“Improving PBMC Isolation: Solving Low Yield, Viability, and Contamination Issues”

You may encounter challenges such as reduced yield, diminished viability, or contamination by granulocyte?during the isolation of peripheral blood mononuclear cells (PBMCs) from human blood samples. In addition to inter-individual variations in blood composition, there exist other potential factors:

1. The isolated PBMC?contains?red blood cells.

The density of a few red blood cells in the blood is similar to that of PBMC (Peripheral Blood Mononuclear Cells). Due to the limitations of density gradient centrifugation, complete removal of these red blood cells is not feasible.?However, when the blood sample is fresh and healthy and the procedures are performed properly, these minimal amounts of red blood cells in PBMC will not impact subsequent experiments such as cell culture and ELISPOT.

If there is an increased presence of red blood cells in PBMC, it may be attributed to the low temperature during centrifugation. This is because the density of the separation solution, primarily composed of polysucrose, tends to rise at lower temperatures under which the aggregation of red blood cells is?also?reduced. Consequently, when compared to room temperature conditions, it becomes challenging for red blood cells and granulocytes at lower temperatures to separate below the layer of separation solution and instead become mixed into the PBMC layer. Therefore, both the blood sample and separation solution should be?equilibrated?to room temperature prior to proceeding with the operation.

The presence of red blood cells in PBMCs may be attributed to the low relative centrifugal force or short centrifugation time, Therefore, it is recommended to set the relative centrifugal force at 800g and ensure a minimum centrifugation time of 15 minutes.

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2. The isolated PBMC?contains?more platelets.

The separation of PBMC may result in the concurrent separation of more plasma due to the presence of platelets in the plasma layer following density gradient centrifugation. In such cases, a lower relative centrifugal force, such as 250g, can be employed during washing procedures. By utilizing a lower relative centrifugal force, platelets with low density will remain within the cleaning agent while PBMC with higher density precipitates at the bottom of the centrifuge tube, thereby preventing platelet mixing.

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3. The isolated PBMC?contains?more granulocytes.

The abnormal density of cells in samples, such as patient blood samples, specialized animal samples, and non-peripheral blood sources, may contribute to this phenomenon. Additionally, prolonged storage of blood samples could also be a factor. According to literature reports[1], compared to PBMC separated from fresh blood, the contamination of isolated PBMC with granulocytes increases by 2.3 times when stored at room temperature for 6-8 hours; it increases by 11.3 times when stored at room temperature for 24-26 hours; and it increases by 84 times when stored under cold storage for 22-26 hours.

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4. The yield of PBMC?is low, but the viability of PBMC?is normal.

The relatively high centrifugation temperature may result in a decrease in the density of the separation solution containing polysucrose as the main component, leading to the entry of some lymphocytes into the separation solution layer. Therefore, it is necessary to restore both the blood sample and the separation solution to room temperature before conducting experiments.

When some individuals are in a sub-healthy or diseased state, their blood may become more viscous, which results in the agglutination of red blood cells and lymphocytes, leading to the sedimentation of lymphocytes along with red blood cells after centrifugation. It is recommended to dilute the blood sample prior to separation.

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5. The viability?of isolated PBMC?is low.

Prolonged blood sample storage before PBMC separation could lead to cell death, including lymphocytes. It is advised to separate PBMC within 2 hours of blood collection. Additionally, improper handling techniques like excessive blowing, high centrifugal force, or extended centrifugation time may also cause cell death.

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The following are the experimental results of BioSci? Density Reagent Separation Medium (Type B), showing the PBMC?isolated under the premise of fresh and healthy blood samples and normal operation.

01 Stratification of blood samples.

02 Counting results of cell analyzer.

This time, the blood sample dosage was 10mL, and the cell re-suspension volume was 820 μ L. AOPI reagent and Cellometer ? K 2 cell analyzer were used for counting, and the cell suspension was not diluted during counting.

03 Flow cytometry analysis.

Are you excited by such an excellent separation effect? For customers whose endotoxin requirements are <0.5EU/mL and require GMP grade separation medium, we would like to recommend using BioSci? Separation Medium.

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[1] Mckenna, K. C. , Beatty, K. M. , ?Miguel, R. V. , & ?Bilonick, R. A. . (2009). Delayed processing of blood increases the frequency of activated cd11b+ cd15+ granulocytes which inhibit cell function. Journal of Immunological Methods, 341(1-2), 68-75.