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From Leukopak to PBMC
What is Leukopak?
Leukopak is a concentrated blood component fraction collected using a apheresis machine, primarily intended for the isolation of white blood cells (WBCs) from peripheral blood. White blood cells are rich in peripheral blood mononuclear cells (PBMCs), which consist mainly of T cells, B cells, NK cells, and monocytes. The concentration of white blood cells in Leukopak is notably high—approximately 4–7 × 10 8 (White blood cells per milliliter) compared with whole blood (average 5–7 × 10^9/L) 6 The white blood cell count (per milliliter) is much higher. In addition, the concentration of red blood cells (RBCs) in the white blood cells is markedly reduced, with a ratio of approximately 1 WBC for every 2 RBCs, whereas in whole blood the ratio is about 1 WBC for every 1,000 RBCs.
Moreover, the purity and quality of leukocytes are significantly superior to those of whole blood, typically containing more than 20-fold the number of mononuclear cells. PBMCs are highly valued for their applications in immunotherapy research, cell therapy process development, and clinical settings. Consequently, leukocytes have become the preferred starting material for cell therapy research and drug development.
High-quality Leukopak represents the perfect balance between purity and yield. Achieving this balance requires not only expertise in blood separation but also meticulous donor management. By providing donors with guidance on diet and lifestyle, we can maximize the potential quality of their leukocytes.
However, even high-quality leukocytes exhibit variability in yield. This inter-donor variability is inherent and often unpredictable. Nevertheless, such natural variability will inevitably be observed in patient populations and must be acknowledged and accounted for during process development.
Peripheral blood mononuclear cells (PBMCs) are nucleated cells found in peripheral blood that originate from hematopoietic stem cells (HSCs) in the bone marrow. HSCs give rise to all blood cells of the immune system through the process of hematopoiesis. HSCs can differentiate into two major lineages: the myeloid lineage (including monocytes, macrophages, granulocytes, megakaryocytes, dendritic cells, and erythrocytes) and the lymphoid lineage (including T cells, B cells, and natural killer cells).
PBMCs primarily comprise lymphocytes and monocytes, with lymphocytes further subdivided into T cells, B cells, NK cells, and others.
Figure 1: Proportions of various cell types in PBMCs
| Cell Name |
Proportion |
| Monocyte |
10-30% |
| Lymphocyte |
70-90% |
| Total T cells |
30-70% |
| B cell |
5-15% |
| NK cells |
5-10% |
| Dendritic cells |
1-2% |
Figure 2. Common surface markers for various cell types in PBMCs
| CD3 |
T lymphocytes |
| CD4 |
Helper T cells |
| CD8 |
Cytotoxic T cells, some NK cells |
| CD14 |
Monocyte |
| CD16 |
NK cells |
| CD19 |
B lymphocytes |
| CD34 |
Hematopoietic stem cells |
| CD45 |
White blood cell |
| CD56 |
NK cells, certain T cells |
| CD66b |
Granulocyte |
03 How to efficiently isolate PBMCs from Leukopak
Typically, PBMCs are isolated from Leukopak by density-gradient centrifugation. Given the varying cell densities in Leukopak, we use a lymphocyte separation medium with a density of 1.077 g/mL. The blood sample is gently layered on top of this medium in a centrifuge tube, and upon centrifugation, four distinct layers are formed:
The lowest layer is a mixture of red blood cells and granulocytes.
Second-layer lymphocyte separation solution
The third layer is composed of PBMCs and platelets.
The top layer is plasma.
During gradient density separation, several technical details can influence the purity, yield, and quality of the isolated PBMCs.
Below, we briefly outline the precautions to be observed during the separation process:
1. Upon receipt of the blood sample, it is recommended to first perform a leukocyte count on the Leukopak to roughly determine the white blood cell count in the sample.
2. Prior to separation, add PBS to the component blood at a 1:2 dilution based on the volume of the component blood (a common recommendation is 1/3 PBS plus 2/3 blood sample).
3. For a standard 50 mL centrifuge tube, it is recommended to add 10–20 mL of diluted blood sample. First add 15–20 mL of lymphocyte separation medium, then gently layer the diluted blood along the wall of the tube on top of the separation medium. (At this stage, neither too much nor too little blood should be added: adding too much increases the likelihood of red blood cell aggregation, which can compromise the purity of the isolated cells; adding too little may result in the loss of some leukocytes, thereby reducing the yield.)
4. During gradient density centrifugation, ensure that the centrifuge temperature is maintained at 20–22°C; temperatures that are too low can severely compromise the yield of PBMCs. In addition, during the separation process, always disable the centrifuge brake to allow the rotor to accelerate at a moderate rate and decelerate slowly.
5. After centrifugation, collect the buffy coat layer containing PBMCs. Since some cells inevitably adhere to the wall of the tube, use a pipette to gently scrape these adherent cells off the wall to ensure a high yield of PBMCs.
6. To remove platelets, add DPBS to the cell pellet, mix thoroughly, and then centrifuge at 250–300 × g for 10 minutes under deceleration settings; however, the platelet-depletion process will inevitably result in some loss of the target cells.
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