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

The overall goal of this procedure is to provide a streamlined protocol that enables rapid eye throughput, simultaneous processing of whole blood samples. In a nonclinical setting, vacutainer processing is performed in an overlapping manner to enable efficient isolation of high quality fractions of D-N-A-R-N-A peripheral blood mononuclear cells or pbmc serum and residual fractions of plasma and red blood cells. Processing begins with centrifugation of the serum vacutainer, removal of an aliquot from one fial containing vacutainer for DNA isolation centrifugation of the fial tubes.

Then completion of DNA isolation after DNA isolation leukocyte derived RNA isolation from the K two EDTA vacutainer can occur. The blood from each vacutainer is passed through a respective filter and washed with PBS, then treated with an RNA stabilization agent returning to the fial containing vacutainer. The residual plasma is drawn off and the mononuclear layer is washed with PBS.

While the tubes are spinning, the residual red blood cells are collected and the separated serum is aliquot. Finally, pbmc are preserved in cell freezing medium. Ultimately, high quality fractions of fresh blood can be produced within two hours of collection and all assay ready specimens can be available within two days.

The main advantage of this technique over existing methods is that the procedures can be performed simultaneously in a nonclinical setting, thus producing, starting material within two days for a multitude of downstream applications, including microarray epigenetic real-time R-T-P-C-R and flow cytometry analyses. Demonstrating the procedure will be Amy Weckl, my laboratory manager To begin whole blood sample processing. Ensure the buffers, centrifuge and heat block have all been prepared.

Document the time of the vacutainer delivery on the sample tracking sheet. After receipt of the samples, document the serum isolation. Start time on the specimen log immediately start processing by centrifuging the seven milliliter serum vacutainer with the brake and acceleration off.

Using a swinging bucket rotor with aerosol caps for 20 minutes at 1300 GS four degree Celsius while the serum vacutainer is being centrifuged. Document the DNA isolation start time in a BSL two cell culture hood. Invert the vacu containers containing the fial gel five times and add 200 microliters of whole blood from the top of one of the vacutainer into each of the two 20 microliter aliquots of protease.

Leave the protease and blood micro centrifuge tubes in the hood and continue to the centrifugation of the call containing vacu containers. Before centrifugation. Document the PBMC isolation start time, which must be within two hours of blood draw.

Invert the FI call containing vacu containers already in the aerosol tight containers eight to 10 times. Then centrifuge the vacutainer with the break and acceleration off for 30 minutes at 1600 Gs 22 degrees Celsius. Return to the DNA isolation samples in the hood and add 200 microliters of buffer al to each of the protease and blood micro centrifuge tubes.

Cap the tubes and remove them from the hood. Then vortex them for 15 seconds. After vortexing immediately flash, spin the samples.

Proceed with DNA isolation as described in the accompanying text protocol until the DNA is alluded from the column upon Ellucian. Pooled the DNA from the two columns per participant for a total yield of 800 microliters per participant. Document the start time of leukocyte RNA isolation.

Pierce the rubber septum of the K two EDTA vacutainer with a transfer spike. Retain the sheath and screw cap for later use following assembly of the K two EDTA tube system. Use the end of a metal spatula to safely unhea the needle and insert the needle into an empty 10 milliliter evacuated blood collection tube.

Invert the K two EDTA vacutainer filter receiver tube assembly to complete the setup After filtration and A PBS wash, withdraw three milliliters of the RNA stabilization agent using a new 10 milliliter syringe. Flush the filter ensuring that the RNA stabilization agent remains attached. Then detach the syringe from the filter without retracting the plunger.

Seal the filter inlet and outlet with the sheath and screw cap retained from the transfer spike and leave the filter saturated with the RNA stabilization agent. Store the filter at minus 80 degrees Celsius for at least two hours until time permits for completion of RA isolation. Once the vacutainer is returned to the BSL two hood, remove the stopper and use a serological pipette to withdraw 1.5 milliliters of the top plasma layer without getting close to the mononuclear layer.

Transfer the plasma from the two vacutainer to a five milliliter cryo vial. Then log the volume collected. Transfer the remaining plasma and the mononuclear layer using a serological pipette to a 15 milliliter conical tube.

Then add enough previously prepared one XPBS to bring the total volume in the conical tube up to 15 milliliters. Cap the tube and invert it five times. Then centrifuge the tube with the break and acceleration off for 15 minutes at 300 GS 22 degrees Celsius.

Return to the fial containing vacu containers in the hood and collect the red blood cells or RBCs. Use a five and three quarter inch pastier pipette to swirl around and loosen the outside of the fial gel layer and remove it. Use a serological pipette to collect and transfer the RBCs to a five milliliter cryo vial.

Then log the volume. Next, transfer the plasma and RBC cryo vials to a controlled rate freezing container and store them at minus 80 degrees Celsius for at least 24 hours. After 24 hours, transfer the samples to a cryo box and return them to minus 80 degrees Celsius for long-term storage.

When the centrifugation is complete, return the conical tube to the hood and aspirate the liquid, leaving 500 microliters of PBS without disturbing the pellet. Add one XPBS to bring the volume to 10 milliliters and resuspend the pellet. Gently cap the tube and invert it five times.

Then centrifuge the sample with the brake and acceleration off for 10 minutes at 300 GS 22 degrees Celsius. After centrifugation, aliquot the top serum layer from the vacutainer into two milliliter cryo vials and document the volume. Use four cryo vials and aliquot 200 microliters into cryo vial.

One 1000 microliters into cryo vial two. Then divide the remaining volume into cryo vials three and four. Transfer the cryo vials to a cryo box and keep them at minus 80 degrees Celsius for long-term storage.

And document the freezer start time. Continue with PBMC isolation. Returning the samples to the hood after centrifugation, aspirate as much supernatant as possible without disturbing the pellet.

We suspend the pellet in 2.5 milliliters of PBMC. Medium one. Add 2.5 milliliters of PBMC, freezing medium two containing DMSO and RPMI.

Medium to the previously prepared cell solution, and gently vortex the sample. Then aliquot 10 microliters of the cell solution into a micro centrifuge tube. Add 10 microliters of 0.4%trip pan blue stain into the micro centrifuge tube and mix the solution by pipetting several times.

Next, pipette 10 microliters of the mixture into a cell counting chamber. Slide and place the slide into the cell counter within three minutes of mixing. Zoom in and focus on the cells.

Then select count cells to obtain A-P-B-M-C Count. If the viable PBMC number is above 3 million cells per milliliter, aliquot the sample into a maximum of five cryo vials at a concentration of at least 3 million cells per milliliter. Each document the cell count per cryo vial on the specimen log.

See the accompanying text protocol for how to proceed if the viable PBMC number is below 3 million cells per milliliter. Finally, transfer the cryo vials to a controlled rate freezing container and place them at minus 80 degrees Celsius for at least 24 hours. After 24 hours, transfer the cryo vials to a cryo box and place the box in the vapor phase of a liquid nitrogen tank for long-term storage.

Document the freezer start time on the specimen log. The leukocyte RNA Isolation and filter processing methods yielded this gel image resulting from capillary electrophoresis. Two distinct bands were produced with minimal degradation representing ATT s and 28 s ribosomal RNA.

While attempting this procedure, it's important to remember that maintaining accurate records is critical. We recommend developing a database to store all of the cryo vial information, including the volume, concentration, quality, barcode, and storage location at the outset. And further recommendations on sample organization can be found in the text protocol.