Complement-Dependent Cytotoxicity Crossmatch

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The complement-dependent cytotoxicity (CDC) crossmatch (CDC-XM) is a technology that can be applied to both T cells and B cells. The former reflects the presence of human leukocyte antigen (HLA) class I antibodies, while the latter reflects both HLA class I and II antibodies. Since B cells express higher amounts of class I antigens, a positive B cell CDC-XM associated with a negative T cell CDC-XM may indicate low levels of class I antibodies. Creative Biolabs offers CDC-XM test to provide direct evidence for the presence of potentially pathologic (cytotoxic) alloantibodies.

CDC

CDC was the first technique routinely used for HLA antibody detection and the crossmatch test. The mechanism of CDC is antibody-coated target cells recruit and activate components of the complement cascade, leading to the formation of a membrane attack complex (MAC) on the cell surface and subsequent cell lysis.

Process of CDC-XM

Firstly, good quality T- and B-lymphocytes are isolated from the donor's blood and incubated separately with the serum of potential recipients. Rabbit complement is subsequently added and after an appropriate incubation period, eosin is added together with formalin to fix the cells. Longer incubation periods and additional wash steps have been introduced to eliminate unbound antibodies before the addition of complement. If donor-specific antibodies are present in recipient serum, these will bind to HLA antigens on donor cells resulting in complement activation via the classical pathway. This finally generates the MAC, which inserts into the lipid bilayer causing cell rupture and uptake of vital dye. These dead cells will be visualized as red when seen under the microscope (shown as Fig.1).

Complement-dependent cytotoxicity crossmatch. Fig.1 Complement-dependent cytotoxicity crossmatch. (Callus, 2017)

Another way to determine the strength of the crossmatch is serial doubling dilutions of the recipient serum. More dilutions are necessary for the test to become negative, indicating a higher level of donor-specific antibodies. The antibodies have to be present in enough quantity to link complement to the Fc-receptor and activate complement-mediated cytotoxicity. The sensitivity of the CDC-XM will be enhanced if anti-human globulin is added before the complement factors. This promotes complement fixation by binding to HLA antibody on donor cells and increases the number of Fc-receptors available for complement binding (shown as Fig. 2).

Anti-human globulin complement-dependent cytotoxicity crossmatch. Fig.2 Anti-human globulin complement-dependent cytotoxicity crossmatch. (Callus, 2017)

Highlights

The CDC-XM assay is a standard method to detect antibodies against HLA antigens of donor cells in a potential recipient.
The readout of this assay is performed by fluorescence microscopy, and the reaction is defined based on a scoring system with values of 0, 2, 4, 6, and 8, according to standard protocols of the National Institute of Health (USA).
According to the Eurotransplant guidelines, crossmatch procedures do not need to use separate T- or B lymphocytes freshly isolated with antibody-coated magnetic beads to enhance the sensitivity of the assay.

Except for classical CDC-XM assay, Creative Biolabs also provides further modified CDC-XM assays such as AHG-enhanced CDC-XM. If you are interested in our CDC-XM assays, please feel free to contact us directly.

References

Callus, R.; et al. Basic concepts in kidney transplant immunology. Br J Hosp Med. 2017, 78(1): 32-37.
Altermann, W.W.; et al. Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure. Histol Histopathol. 2006, 21(10): 1115-24.