amount of the platelet mixture into the tubing, filling about 4 cm of the tubing. This ensures platelets in both the tubing and illumination container undergo the pathogen inactivation treatment.</li>
	<li>Seal the tubing between the illumination container and amotosalen container. Leave no more than approximately 4 cm of tubing extending from the illumination container. Remove and discard the empty platelet and amotosalen containers and close the clamps on the sampling pouches.</li>
	<li>Place the processing set into the illuminator with the illumination container in the large compartment on the left and the organizer in the smaller compartment on the right side.</li>
	<li>Use the hand-held barcode device to enter the donation ID, product code, and processing set lot number into the illuminator. Close the metal cover and when prompted on the illuminator graphical interface, close the drawer. Press "Start" to start the Illumination.</li>
	<li>After illumination, remove the processing set from the illuminator. The illuminator automatically prints the treatment report for the treated platelet unit(s).</li>
	<li>Unwrap the containers from the organizer and hang the platelets and processing set, break the cannula at the outlet of the illumination container, and allow the platelets to flow into the compound adsorption device (CAD) container.</li>
	<li>Taking care not to bend the CAD wafer, express the air from the CAD container into the illumination container using a plasma extractor.</li>
	<li>Seal the tubing near to the inlet port of the CAD container. Remove and discard the empty illumination container.</li>
	<li>Place the CAD container with the attached storage containers on a platelet agitator for at least 6 hr, but no more than 16 hr. This will result in reduction of residual amotosalen to a concentration of &le; 2 &mu;M.</li>
	<li>After CAD treatment, remove the platelet units from the agitator. Hang the platelets. Break the cannula and allow platelets to flow into the storage containers.</li>
	<li>Express the air from the storage containers into the CAD container. Let the residual platelet concentrate flow back into the storage containers by gravity. Seal the tubing above the Y-fitting and remove the empty CAD container.</li>
	<li>Redistribute the volume between the storage containers as needed. Volume adjustments are made by weighing the storage containers. Seal the tubing to each storage container a few centimeters above the inlet of the storage container; this facilitates obtaining a sterile sample from the final product as described in 5.2.</li>
</ol>
5. Product Sampling
<ol>
	<li>For routine QC testing, the final storage containers can be sampled once by using the sampling pouch on the storage containers. To do so, ensure the platelet unit is well mixed, then open the clamp to the pouch and squeeze several times. Seal the tubing after the pouch has filled with platelets. Transfer the platelet sample into an appropriate laboratory tube and perform assays immediately.</li>
	<li>To obtain samples at multiple time points over the course of storage, such as for a validation study, sterile connect a new sampling container to the tubing of the platelet storage container. Ensure that platelets are well mixed prior to transfer to the sampling container.</li>
</ol>
6. In vitro Function Evaluation
<ol>
	<li>For this validation study, in vitro evaluation was performed after CAD treatment (Day 1 or Day 2, depending on CAD duration) and again on Day 4 (or Day 5) and Day 7. In vitro measurements included volume, platelet count, pH, blood gas (pO2, pCO2), glucose, lactate, and swirling.</li>
	<li>Volume was determined by weight using 1.01 g/ml as the specific gravity of platelets in additive solution.</li>
	<li>Hemoglobin contamination was evaluated visually using comparison to a color chart.</li>
	<li>Swirling was determined visually.</li>
	<li>See "Table of equipment" for methodology of other assays.</li>
</ol>
7. Representative Results
The process of producing double dose buffy coat platelets begins with the production of individual buffy coats which meet target specifications for volume and hematocrit. As it is not practical to measure the hematocrit of the individual buffy coats during the final process validation, we began by undertaking a separate effort to optimize our buffy coats to ensure we could consistently meet target volume and hematocrit. As shown in Table 1, our optimized buffy coats compared favorably to the target values for both volume and hematocrit at 46 &plusmn; 2 ml and 37 &plusmn; 3%, respectively.
After pooling, the volume and hematocrit of the buffy coat pool should be approximately 600 ml and 20%, respectively, prior to the soft spin centrifugation. As illustrated in Table 2, our buffy coat pools averaged 615 &plusmn; 5 ml; the hematocrit averaged 19 &plusmn; 1%.
The soft spin centrifugation results in a double dose platelet concentrate that will meet the input requirements for pathogen inactivation utilizing the INTERCEPT Blood System DS processing set. Key input parameters for INTERCEPT treatment include volume, platelet count, plasma ratio, and RBC content. In addition, we aim to recover &ge; 75% of the platelets in the platelet concentrate as compared to the buffy coat pool. Per local requirements, the white blood cell (WBC) contamination must be &lt;1x106/unit. The platelet concentrates in our validation met the key parameters for INTERCEPT treatment as well as the targets for WBC contamination and platelet recovery as shown in Table 3.
The INTERCEPT process for pathogen inactivation is performed on the double dose platelet concentrate using a single INTERCEPT processing set. The processing set contains two integral storage containers which allow the treated unit to be split into two individual therapeutic platelet doses at the completion of the pathogen inactivation process. European guidelines require that 75% of units tested contain &ge; 200x109 platelets per therapeutic dose1; local requirements in Sweden require that 75% of the units tested contain &gt;240x109 platelets per dose. After INTERCEPT treatment, the average platelet content was 265 &plusmn; 22 x109 (n=24). Furthermore, 88% of units met or exceeded 240x109 platelets per therapeutic dose; this is well within both the European guidelines and Swedish regulations. See Figures 1 &amp; 2 for an illustration of the double dose buffy coat preparation and INTERCEPT treatment processes respectively.
