Accurately Count PBMC and Measure Viability in Presence of Residual RBC

 

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Accurately Count PBMC and Measure Viability in Presence of Residual RBC

Importance of Accurate PBMC Counts and Viability Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically in immunology research, PBMCs have been utilized to monitor immunological functions in different patients. The ability to monitor concentration, viability, proliferation, and cytokine production of PBMCs is critical for both clinical trials and biomedical research. PBMCs have been commonly isolated from whole blood and umbilical cord blood collected from patients, which routinely requires the use of Ficoll to separate the PBMCs from plasma, platelets, and red blood cells (RBC). After the isolation of PBMCs, a routine viability and concentration measurement is usually performed to confirm the quality of the sample before cryopreservation process for storage or various immunological assays. One of the major issues of PBMC isolation is RBC contamination, which can be sample-dependent. RBC contamination in a PBMC sample can introduce error to the measured concentration and viability, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. Method to Eliminate Red Blood Cell Induced Counting Error Identification of Residual RBC in Fresh Human PBMC Samples Fifteen freshly isolated leukocyte samples were manually counted using captured bright-field images of Cellometer Vision. The unique optical design allows the visualization of the bi-concave morphology of the red blood cells (RBCs), thus both PBMCs and RBCs were visually identified and counted in the images.  

 

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An example of the bright-field image used for manual counting is shown in Figure 1. The red circled cells represented the bi-concave morphology of RBCs, while blue circled cells represented PBMCs. RBC contamination percentages are calculated using the equation “RBC Contamination % = RBC count/Total count x 100”. The results showed 4 samples with high contaminations (greater than 30%), 6 samples with medium contaminations (10% to 30%), and 4 samples with low contaminations (less than 10%), which indicated the variability of donor-dependent RBC residuals from sample to sample (Figure 2).

Figure 1 (c) Mannually circle red blood cells in red color and leukocytes in blue color.

Figure 1 (a) Image human blood at 2000 fold dilution using PBS. Adjust Cellometer Vision to focus on the bi-concave morphology of the red blood cells.

Figure 1(b) Image human PBMC at 1 to 1 dilution using fluorescence staining dye solution. The red arrows indicate the bi-concave morphology of the red blood cells, while the green arrows indicate leukocytes.

Figure 2.

 

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AO/PI dual staining method for automated live/dead cell enumeration Acridine Orange (AO) and Propidium Iodide (PI) are fluorescent nucleic acid stains used to determine viability of a cell sample (Figure 3). AO is a membrane permeable nuclear dye that stains both live and dead cells green, while PI is a membrane impermeable nuclear dye that stains dead cells orange. When dead cells contained both AO and PI, fluorescence resonance energy transfer (FRET) occurred, where AO emission was absorbed by PI molecules, thus only live cells fluoresced green. In addition, since platelets and mature RBCs do not have a nucleus, thus they are not stained by the fluorescent stains. The AO/PI solution (20 µl) is mixed 1:1 with the leukocyte sample (20 µl), and immediately analyzed using the Cellometer image cytometer for PBMC concentration measurement. Validation of AO/PI dual staining method for PBMC measurement In order to validate the AO/PI method for accurate total PBMC measurement, five samples of freshly isolated leukocytes were subjected to 3% acetic acid lysing and stained with trypan blue. The trypan blue stained nuclei are counted manually and compared directly to the automated AO/PI dual staining method (Figure 4).

Figure 3.

 

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The results showed comparable cell count between the acetic acid lysing method for total nuclei counting and the AO/PI dual staining method, which confirmed that AO/PI method can be utilized to measure PBMC count and viability without the need for RBC lysing.

Elimination of RBC-induced counting error AO/PI dual staining method In order to show the ability to eliminate RBC-induced counting error using AO/PI, the 15 leukocyte samples were measured using the manual method to identify PBMCs and RBCs, and AO/PI method is used simultaneously with Cellometer image cytometer. The results showed comparable cell count between the manual (blue bars) and automated AO/PI (green bars), shown in figure 5. The samples with high percentages of RBC contamination did not affect the results of AO/PI method, which still produced a comparable data compared to manual counting. Conclusion RBC contamination can greatly affect the accuracy of concentration and viability measurement of isolated PBMC samples. The level of RBC contamination can vary among different patient samples, which may not be identified in the standard PBMC process protocol. Fluorescence-based image cytometry can be utilized to eliminate the RBC-induced error in these patient samples, which can improve accuracy and efficiency of PBMC measurement. More importantly, it can aid in the advancement of immunological research.

Figure 4.

Figure 5.