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±2 °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±2 °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 ≤ 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 ≤ 2 μM.</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 ± 2 ml and 37 ± 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 ± 5 ml; the hematocrit averaged 19 ± 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 ≥ 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 ≥ 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 ± 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 ± SD</td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>~ 48</td>
			<td>46 ± 2</td>
		</tr>
		<tr>
			<td>Hematocrit (%)</td>
			<td>~ 37</td>
			<td>37 ± 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 ± SD</td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>~ 600</td>
			<td>615 ± 5</td>
		</tr>
		<tr>
			<td>Hematocrit (%)</td>
			<td>~ 20</td>
			<td>19 ± 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 ± SD</td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>370 - 420</td>
			<td>404 ± 8</td>
		</tr>
		<tr>
			<td>Platelet count (x109/unit)</td>
			<td>250 - 700</td>
			<td>577 ± 62</td>
		</tr>
		<tr>
			<td>Mean Platelet recovery (%)</td>
			<td>≥ 75</td>
			<td>75 ± 4</td>
		</tr>
		<tr>
			<td>Plasma ratio (%)</td>
			<td>32 - 47</td>
			<td>38 ± 1</td>
		</tr>
		<tr>
			<td>RBC (x106/ml)</td>
			<td>≤ 4</td>
			<td>1.2 ± 0.4</td>
		</tr>
		<tr>
			<td>WBC (x106/unit)</td>
			<td>≤ 1</td>
			<td>0.11 ± 0.1</td>
		</tr>
		<tr>
			<td>Hold time before INTERCEPT (hr)</td>
			<td>≤ end of day 1</td>
			<td>≤ 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> </td>
			<td>Mean ± SD </td>
			<td>Min </td>
			<td>Max </td>
		</tr>
		<tr>
			<td>Volume (ml)</td>
			<td>199 ± 16</td>
			<td>182</td>
			<td>236</td>
		</tr>
		<tr>
			<td>Platelet count (x109/unit)</td>
			<td>265 ± 22</td>
			<td>225</td>
			<td>292</td>
		</tr>
		<tr>
			<td>pH (22 °C)</td>
			<td>6.9 ± 0.1</td>
			<td>6.8</td>
			<td>7.0</td>
		</tr>
		<tr>
			<td>pO2 (kPa)</td>
			<td>21.4 ± 4.8</td>
			<td>12.8</td>
			<td>27.3</td>
		</tr>
		<tr>
			<td>pCO2 (kPa)</td>
			<td>4.3 ± 0.4</td>
			<td>3.3</td>
			<td>4.9</td>
		</tr>
		<tr>
			<td>Lactate (mmol/l)</td>
			<td>9.1 ± 1.7</td>
			<td>6.8</td>
			<td>12.4</td>
		</tr>
		<tr>
			<td>Glucose (mmol/l)</td>
			<td>5.3 ± 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="Buffy coat platelet preparation process, leukoreduction diagram; centrifuge, filter, store method." src="/files/ftp_upload/4414/4414fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Production of a double dose platelet concentrate from a pool of 7 buffy coats.
<img alt="Double dose platelet processing diagram; steps: add, inactivate, remove amotosalen; INTERCEPT setup." src="/files/ftp_upload/4414/4414fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Pathogen inactivation of a double dose platelet concentrate with the INTERCEPT Blood System.
<img alt="Platelet dose chart comparing INTERCEPT treatment effects on storage; bar graph, statistical analysis." src="/files/ftp_upload/4414/4414fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Mean platelet dose before and for 7 days after INTERCEPT treatment (n=12 before INTERCEPT; n=24 after INTERCEPT).
<img alt="Mean platelet pH chart over 7 days storage; data analysis of pH variability at 22°C." src="/files/ftp_upload/4414/4414fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Mean platelet pH ±SD after INTERCEPT treatment (n=24).
<img alt="Bar chart showing mean pO2 changes over 7 days of storage; error bars indicate variability." src="/files/ftp_upload/4414/4414fig5a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5A"> 
Figure 5A. Mean pO2 ±SD after INTERCEPT treatment (n=24).
<img alt="Mean pCO₂ levels over 7 days; bar chart; storage duration impact; CO₂ measurement; chart analysis." src="/files/ftp_upload/4414/4414fig5b.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5B"> 
Figure 5B. Mean pO2 ±SD after INTERCEPT treatment (n=24).
<img alt="Mean lactate levels chart over 7 days; bar graph; lactate increase analysis; storage study." src="/files/ftp_upload/4414/4414fig6a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6A"> 
Figure 6A. Mean lactate levels ±SD after INTERCEPT treatment (n=24).
<img alt="Glucose levels over 7 days, bar chart; measures mg/dL with error bars; stability monitoring." src="/files/ftp_upload/4414/4414fig6b.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6B"> 
Figure 6B. Mean glucose levels ±SD after INTERCEPT treatment (n=24).

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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><tbody><tr><td colspan="4">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></td></tr><tr><td>Blood mixer and balance system</td><td>Baxter</td><td>Easymix V3</td><td></td></tr><tr><td>Platelet leukocyte filtration set</td><td>Fenwal</td><td>K4R7042</td><td></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></td></tr><tr><td colspan="4">INTERCEPT treatment &amp;amp; storage</td></tr><tr><td>INTERCEPT processing set</td><td><a href="http://www.interceptbloodsystem.com/" target="_blank"><img src="/img/new_window.png"> Cerus</a></td><td>INT2503</td><td>Dual Storage (DS) set</td></tr><tr><td>INTERCEPT Illuminator</td><td><a href="http://www.interceptbloodsystem.com/" target="_blank"><img src="/img/new_window.png"> Cerus</a></td><td>INT100</td><td></td></tr><tr><td>PC sample pack</td><td>Fenwal</td><td>FTX 1122</td><td></td></tr><tr><td>Incubator</td><td>Helmer</td><td>PC2200/PC3200</td><td></td></tr><tr><td>Agitator</td><td>Helmer</td><td>PF48H/PF96H</td><td></td></tr><tr><td colspan="4">Evaluation of &lt;em&gt;in vitro&lt;/em&gt; 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 ...

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Research

JoVE Journal

Medicine

28.6K Views.  Örebro University Hospital. This article describes the process used by Örebro 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.

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

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

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

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

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

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

Bioengineering

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Immunology and Infection

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

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

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

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

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

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 ...

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

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

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

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

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Biology

Isolating Quiescent Platelets for Metabolic Research

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%&#8211;40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

Platelet metabolism is of interest, particularly as it relates to the role of platelet hyper- and hypoactivity in bleeding and thrombotic disorders. Isolating platelets from plasma is necessary for some metabolic assays; presented here is a method for isolating of intracellular metabolites from washed platelets.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated ...

Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

Source: Mily, A., et al. <a href="https://app.jove.com/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further analysis.

Source: Mily, A., et al. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

A Riboflavin and UV Light Treatment of Platelet Products to Eliminate Bacteria

Source: Keil, S. D., et al. <a href="https://app.jove.com/v/52820">Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination.</a> J. Vis. Exp. (2015).
This video demonstrates the riboflavin and UV light-based pathogen reduction process against high-titer bacterial contamination in human platelet products. This process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

1. Platelet Product Preparation

	Collect a standard apheresis human platelet product at an accredited blood bank. The platelet product must meet the ...

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

Summary

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

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

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

Routine Screening Method for Microparticles in Platelet Transfusions

Microfluidics in Assessing Platelet Function

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

A Riboflavin and UV Light Treatment of Platelet Products to Eliminate Bacteria