Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Human PBMCs are versatile cell types used for numerous assays; however, isolation throughput is often a limitation in many laboratories, including biobanks¹⁶. Previously, the NSW Health Statewide Biobank isolated PBMCs using the density gradient method. Automation was investigated for the density gradient separation method to increase processing capacity, but barriers to implementation were identified, including (i) the cost of a liquid handler with a centrifugation unit to be fully automated, with the added requirement for a HEPA unit to produce a sterile product, (ii) trained personnel for programming, and (iii) time required for protocol testing. Therefore, this study explored alternative methods and identified the commercially obtained Human PBMC kit that could be used for manual and automated processing. Sterility is ensured as the equipment required for processing fits within a standard (length of 1.2 m) biological safety cabinet. This paper details modifications made to the manufacturer's recommended protocol¹⁴, to increase efficiency and lower reagent costs without sacrificing quality. Additionally, the manufacturer's protocol was extended to detail steps to biobank specimens for future research, including whole blood (step 1.2), plasma (step 2.2), and RBC (step 2.5) aliquoting from the original blood tube, as well as cell counting and cryopreservation.

In this study, three PBMC isolation protocols were compared: density gradient separation, manual, and automated bead-based isolation. Modifications to the manufacturer's manual bead-based protocol were made, including the removal of buffy coat dilution prior to PBMC isolation, and elimination of the requirement for break OFF for centrifugations, allowing investigators to follow an adaptable, cost-effective, and high throughput PBMC isolation protocol. First, eight matched whole blood samples were used to isolate PBMCs to compare the density gradient separation and manual bead-based technique. Importantly, cell population distributions, cell viability, and recovery of PBMCs were not significantly different between the two methods compared, as shown in Figure 2A-F, Figure 3A, and Supplementary Figure 1, respectively. In the representative data, cell counts were higher for the density gradient separation method using the trypan blue exclusion method but not when a hematology cell analyzer was used. The PBMC cell type settings on the cell counter employed a cell diameter range of 8-50 µm, and therefore, counts will include PBMCs as well as granulocytes (approximately 12-15 µm in diameter) when using the trypan blue exclusion method¹⁷. While the hematological cell counter offered higher specificity than the trypan blue exclusion method, some recovery calculations were higher than 100%, reflecting the instrument's margin of error (see Supplementary Figure 1). It is, therefore, recommended that investigators apply a combination of cell counting techniques when comparing the yields from PBMC isolation protocols, as most techniques do not offer both specific and highly sensitive differential cell counts. Further, assays were not performed to compare the functional activity of PBMCs yielded from either technique, which is a limitation of our analysis.

Next, the manual and automated bead-based methods were compared, and no significant differences between PBMC yield or viability were identified from 8 matched samples (Figure 4A,B). Cell populations were not individually compared, as the same antibody isolation cocktail was used for both methods. Importantly, the hands-on time to process 8 samples was reduced from 43 min to 22 min using the automated protocol (Figure 4C). While throughput, prevention of technician burnout, and consistency of specimen processing are ensured using automation, the cost of reagents and consumables are significantly higher at 3-4 fold more than the manual bead-based method. This is after making modifications to the manufacture's protocol to use 1 mL of buffy coat (from a whole blood volume of 10 mL) instead of the recommended range of 2-5 mL (from a whole blood volume of 10 mL and/or greater). If budget is a limitation, opting for the manual method can reduce processing time by ~25% while maintaining reagent and consumable costs comparable to those of the density separation method. It is recommended to process no more than 8 samples at a time per technician to ensure adequate staggering of samples (~30 s/sample) within the 5 min incubation periods (steps 3.7, 3.11, and 3.14).

In both the manual and automated bead-based methods, the removal of the buffy coat is a critical step to ensure optimal PBMC isolation. It is important to note that a buffy coat rather than whole blood is used in this protocol as the volume of reagents are based on starting material volumes¹⁰. Effectively removing the entire buffy coat volume can be technically challenging. Initially, this method detailed removing 0.5 mL buffy coat, however it was increased to 1 mL to improve recovery. To ensure adequate and consistent recovery of buffy coat, detailing this process is important in protocol documentation and training. It is recommended to swirl a pipette tip whilst aspirating the buffy coat while taking care not to aspirate too many RBCs from the layer below (step 2.3). It is important not to saturate the antibody cocktail that binds unwanted cells (i.e., granulocytes and red blood cells), which can impact yield and purity¹⁰. To minimize RBCs collected during the buffy coat extraction, the pipette tip should be inbetween the plasma and buffy coat layer. The volume of the buffy coat can be increased from 1 mL; however, care must be taken as described above to ensure that no more than 10% of the collected volume contains RBCs. Alternatively, an automated liquid handler can be used to collect buffy coats consistently¹⁸. However, the dedicated hours required to calibrate and troubleshoot liquid handling instrument protocols, especially considering the expense, may not be feasible for most laboratories.

The transition and application of the automation bead-based protocol was essential given the NSW Health Statewide Biobank's aim to process 23,000 PBMCs over the next 3 years. Here, it was demonstrated that PBMCs can be isolated from ACD tubes up to 9 days from collection with mean viabilities >70%. While yields were optimal 24 h post-collection, processing within this timeframe is not always feasible as samples may need to be transported. It was shown that PBMCs isolated with either the manual or automated bead-based method may have yields of >3 x 10⁵ cells/mL whole blood when isolated within 4 days of collection and >1 x 10⁵ cells/mL whole blood when isolated within 10 days of collection. Low numbers for samples over 5 days are noted as a limitation of this analysis. Further, data was not separated based on participant ages, genders, and clinical history, as this information was not made available. Although a comparative analysis is needed to examine the effects of delays in processing times for both methods, it has previously been reported that delays in PBMC isolation using the density gradient method decrease cell quality and significantly increase RBC contamination^19,20. In addition, granulocyte proportions increase if processing is delayed, and hence, specimens of similar "ages" should be grouped for downstream analyses^20,21,22. Of note, this experiment was performed using blood anticoagulated with acid citric dextrose (i.e., trisodium citrate, citric acid, and dextrose); however, yield and/or proportion of cell types may vary if other anticoagulants are used²³; therefore investigators are advised to choose an appropriate anticoagulant based on the intended downstream PBMC analyses.

In summary, a protocol for PBMC isolation using magnetic beads that is adaptable to high throughput workflows is detailed to fulfill the requirements for scaling without compromising cell viability. Both the manual and automated methods can be optimized to produce specific cell concentrations by changing starting and resuspension volumes. The NSW Health Statewide Biobank has transitioned from extracting ~60 PBMCs per month using the traditional density gradient separation technique to ~300 PBMCs per month using this automation-compatible bead-based method. The authors' next aim is to use the automated platform to process up to 1200 PBMCs per month and further compare PBMCs isolated by both magnetic bead-based (manual and automated) and density gradient techniques to guide the implementation of this technique to other laboratories with a particular focus on biobanks.