For this validation, we measured the in vitro characteristics of the platelet concentrates after INTERCEPT treatment (i.e. after the split into the individual storage containers); parameters were measured over 7 days of storage. Mean and standard deviation were collected for platelet dose, pH, pO2, pCO2, lactate production and glucose consumption.
Figure 3 shows the mean starting platelet dose of the double dose platelet concentrate before INTERCEPT treatment and the mean platelet dose in each of the split products after INTERCEPT treatment over 7 days of storage. Platelet loss during storage was approximately 9%. This reduction is not different from the expected loss of platelets during storage of conventional platelets.2
Table 4 summarizes the in vitro characteristics of the INTERCEPT platelets after treatment and splitting into single units.
Per European requirements, the pH of platelets must remain above 6.4 through the end of shelf life. During processing, the pH of the platelet concentrates drops slightly based on the platelet concentration, volume, and the gas permeability of the platelet storage container. Figure 4 illustrates the pH of the split platelet products over 7 days of storage. During storage, the pH is stable and well maintained within the processing requirements.
As shown in Figures 5A and 5B, the platelet O2 consumption and the CO2 production indicate continued respiration by the INTERCEPT platelets in the PL2410 container during 7 days of storage. Figures 6A and 6B show the lactate and glucose levels over 7 days of storage. Platelet glucose consumption and lactate production are consistent with each other during the 7 days of storage. 5 units had glucose level less than 1.11 mmol/l (the lower limit for the glucose assay).
The buffy coat preparation and pooling validation produced platelet concentrates that met the input criteria for INTERCEPT treatment; specifically, volume, platelet count, plasma ratio, and red blood cell contamination. The final units met the validation criteria over 7 days of storage including platelet dose and pH.
<table border="1">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Target Range</td>
			<td>Results Mean &plusmn; SD</td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>~ 48</td>
			<td>46 &plusmn; 2</td>
		</tr>
		<tr>
			<td>Hematocrit (%)</td>
			<td>~ 37</td>
			<td>37 &plusmn; 3</td>
		</tr>
		<tr>
			<td>Hold time before pooling</td>
			<td>Overnight on agitator</td>
			<td>Overnight on agitator</td>
		</tr>
	</tbody>
</table>
Table 1. Characteristics of the Individual Buffy Coats (n=19).
<table border="1">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Target Range</td>
			<td>Results Mean &plusmn; SD</td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>~ 600</td>
			<td>615 &plusmn; 5</td>
		</tr>
		<tr>
			<td>Hematocrit (%)</td>
			<td>~ 20</td>
			<td>19 &plusmn; 1</td>
		</tr>
		<tr>
			<td>Hold time before centrifugation</td>
			<td>1 hr on agitator</td>
			<td>1 hr on agitator</td>
		</tr>
	</tbody>
</table>
Table 2. Characteristics of the Buffy Coat Pools (n=12).
<table border="1">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Target</td>
			<td>Results Mean &plusmn; SD</td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>370 - 420</td>
			<td>404 &plusmn; 8</td>
		</tr>
		<tr>
			<td>Platelet count (x109/unit)</td>
			<td>250 - 700</td>
			<td>577 &plusmn; 62</td>
		</tr>
		<tr>
			<td>Mean Platelet recovery (%)</td>
			<td>&ge; 75</td>
			<td>75 &plusmn; 4</td>
		</tr>
		<tr>
			<td>Plasma ratio (%)</td>
			<td>32 - 47</td>
			<td>38 &plusmn; 1</td>
		</tr>
		<tr>
			<td>RBC (x106/ml)</td>
			<td>&le; 4</td>
			<td>1.2 &plusmn; 0.4</td>
		</tr>
		<tr>
			<td>WBC (x106/unit)</td>
			<td>&le; 1</td>
			<td>0.11 &plusmn; 0.1</td>
		</tr>
		<tr>
			<td>Hold time before INTERCEPT (hr)</td>
			<td>&le; end of day 1</td>
			<td>&le; end of day 1</td>
		</tr>
	</tbody>
</table>
Table 3. Characteristics of the Double-Dose Platelet Concentrates (n=12).
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Mean &plusmn; SD </td>
			<td>Min </td>
			<td>Max </td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>199 &plusmn; 16</td>
			<td>182</td>
			<td>236</td>
		</tr>
		<tr>
			<td>Platelet count (x109/unit)</td>
			<td>265 &plusmn; 22</td>
			<td>225</td>
			<td>292</td>
		</tr>
		<tr>
			<td>pH (22 &deg;C)</td>
			<td>6.9 &plusmn; 0.1</td>
			<td>6.8</td>
			<td>7.0</td>
		</tr>
		<tr>
			<td>pO2 (kPa)</td>
			<td>21.4 &plusmn; 4.8</td>
			<td>12.8</td>
			<td>27.3</td>
		</tr>
		<tr>
			<td>pCO2 (kPa)</td>
			<td>4.3 &plusmn; 0.4</td>
			<td>3.3</td>
			<td>4.9</td>
		</tr>
		<tr>
			<td>Lactate (mmol/l)</td>
			<td>9.1 &plusmn; 1.7</td>
			<td>6.8</td>
			<td>12.4</td>
		</tr>
		<tr>
			<td>Glucose (mmol/l)</td>
			<td>5.3 &plusmn; 0.9</td>
			<td>3.7</td>
			<td>6.5</td>
		</tr>
	</tbody>
</table>
Table 4. Characteristics of the INTERCEPT Treated Platelets Day 1/2 (Single Units, n=24).
<img alt="Figure 1" src="/files/ftp_upload/4414/4414fig1.jpg" /> 
Figure 1. Production of a double dose platelet concentrate from a pool of 7 buffy coats.
<img alt="Figure 2" src="/files/ftp_upload/4414/4414fig2.jpg" /> 
Figure 2. Pathogen inactivation of a double dose platelet concentrate with the INTERCEPT Blood System.
<img alt="Figure 3" src="/files/ftp_upload/4414/4414fig3.jpg" /> 
Figure 3. Mean platelet dose before and for 7 days after INTERCEPT treatment (n=12 before INTERCEPT; n=24 after INTERCEPT).
<img alt="Figure 4" src="/files/ftp_upload/4414/4414fig4.jpg" /> 
Figure 4. Mean platelet pH &plusmn;SD after INTERCEPT treatment (n=24).
<img alt="Figure 5A" src="/files/ftp_upload/4414/4414fig5a.jpg" /> 
Figure 5A. Mean pO2 &plusmn;SD after INTERCEPT treatment (n=24).
<img alt="Figure 5B" src="/files/ftp_upload/4414/4414fig5b.jpg" /> 
Figure 5B. Mean pO2 &plusmn;SD after INTERCEPT treatment (n=24).
<img alt="Figure 6A" src="/files/ftp_upload/4414/4414fig6a.jpg" /> 
Figure 6A. Mean lactate levels &plusmn;SD after INTERCEPT treatment (n=24).
<img alt="Figure 6B" src="/files/ftp_upload/4414/4414fig6b.jpg" /> 
Figure 6B. Mean glucose levels &plusmn;SD after INTERCEPT treatment (n=24).

