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

In this video, a double dose platelet concentrate will be prepared from a pool of seven Buffy coats and subsequently treated with intercept. After watching this video, you should have a good understanding of how to produce a pathogen inactivated double-dose platelet concentrate. First, we present a schematic overview followed by a demonstration.

First seven units of donated whole blood are centrifuged and separated to yield seven buffy coat units that meet volume and hematocrit targets. The Buffy coats are connected, pooled, and centrifuged. The platelet suspension is then separated from the residual red blood cells to produce a double dose platelet concentrate to inactivate pathogens and leukocytes.

The double dose platelet concentrate is treated using the intercept system, which cross-links nucleic acids. Ultimately, this procedure yields two therapeutic platelet doses with acceptable in vitro quality over seven days of storage, as determined by assessment of pH glucose lactate, PO two and PC O2, And shallot doru. A technician from my blood center will demonstrate the procedure, the processing requirements we will describe.

First, ensure that the double dose platelet concentrate meets the intercept processing specifications of 300 to 420 milliliters. Volume 2.5 to seven times 10 to the 11 the platelet dose, and less than four times 10 to the six per milliliter. Red cells Begin this procedure with seven units of whole blood collected in 450 milliliter top bottom collection sets.

Place the blood units in centrifuge cups and perform a hard spin at 4, 880 RCF for 11 minutes at room temperature with break setting B five according to the manufacturer’s instructions following the spin, the blood will be separated into three layers. Red blood cells, buffy coat and plasma. Express the plasma into the top satellite bag and the red blood cells into the bottom satellite bag, leaving the buffy coat of approximately 48 milliliters and 37%hematocrit in the collection container.

Place the Buffy coats on a platelet agitator at room temperature overnight sterile connect, seven buffy coats and 300 milliliters of SSP plus platelet additive solution. In a train configuration with the additive solution at the top, clamp the line between the additive solution and the first buffy coat as shown here. Open the welds between the Buffy coat units and allow them to drain into the last buffy coat container.

Next, open the clamp and the weld between the additive solution and the first buffy coat, and allow approximately one third of the additive solution approximately 100 milliliters to rinse through each of the Buffy coat containers sequentially before closing the clamp, repeat the wash two more times each time using approximately 100 milliliters of the additive solution. Disconnect the pooled buffy coat container from the empty containers. The Buffy coat pool volume, including the additive solution, should be approximately 600 milliliters.

Place the pooled Buffy coats on an agitator for one hour at room temperature. Then connect a platelet storage container with an integrated leco reduction filter to the bag containing the Buffy coat pool. Place the pooled buffy coats and platelet storage container in a centrifuge and spin at 462 RCF for nine minutes and 20 seconds.

Following the spin, express the platelet suspension through the Luca reduction filter into the platelet storage container. The result is a double dose platelet concentrate containing approximately seven times 10 to the 11th platelets in 420 milliliters, which can undergo pathogen inactivation with intercept for pathogen inactivation. The double dose platelet concentrate is treated using an intercept processing set, which consists of antoin container and illumination container and dual storage containers.

Connect the platelet suspension container to the Amin container on the intercept processing set. Then hang the platelets. Break the bottom cannula on the amin container to allow the solution to flow into the illumination container.

Then break the top cannula on the Amin container to allow the platelets to flow through the Amin container into the illumination container. Gently mix the platelet and am tosin mixture. Lightly squeeze the illumination container to express the air from the illumination container.

Seal the tubing between the illumination and amato and container so that no more than four centimeters of tubing extends from the illumination container. Remove and discard the empty containers. Place the processing set in the illuminator with the illumination container in the large compartment on the left and the organizer in the smaller compartment on the right side.

Scan the donation id. Product code and processing. Set lot number into the illuminator.

Close the metal cover and the drawer. Press start to begin the illumination. The illuminator provides a controlled dose of UVA light to the platelet amin mixture in the illumination container.

Amin docks between the nucleic acid base pairs of pathogens, which may be present in the platelet unit. Upon illumination, permanent crosslinks are formed locking the DNA or RNA together so the pathogens can no longer replicate and cause disease in the recipient of the blood product. After illumination, remove the processing set.

Next, unwrap the containers from the organizer, hang the platelets and processing set. Break the cannula at the outlet of the illumination container and allow the platelets to flow into the CAD container. Inside the CAD container is a wafer with immobilized macroporous polystyrene beads that reduce the levels of residual amin in the blood product.

Taking care not to bend the CAD wafer. Express the air from the CAD container into the illumination container. Seal the tubing near the inlet port of the CAD container.

Remove and discard the empty illumination container. Place the CAD container with the attached storage containers on a platelet agitator for six hours to 16 hours. This will result in a reduction of residual amlin to a concentration of less than or equal to two micromolar.

After CAD treatment, remove the platelet units from the agitator, hang the platelets, break the cannula at the outlet of the CAD container and allow platelets to flow into the two storage containers. Next, express the air from the storage containers. Seal the tubing above the Y fitting and remove the CAD container.

The result is two pathogen inactivated platelet concentrates to assess in vitro platelet function. The platelet dose pH PO two PC O2 lactate production, and glucose consumption of the intercept. Platelet concentrates were determined through day seven of storage.

This figure shows mean starting platelet dose of the double dose platelet concentrate before intercept treatment, and the mean platelet dose in each of the treated split products through day seven of storage. As can be seen here, platelet loss during storage was approximately 9%This reduction is not different from the expected loss of platelets during storage of conventional platelets. Per European requirements, the pH of platelets must remain above 6.4 through the end of shelf life.

During processing, the pH of platelet concentrates drops slightly based on the platelet concentration volume and the gas permeability of the platelet storage container. This figure shows the pH of the split platelet products over seven days of storage during storage, the pH is stable and well-maintained within the processing requirements. Aerobic metabolism should continue to take place in stored platelets and is an indicator of the ability of the platelets to function in vivo after transfusion into a patient.

The platelet O2 consumption and the CO2 production indicate continued respiration by the intercept platelets in the PL 24 10 container during seven days of storage. Likewise, anaerobic metabolism should continue in stored platelets. Thus glucose consumption and lactate production are also indicators of the platelets ability to function following transfusion.

These two figures show the lactate and glucose levels over seven days of storage. Platelet glucose consumption and lactate production are consistent with each other. During the seven days of storage, five units had glucose levels of less than 1.11 millimoles per liter, which is the lower limit for the glucose assay.

The Buffy code preparation and pooling validation produced platelet concentrates that meet the input criteria for intercept treatment, specifically volume, platelet count, plasma ratio, and red blood contamination. The final units meet the validation criteria over seven days of storage, including platelet dose and pH. The main advantage of this technique over conventional buffer code preparation method, which produces a single dose platelet concentrate, is that it maximizes component yield from blood collections, reduces operating expenses by using a double dose technique and improves patient safety by inactivating transfusion transmitted pathogens.