Case Study

To improve the efficiency of cell preparation for the flow cytometric crossmatch (FCXM) assay, the University of Miami Immunomonitoring Laboratory has adopted an automated immunomagnetic cell separation system. This technical bulletin presents a case study of how this automated cell separation system frees up valuable technician time by minimizing sample handling and reducing the overall time to obtain cells for the FCXM assay, all without compromising assay sensitivity.

Background

The FCXM assay is part of a pre-transplant risk assessment to facilitate organ transplantation. The assay assesses whether the organ recipient has antibodies, also known as donor specific antibodies (DSAs), against the donor’s human leukocyte antigen (HLA). The assay involves mixing donor lymphocytes with recipient serum and detecting DSAs by flow cytometry. For analysis by flow cytometry, samples are stained with fluorochrome-conjugated antibodies that recognize T cells and B cells, and an anti-human IgG antibody to recognize DSAs.

Using high purity donor lymphocytes is critical to obtaining reliable FCXM assay results.¹ However, traditional methods for isolating lymphocytes from donor blood and tissue—such as density gradient centrifugation (DCG)—can be time consuming, be difficult to automate, and result in inconsistent cell purities.² This technical bulletin discusses how the University of Miami Immunomonitoring Laboratory has adopted an automated immunomagnetic cell preparation method that uses EasySep™ Direct reagents and the RoboSep™ instrument to make the lymphocyte isolation step more efficient.³

Methods

Lymphocytes were isolated from buffy coat by two different methods and their performance in the FCXM assay was compared. The first method—Manual DGC—employed cell isolation by DGC, using Ficoll-Paque® and Leucosep™ tubes, followed by a manual immunomagnetic granulocyte depletion step using Dynabeads® CD15. The second method—Automated RoboSep™ —involved fully automated immunomagnetic cell isolation with the RoboSep™ instrument (Catalog #21000) and the EasySep™ Direct Human Total Lymphocyte Isolation Kit (Catalog #19655). Cell preparations obtained using both methods were evaluated by flow cytometry for cell purity and viability. Platelet contamination was determined by complete blood count using a Sysmex® XN-3000™. FCXM assays were performed according to the laboratory's pronase-free protocol, using the cell preparations and negative and positive control sera samples.

Results & Discussion

The University of Miami Immunomonitoring Laboratory group showed that, compared to the Manual DGC method, the Automated RoboSep™ method resulted in cell preparations with higher lymphocyte purity (Figure 1), without the need to perform additional red blood cell lysis and granulocyte depletion steps. The Automated RoboSep™ method thereby reduced the average cost of reagents required per sample, leading to significant cost savings for the laboratory.

Compared to the Manual DGC method, using cells isolated by the Automated RoboSep™ method had no impact on FCXM assay sensitivity (Figure 2 and 3). All Mean Fluorescence Intensity (MFI) values were within the expected ranges for positive and negative controls. There was a trend of higher MFI values when using cell preparations obtained with the Automated RoboSep™ method (Figure 2). The group at the University of Miami Immunomonitoring Laboratory attributes this trend to cell preparations with higher lymphocyte purity, although more in-depth studies are needed to confirm this.

In addition to providing samples with higher lymphocyte purity, the Automated RoboSep™ method took less time: ~1 hour for Automated RoboSep™ compared to ~2.5 hours for the Manual DCG (Table 1). Most importantly, the Automated RoboSep™ method reduced the active time spent handling samples and performing cell isolation to ~0.5 hours from ~2 hours, thus reducing the hands-on time by more than 70%. This means that using the Automated RoboSep™ method can save a technician's valuable time and contribute to increased laboratory sample throughput and productivity.

Summary

The authors concluded that using the Automated RoboSep™ method, i.e. EasySep™ Direct Human Total Lymphocyte Isolation Kit on the RoboSep™ instrument, resulted in the following:

• No change in FCXM results compared to their Manual DGC method.
• Increased lymphocyte purity without the need for additional red blood cell lysis and granulocyte depletion steps.
• More than 50% reduction in the overall time to obtain cells: from ~ 2.5 hours to ~1 hour.
• Minimized sample handling, thereby reducing total technician hands-on time by more than 70%: from ~2 hours to ~0.5 hours.
• Significant cost savings and overall increased sample processing efficiency.

Automated Lymphocyte Isolation for the Flow Cytometric Crossmatch Assay

RoboSep™ instruments work with EasySep™ Direct to isolate untouched and highly purified cells directly from whole blood without density gradient centrifugation, sedimentation or red blood cell (RBC) lysis. Highly purified cells are immediately ready for the FCXM assay. Cell isolation with RoboSep™ and EasySep™ Direct is gentle, fast, and efficient and works well on older blood samples.

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