Technote: Whole Blood ELISpot

There may be times when isolating PBMCs may not be practical and alternative methods for assessing antigen-specific T cell responses are needed. In the following technote, learn how our R&D team investigated using RBC lysis as a convenient and efficient approach for running ELISpot. We provide the step-by-step protocol so you can implement it in your own lab!

Purpose

The present study aimed to evaluate the use of RBC lysis of whole blood as an alternative to isolated PBMCs for measuring antigen-specific T cell responses in ELISpot. Mabtech’s R&D team sought to determine if RBC lysis in whole blood samples could provide a convenient and efficient method for assessing T cell responses.

Methods

RBC-Lysis protocol:

Transfer the whole blood (WB) from Heparin/EDTA tubes to a sterile 50 ml Falcon tube.
For each 1 ml of whole blood, add 10 ml of 1x RBC lysis buffer to the whole blood.
Incubate at room temperature for approximately 10 minutes and check the lysis process after the first 3 minutes and then every minute afterward for changes in turbidity. Observe the WB sample transition from opaque to clear during RBC lysis, while still maintaining a red color.
Once the WB becomes clear, stop the reaction by adding 30 ml of sterile PBS.
Centrifuge the tubes at 400-500 g at 2-8° C for 7 minutes.
Carefully remove the supernatant using a pipette (avoid pouring).
Resuspend the cell pellet with RPMI supplemented with 10% FBS to the ELISpot starting volume (e.g., 1 ml).
Count cells and check viability (e.g., a Muse Cell Analyzer with ViaCount solution).

Preparation of PBMCs from WB using Ficoll-Paque:

Transfer freshly collected WB from the Heparin/EDTA collection tube to a 15 ml tube.
Rinse the original blood tube with RPMI, and add to the 15 ml tube to bring the final volume to 8 ml.
Carefully layer the blood/RPMI mixture over 5 ml of Ficoll-Hypaque in a separate 15 ml tube.
Centrifuge the tube at 1200g for 20 minutes with no brake applied.
After centrifugation, discard most of the upper layer with a pipette, leaving behind the interface layer.
Collect the interface using a 2 ml pipette and transfer it to a new 15 ml tube.
Add RPMI to bring the volume up to 14 ml and wash the cells by centrifuging at 1000g for 10 minutes.
Wash a second time with RPMI (14 ml) and centrifuge at 400g for 10 minutes.
Count cells and check viability (e.g., a Muse Cell Analyzer with ViaCount solution).
Dilute the PBMCs to a concentration of 2.5-5 million cells/ml in RPMI supplemented with 10% FBS for ELISpot.

Assess IFN-γ release with ELISpot Plus: Human IFN-γ (ALP) according to the kit protocol with proper controls and desired peptide pools.

In our evaluation of the WB ELISpot assay, the lysed WB-RBC and PBMC samples were stimulated with the following Mabtech peptide pools that induced cytokine secretion from Ag-specific T cells:

Following ELISpot development, spot-forming units (SFU) can be enumerated with Mabtech IRIS^TM 2 using the Mabtech Apex^TM software.

Results

Our R&D team compared Ag-specific CD4 and/or CD8 IFN-γ responses measured by ELISpot in PBMC and RBC-lysed WB samples. Our findings demonstrated that RBC lysis of WB can effectively measure specific CD4 and/or CD8 T-cell responses using the IFN-γ ELISpot. Stimulation with CEFTA, CEFex, and EBV peptide pools generated clear and measurable SFU in both PBMC and RBC-lysed WB samples.

Figure 1 | Comparison of IFN-γ SFU from RBC-lysed WB (lower images) and PBMCs (upper images) from a healthy donor stimulated with Ag-specific peptide pools (CEFTA, CEFex or EBV: 250,000 or 500,000 cells/well).

CEFTA, CEFex, and EBV pools are commonly used peptide pools as positive controls to confirm T cells’ ability to respond to specific antigens. The team next assessed T cell responses to an SNMO SARS-CoV-2 peptide pool in recovered COVID-19 donors. IFN-γ SFUs were readily detectable in both RBC-lysed WB and PBMC samples incubated overnight. IFN-γ SFUs increased in both samples with 2-day stimulation compared to overnight stimulation when assessing the SNMO-specific T cells.

Figure 2 | Comparison of IFN-γ SFU from RBC-lysed WB and PBMCs from two healthy donors stimulated with SARS-CoV-2 peptide pools: 250,000 or 500,000 cells/well.

Enumerating viable cells in lysed WB samples can prove difficult and inaccurate using Trypan blue staining. By comparing SFUs, our team concluded that 100 µl of RBC-lysed WB samples corresponded to roughly 350,000 leukocytes per ELISpot well. These counts are highly donor-specific, but after repeated experiments, it was generally assumed that 300,000-500,000 RBC-lysed WB cells are roughly comparable to 250,000 PBMCs.

Figure 3 | Comparison of IFN-γ SFU from RBC-lysed WB and PBMCs from 13 healthy donors stimulated with CEFTA and CEFex peptide pools: 250,000 or 500,000 cells/well.

Conclusions

RBC lysed whole blood can be used when purification of PBMCs is impractical or unavailable to assess antigen-specific responses in human IFN-γ ELISpot.

The use of RBC lysis buffer had minimal impact on lymphocytes.
This approach significantly decreased handling time and allows for streamlined and automated analysis of multiple samples simultaneously.

Note:

PBMCs are ideal for ELISpot assays, and this protocol is not a substitute for proper PBMC isolation when feasible. However, in scenarios where PBMC isolation is impractical or unavailable, a whole blood ELISpot method can be utilized.
It's important to note that data from whole blood ELISpot and PBMC ELISpot are not directly comparable, and study designs should commit to one method.
Additionally, RBC lysis does not eliminate granulocytes, which can affect T cell functionality and distort results. Therefore, cell counts from RBC-lysed whole blood should not be equated with those from isolated PBMCs.