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	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the preparation, use and quality assurance of blood components. , (2010).</li><li>Van Rhenen, D. J., Vermeij, J., Mayaudon, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characteristics+of+S-59+photochemically+treated+platelet+concentrates+derived+from+buffy+coats.">Functional characteristics of S-59 photochemically treated platelet concentrates derived from buffy coats.</a> Vox Sang. 79, 206-214 (2000).</li><li>Wollowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+and+RNA+in+pathogens:+mode+of+action+of+amotosalen+HCl.">Targeting DNA and RNA in pathogens: mode of action of amotosalen HCl.</a> Transfus. Med. Rev. 31, 11-16 (2004).</li><li>Irsch, J., Lin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+Inactivation+of+Platelet+and+Plasma+Blood+Components+for+Transfusion+Using+the+INTERCEPT+Blood+SystemTM.">Pathogen Inactivation of Platelet and Plasma Blood Components for Transfusion Using the INTERCEPT Blood SystemTM.</a> Transfus. Med. Hemother. 38, (2011).</li><li>Lin, L., Dikeman, R., Molini, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemical+treatment+of+platelet+concentrates+with+amotosalen+and+long-wavelength+ultraviolet+light+inactivates+a+broad+spectrum+of+pathogenic+bacteria.">Photochemical treatment of platelet concentrates with amotosalen and long-wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria.</a> Transfusion. 44, 1496-1504 (2004).</li><li>Lin, L., Hanson, C., Alter, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+viruses+in+platelet+concentrates+by+photochemical+treatment+with+amotosalen+and+long-wavelength+ultraviolet+light.">Inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light.</a> Transfusion. 45, 580-590 (2005).</li><li>Allain, J. 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Production and Free Access to this article is sponsored by Cerus.

