Flow Cytometric Crossmatch Service

Except for analyzing the cell types, flow cytometry can be applied to immunology to ensure that, before a transplant procedure, interactions between donor and recipient immune components do not cause an adverse immune reaction. Creative Biolabs provides flow cytometric crossmatch (FCXM) test to detect donor-specific antibodies pre- or post-transplant (donor cells as targets) and autoantibodies (recipient cells as targets).

Fig.1 HLA class I (left) and HLA class II (right). Distributed under CC BY 2.5, left from Wiki, right from Wiki, without modification.

The Role of Human Leukocyte Antigen (HLA) Proteins

The HLA proteins are crucial for the body's immune defense against potentially harmful foreign substances. HLA class I and class II proteins are the ones involved with the immune response and transplantations.

The HLA-peptide complex (composed of HLA proteins and peptides) subsequently interacts with effector T-cells causing intracellular signals in both cells, which determines if a specific immune response occurs.

Three genes contribute to the formation of HLA I proteins, and six genes contribute to the formation of HLA II proteins, which leading the high specificity of HLA proteins.

Fig.2 Flow cytometry crossmatch technique.¹

Technologies

• Flow cytometry

Flow cytometry is used to study both the physical and chemical properties of cells and/or particles. During flow cytometry, the sample is suspended in a fluid and injected into the flow cytometer. Usually, only one cell at a time is passed through a laser beam for analysis purposes. The scattering of light gives information on the characteristics of the cells of the sample.

The cells are fluorescently labeled before being passed through the cytometer. The labels contain antibodies that are attached to fluorochromes. Separate labels can be used for different cells within a sample, which allows for a heterogeneous population to be analyzed.

• FCXM

FCXM involves mixing donor lymphocytes, the recipient's immune serum, and fluorescent-labeled antibodies into a sample. The antibodies used are specific to the donor HLA and various T-cell and B-cell specific markers (e.g. CD3, 5, and 8 for T-cells, and CD19, 20, and 21 for B-cells).

Then, the sample runs through a cytometer, so the lymphocytes can interact with the antibodies in the recipient's serum. If there are donor-specific HLA antibodies in the serum, they will bind to the donor lymphocytes, which allows the fluorescently labeled antibodies to bind, giving a positive cross-match in turn.

Highlights

• FCXM enables semiquantitative identification of anti-class I and class II antibodies by independently and simultaneously assessing both T and B cells. Also, FCXM offers definitive antibody and class identification at much lower levels than previously obtained.
• Three-color FCXM can detect the presence or absence of IgG antibodies directed towards donor lymphocyte-specific antigens.
• Donor lymphocytes are obtained using various methods that will yield highly purified lymphocytes (free from platelets, monocytes, and neutrophils) for optimal sensitivity, leading to getting more accurate crossmatch test results.
• FCXM allows for donor antigens and host antibodies to be checked before a transplant to avoid a host immune response. This reduces the chances of acute or chronic allograft rejection.
• Crossmatch test shows negative or positive results. Also, it displays the strength of the crossmatch in median channel shift (MCS), the strength of donor-specific HLA antibodies, and a comment indicating the risk of accelerated rejection.

As a leader in HLA antibody detection, Creative Biolabs offers a full range portfolio of assays for low- to high-resolution detection. We also offer the most cost-effective multiplex assays and a wide range of singleplex kits. If you are interested in our FCXM test, please feel free to contact us directly.

Reference

1. Kumar, A., et al. "An update on crossmatch techniques in transplantation." J Kidney 3.4 (2017): 1-5. Distributed under Open Access license CC BY 4.0, without modification.