Routine Screening Method for Microparticles in Platelet Transfusions

The overall goal of this method is to characterize platelet tranfusions by routine tests that can be performed in donor centers or hospital blood banks to optimize the use of these blood products for bleeding or blood cancer patients. This method can help answer key questions in transfusion medicine, such as, whether a platelet transfusion contains activated or non-activated platelets. The main advantage of this technique is that it provides a quick and accurate screen for microparticle content as a proven indicator of platelet activation.

Individuals new to this method, will need to learn how to take samples from tubing segments of bags, because this is not routinely done right now. We first had the idea for this method when we saw that platelets from different donors had high variability in the activation status and this seemed to have an impact on the patient. There are three methods to obtain sample from a platelet concentrate.

To sample from a pouch, mix the content of the platelet bag well by gentle horizontal movement for five seconds, tipping from end to end, five times. Open the clamp to the pouch, the pouch is evacuated and will fill by itself. It is not necessary to fill the pouch completely, as it is only a 100 microliter sample will be required for Dynamic Light Scattering or DLS testing.

Disconnect the pouch from the bag by heat sealing the connecting tubing with a tube sealer. When sampling from empty tubing, verify that the platelet bag tubing has been stored empty and that the tubing block is still in place. Visually inspect the tubing to verify that there is no platelet concentrate in the tubing.

Close the tube stripper on the tubing, as close to the tubing block as possible, by compressing the handles to squeeze the tubing between the rollers while keeping the stripper closed, release the tubing block. Slowly pull the stripper down the empty tubing, allowing for the tubing to slowly fill behind the stripper. Continue until the section below the stripper has fully inflated or until the stripper is within one inch of the end of the tubing.

It is critical to compress the handles of the tube stripper firmly and slowly pull the stripper down the tubing. Once the stopping point is reached with the stripper, use the tube sealer to heat seal the tubing one inch above the stripper. Now, release the handles of the manual tube stripper.

Heat seal again, one to two inches above the previous seal to create the testing segment. Then, cut off the testing segment from the platelet unit. This segment will be used for DLS testing.

When sampling from tubing that is not empty, hang the bag vertically and release the tubing block, if in place. Visually inspect the tubing to determine if there are any significant solid clumps. If solid clumps do exist, seal above them, such that they cannot be stripped into the bag.

Next, close the tube stripper on the tubing, as close to the sealed end of the tubing as possible. Strip the contents of the tubing, into the bag, by compressing the handles to squeeze the tubing between the rollers. While maintaining the clamping force, move the tube stripper along the tubing towards the bag.

Remove the platelet bag from the hanging hook and gently mix the bag for five seconds, by tipping it from end to end five times, while keeping the stripper closed. Hang the bag vertically, while keeping the stripper closed. Slowly pull the stripper down the empty tubing, allowing for the tubing to slowly fill behind the stripper.

Continue until the section below the stripper has fully inflated, or until the stripper is within one inch of the end of the tubing. Once the stopping point is reached with the stripper, use the tube sealer to heat seal the tubing, one inch above the stripper. After releasing the stripper, heat seal again one to two inches above the previous seal to create the testing segment.

Cut off and discard the last portion of the tubing. Then, cut off the testing segment from the platelet unit. This segment will be used for DLS testing.

Using a splash shield and clean, dry scissors, cut one end of the pouch tubing or testing segment. Immediately fill the capillary by using the sampling tool to draw the sample directly from the opened pouch, or tubing segment into the capillary. Seal the bottom of the filled capillary by gently pushing it into the capillary tube sealant, while applying a gentle twist and some pressure against the tray.

Then, disconnect the 100 microliter fixed volume pipette and tip from the capillary, insuring the capillary remains firmly embedded in the capillary tube sealant. Remove the capillary from the capillary tube sealant tray and wipe with an isopropyl alcohol pad. Insure that no air bubbles are trapped by gently flicking the bottom of the capillary.

Finally, place the capillary into the DLS system. To begin, start up the console, follow the instructions on the touch screen to login and select the microparticles or MP test option to initiate a new DLS test for measuring the microparticle content of a platelet sample. Enter the sample information using a barcode scanner, or enter it manually.

Place the capillary into the DLS system when instructed and start the test. When the test is completed, remove the sample from the capillary holder and appropriately dispose of the consumables according to facility guideline. Tag the platelet bag with the color corresponding to the result.

For example, orange for non-activated and pink for activated. Dynamic Light Scattering measures the speed of particles moving in suspension by Brownian motion. Based on the different speeds, small particles move fast and large particles move slowly.

This technology determines the size distribution of a sample. DLS determine that this transfusion contains non-activated platelets based on less than 15%microparticle content.Indeed. Mostly discoid platelets with very few microparticles were visible by microscopy.

However, a different platelet transfusion, which to the naked eye, looks exactly the same as the first bag, contained activated platelets, based on more than 15%microparticle content. In this sample, various platelet morphologies, small aggregates and many microparticles were visible by microscopy. After watching this video, you should have a good understanding of how you do noninvasively take a sample from a platelet bag, fill the capillary, and test the sample for microparticle content, to allow for optimize use of a platelet inventory.

While attempting this procedure, it's important to mix the bag well in order to get a good, representative sample. And following this procedure, you could do other methods, such as the flow cytometry, to answer questions as to the cellular origin of microparticles. Once mastered, this technique can be performed in about two and a half minutes.

Together with the five minute test, platelet transfusions can be characterized in less than eight minutes. After it's development, this technique allowed hospital blood banks to screen their platelet inventory, treat hematology/oncology patients only with non-activated platelets and observe a reduction in platelet transfusions used. As we say, the safest transfusion is the one not given.

Although this method provides insight into characterization of platelets prior to transfusion, it can also be used to test platelet rich plasma directly from the donors. The implications of this technique extent toward the treatment of blood cancer patients because they benefit from non-activated platelet transfusions, which reduce the risk of becoming refractory.