Buffy coat platelets require several processing steps that result in post-collection costs, including personnel time, consumables, equipment, and waste, which must be factored into the overall cost of producing the platelet units. Improving the platelet yield from each buffy coat (via optimizing the buffy coat volume and hematocrit) allows the production of a double dose buffy coat platelet unit from a pool of seven buffy coats. When this optimization is performed, the number of buffy coats required to produce a fixed number of platelet doses may be reduced, thereby improving the overall utilization of buffy coats and enabling the production of additional platelet concentrates (PCs). Personnel time as well as consumable and equipment costs such as pooling sets, platelet additive solution, sterile connection wafers, filter sets, and centrifugation procedures are reduced by up to 50%. In addition to optimizing platelet production and reducing associated costs, this pooling method can generate a platelet concentrate (PC) that meets the input requirement for use with the INTERCEPT dual storage container processing set, allowing us to provide enhanced protection to our transfusion recipients as well.
The INTERCEPT Blood System for pathogen inactivation uses amotosalen and ultraviolet A (UVA) light to covalently cross-link DNA and RNA, preventing nucleic acid replication and rendering pathogens unable to cause disease.3 It effectively inactivates a broad spectrum of pathogens including viruses, bacteria, parasites, and contaminating donor white blood cells.4-6 INTERCEPT provides an alternative to the current testing paradigm for emerging pathogens, which historically involves a substantial delay while a new test is developed and ultimately requires a significant financial investment to implement when a test becomes available.7,8 It can also provide an alternative to redundant safety measures such as bacterial detection9 and gamma irradiation.10-12 In addition, INTERCEPT allows us to treat double dose platelet units which benefits our production efficiency and helps us adhere to our budgets.
INTERCEPT and the double dose buffy coat method can be adapted to a variety of blood center workflows. As examples, whole blood collections can be increased to 500 ml; collections could also be stored overnight at 22 &deg;C prior to separation. In addition, an alternate buffy coat pooling method is possible (e.g. an octopus method instead of a train method) and/or an automated device (e.g. TACSI) can be utilized for the second centrifugation and separation of the platelet suspension from the remaining red cells.
If increased platelet content is needed, several techniques are available including pre-selection of donors based on platelet count, overnight incubation of the whole blood, adjustment of centrifugation settings, or utilizing a pool of 8 buffy coats in lieu of 7.
Due to the limitation of pooling sets currently available and the capacity of centrifuge buckets, the total volume of the buffy coat pool should be approximately 600 ml. The centrifugation process and settings described in this protocol have been optimized to yield product that meets the INTERCEPT parameters for a pool of 7 buffy coats. The centrifugation settings must be modified if the number of buffy coats in the pool is modified.
In some countries, the minimum platelet dose is higher and the QC requirements are more stringent than in Sweden. As such, our results may not be universally applicable. In these countries, the double dose PC prior to INTERCEPT treatment will need to contain a larger number of platelets in order to meet local requirements after INTERCEPT treatment and split. Because the maximum platelet content and treatment volume for INTERCEPT are 7x1011 platelets and 420 ml respectively, the percentage of units which exceed the INTERCEPT processing requirements or have insufficient platelet content to be split into two therapeutic doses, will vary depending on factors such as local requirements for platelet dose, QC criteria, buffy coat optimization results, and production stability.
If a double dose PC exceeds the INTERCEPT processing requirements, it may be manually adjusted to meet the requirements and subsequently treated. In those few occasions where the 7-buffy coat pool yields insufficient platelets for a double dose product after INTERCEPT, we elect not to perform the INTERCEPT treatment. Alternatively, other blood centers may choose to treat the PC with a single dose INTERCEPT processing set (i.e. the Large Volume set) and store the PC as a single larger therapeutic dose, thereby performing pathogen inactivation on all units. In order to ensure we meet our QC requirements and to pathogen inactivate as many of our platelet units as possible, we select buffy coats based on the donor platelet counts, which, when pooled, will result in a minimum platelet content of 5.6x1011 platelets in the pool. This ensures sufficient platelet content to produce two therapeutic doses after INTERCEPT treatment.
Our validation demonstrates that 7 whole blood derived buffy coat units can be successfully pooled and treated with the INTERCEPT process for platelets, resulting in 2 pathogen inactivated platelet products that meet the acceptance criteria for manufacturing (Swedish requirements and European guidelines) and for support of patients requiring platelet transfusions according to clinical practice guidelines and standard platelet infusion methods in Sweden.

