Improved flow cytometry crossmatching in kidney transplantation

Abstract

Flow cytometry crossmatching (FC‐XM) assay is the most sensitive cell‐based method for detecting donor‐specific antibodies (DSAs). However, the use of FC‐XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement‐fixing and noncomplement‐fixing antibodies. FC‐XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B‐cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC‐XM. Pronase treatment (0.5‐1 mg/mL) prevents false‐positive B‐cell FC‐XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B‐cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti‐rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin‐D (7‐AAD) or fluorochrome‐conjugated C4d Ab, after complement incubation, allows complement‐fixing antibodies to be distinguished from noncomplement‐fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab‐binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.

Crossmatch assays in transplantation: Physical or virtual?: A review

“…Lymphocytes express many surface antigens besides HLA molecules, and some of these antigens can bind antibodies circulating in the recipient's serum, irrespective of their specificity. [11] In FCXM have been described different causes of false-negative results, including: (a) low levels of HLA expression on donor cells, (b) excess number of cells, (c) low serum volume (which is generally a bad cell-to-antibody ratio), (d) low DSA levels, (e) low purity of the lymphocytes, and (f) high background in the negative control serum. False-positive results may be due to: (a) IgG binding to FcR in B cells, (b) low background in the negative control, (c) insufficient washing after incubations with antibodies, (d) autoantibodies, and (e) the use of therapeutic antibodies such as anti-thymocyte globulin, rituximab (anti-CD20), alemtuzumab (anti-CD52), basiliximab (anti-CD25), and daclizumab (anti-CD25).…”

“…False-positive results may be due to: (a) IgG binding to FcR in B cells, (b) low background in the negative control, (c) insufficient washing after incubations with antibodies, (d) autoantibodies, and (e) the use of therapeutic antibodies such as anti-thymocyte globulin, rituximab (anti-CD20), alemtuzumab (anti-CD52), basiliximab (anti-CD25), and daclizumab (anti-CD25). [10,11] Correlating crossmatch results with patient history, sensitizing events, and DSA history is important when interpreting possible false-positive or false-negative scenarios. Therefore, an adequate analysis will allow us to define if they are DSAs and, therefore, a more precise immunological risk based on the FCXM.…”

Kidney transplantation in highly sensitized recipients

“…As a significant proportion of patients reportedly presented an uneventful clinical course, although they were FCXM-positive, FCXM is considered a test with high sensitivity and low specificity in early graft dysfunction and antibody-mediated rejection (ABMR) [ 23 ]. Furthermore, FCXM can be impacted by several variable factors, including flow cytometers, fluorochrome reagents, cell-to-serum ratio, and incubation conditions, rendering standardization difficult.…”

“…It is necessary to establish individual cutoff values for each laboratory [ 24 ]. Moreover, the problem of false positives induced by non-HLA antibodies, autoantibodies, and rituximab tends to persist, which are disadvantages associated with traditional CDC crossmatch [ 23 ].…”

T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests

“…In a landmark publication, Patel and Terasaki reported that 24 of 30 kidneys transplants performed across a positive complement dependent cytotoxicity crossmatch (CDC-XM) failed in an accelerated fashion 1 . Test systems to detect circulating antibodies at a higher sensitivity than CDC-XM have been developed, and the flow cytometry crossmatch (FCXM) is in widespread use to identify donor specific IgG antibodies (DSA) [2][3][4][5] . Pre-transplant FCXM results are used in many centers for optimizing donor selection, and pre-transplant T and B cell FCXM results have been associated with allograft outcomes [6][7][8][9][10] .…”