In this technical bulletin, we describe a method to automate isolation of lymphoid (T and B cells) and myeloid lineages from human whole blood samples for chimerism analysis.

Background

Lineage-specific chimerism analysis is an important tool for monitoring the outcome of allogeneic hematopoietic cell transplantations (allo-HCT). It involves investigating the presence of donor leukocytes within specific cell subsets of the host’s peripheral blood or bone marrow.^1,2 As patients can show complete chimerism in one cell lineage and partial chimerism in another, lineage-specific chimerism analysis is becoming an increasingly common practice that offers several advantages over analyzing the entire leukocyte population.^3,4 Performing chimerism analysis requires sensitive and reliable techniques, and ensuring that cross-contamination between samples does not occur is critical to the success of downstream chimerism analyses.

Chimerism analysis is typically performed on small blood samples, and analysis of purified cell subsets often requires isolation of more than one cell type from a single starting sample. Chimerism analysis can be facilitated with EasySep™, a column-free immunomagnetic cell separation platform, which provides a fast and easy method to obtain highly purified cells (Figure 1). In addition, EasySep™ reagents have flexible protocols to isolate multiple cell types from the same sample.

To help large routine laboratories save time and increase throughput, EasySep™ can be automated using RoboSep™ instruments. RoboSep™ uses individual separation magnets and disposable pipette tips to eliminate and provide highly purified cells in sufficient quantities for downstream flow cytometry and DNA analyses.

Method: Automated Separation of Multiple Cell Types from a Single Sample

Below, we describe a method to sequentially isolate lymphoid (T and B cells) and myeloid lineages from human whole blood samples using RoboSep™-S, the fully automated cell isolation platform. The RoboSep™ -S instrument uses EasySep™ column-free immunomagnetic technology and automates all the labeling and separation steps. After completion of the cell separation cycle, the cells of interest are immediately available for any application.

The carousel inside the RoboSep™-S instrument is divided into four quadrants (see page 3, bottom), each containing an EasySep™ magnet and designated space for loading sample and waste tubes and EasySep™ reagents, as well as a filter tip rack. The quadrant system allows RoboSep™-S to be programmed to separate cells simultaneously from up to four samples or to sequentially isolate up to four different cell types from a single starting sample.

Sample Preparation

Samples of whole blood need to be prepared before loading them into the RoboSep™-S instrument for automated cell isolation, which can be performed on whole blood or buffy coat. To prepare whole blood, first collect your sample in a blood collection tube containing anticoagulant. Transfer a maximum of 4.5 mL of whole blood to a 14 mL polystyrene round-bottom tube and add 1X EasySep™ RBC Lysis Buffer at a ratio of 1:1. The sample is now ready to be loaded into the RoboSep™-S instrument.

To prepare a buffy coat sample, add an equal volume of buffer (PBS with 2% FBS and 1mM EDTA) to the whole blood. Centrifuge the tube at 800 x g for 10 minutes at room temperature (15 - 25°C) with the brake off. Collect the concentrated leukocyte band (this is the buffy coat), plus a small portion of the plasma and concentrated red blood cells (RBCs). The target is to concentrate the leukocytes approximately 5-fold while maintaining the same hematocrit (e.g. collect 2 mL of buffy coat when starting with 10 mL of whole blood). Proceed with transferring and diluting the buffy coat sample, as described above for whole blood.

Automated Cell Isolations with RoboSep™

The labeling and sequential separation of B cells (CD19+), T cells (CD3+), and myeloid cells (CD33+CD66b+) can be fully automated with RoboSep™ (see Figure 2). This 3-step process uses EasySep™ reagents for positive selection from an undivided sample of whole blood or buffy coat. First, the cells in the sample in quadrant 1 are labeled with antibody complexes targeting CD19+ cells. After a short incubation, magnetic particles are added to the sample, followed by another short incubation. The sample with labeled CD19+ cells linked to magnetic particles is then transferred to the tube sitting inside the magnet in quadrant 1. The labeled cells are held in the tube by the magnetic fi eld and the supernatant containing unlabeled cells is transferred to the sample tube in quadrant 2. The fraction left behind in the tube sitting inside the magnet in quadrant 1 now contains the isolated CD19+ cells.

Next, the CD19-depleted supernatant in quadrant 2 is labeled with anti-CD3 antibody complexes and magnetic particles, and transferred to the tube sitting inside the magnet in quadrant 2. After a short incubation of the sample in the magnet, the supernatant, which is now depleted of CD19 and CD3 cells, is transferred to the sample tube in quadrant 3, leaving highly purifi ed CD3+ cells in the tube sitting inside the magnet in quadrant 2.

Finally, the last fraction containing the myeloid cells is labeled with a combination of anti-CD33, anti-CD66b antibody complexes and magnetic particles, and is transferred to the tube sitting inside the magnet in quadrant 3. Unlabeled cells are removed to the waste tube, leaving highly enriched myeloid cells in the tube sitting inside the magnet in quadrant 3. To perform the cell isolation as described above, use the following reagents: EasySep™ HLA Chimerism Whole Blood CD19 Positive Selection Kit (Catalog #17874), EasySep™ HLA Chimerism Whole Blood CD3 Positive Selection Kit (Catalog #17871), and EasySep™ HLA Chimerism Whole Blood Myeloid Positive Selection Kit (Catalog #17884).

Results

Automated sequential cell isolations with RoboSep™-S from a single sample provides final isolated fractions with high cell purity and recovery rates (Figure 3, Table 1). Small starting samples can provide sufficient genomic DNA for downstream chimerism analysis (Table 2).

Summary

• Up to four cell types can be isolated sequentially from the same sample of whole blood using RoboSep™.
• No columns are required.
• Entire procedure takes less than 2 hours.
• All isolated cell types can be collected after a single loading of the RoboSep™ machine with the whole blood sample and reagents.
• No cross contamination occurs during enrichment as different filter tips are used to isolate each cell sample.
• No layering over Ficoll™ or post-enrichment lysis step is required.
• High cell purity and viability (96 - 99%) with good recovery of desired cells can be achieved.
• Small volumes of blood (0.5 - 4.5 mL containing 2 x 10⁶ - 3 x 10⁷ cells) yield sufficient cells to run flow cytometry and DNA analysis.

RoboSep™ instruments fit easily into the workflow of any lab that needs the multi-sample processing capacity, speed, reliability, and convenience of automated cell isolation.