<table border="1">
	<tbody>
		<tr>
			<td>Material / Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Whole blood donation, primary separation, and platelet production</td>
		</tr>
		<tr>
			<td>Blood collection pack</td>
			<td>Fenwal</td>
			<td>R6485</td>
			<td>Top/Bottom set</td>
		</tr>
		<tr>
			<td>Automated component extractor</td>
			<td>Fenwal</td>
			<td>Optipress-II</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blood mixer and balance system</td>
			<td>Baxter</td>
			<td>Easymix V3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Platelet leukocyte filtration set</td>
			<td>Fenwal</td>
			<td>K4R7042</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich</td>
			<td>Roto Silenta 63 RS</td>
			<td>Version 5.5</td>
		</tr>
		<tr>
			<td>Platelet additive solution - SSP+</td>
			<td>MacoPharma</td>
			<td>SSP2030U</td>
			<td>300 ml</td>
		</tr>
		<tr>
			<td>Sterile tubing welder</td>
			<td>Terumo</td>
			<td>T-SCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>INTERCEPT treatment &amp; storage</td>
		</tr>
		<tr>
			<td>INTERCEPT processing set</td>
			<td>Cerus</td>
			<td>INT2503</td>
			<td>Dual Storage (DS) set</td>
		</tr>
		<tr>
			<td>INTERCEPT Illuminator</td>
			<td>Cerus</td>
			<td>INT100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PC sample pack</td>
			<td>Fenwal</td>
			<td>FTX 1122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Helmer</td>
			<td>PC2200/PC3200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agitator</td>
			<td>Helmer</td>
			<td>PF48H/PF96H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Evaluation of in vitro Platelet Function</td>
		</tr>
		<tr>
			<td>Blood gas analyzer</td>
			<td>Radiometer</td>
			<td>ABL 735</td>
			<td>Used for pH, blood gases, and lactate measurement</td>
		</tr>
		<tr>
			<td>Chemistry system</td>
			<td>Ortho Clinical Diagnostic</td>
			<td>Vitros 5.1</td>
			<td>Used for glucose measurement</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Boule Medical AB</td>
			<td>Medonic CA620-Cellguard</td>
			<td>Used for platelet count measurement</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>FACSCanto</td>
			<td>Used for white blood cell measurement</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System at JoVE.com

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

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 ...

preparation-pathogen-inactivation-double-dose-buffy-coat-platelet

Research

JoVE Journal

Medicine

Minimal Erythema Dose (MED) Testing

Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual's skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual's skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.

Minimal Erythema Dose MED Testing

This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.

Ultraviolet radiation (UV) therapy is  sometimes used as a treatment for various common skin conditions, including  psoriasis, acne, and eczema. The ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

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 ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

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.

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 ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

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 ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...