In order to select recipients without donor-specific anti-HLA antibodies, the complement-dependent cytotoxicity crossmatch (CDC-CM) was established as the standard procedure about 40 years ago. However, the interpretability of this functional assay strongly depends on the vitality of isolated donors’ lymphocytes. Since the application of therapeutic antibodies for the immunosuppressive regimen falsifies the outcome of the CDC-crossmatch as a result of these antibodies’ complement-activating capacity in the recipients’ sera, we looked for an alternative methodical approach. We here present 27 examples of AB0 blood group-incompatible living kidney allograft recipients who, due to their treatment with the humanized chimeric monoclonal anti-CD20 antibody Rituximab, did not present valid outcomes of CDC-based pretransplant cross-matching. Additionally, four cases of posttransplant cross-matching after living kidney allografting and consequent treatment with the therapeutic anti-CD25 antibody Basiliximab (Simulect) due to acute biopsy-proven rejection episodes are presented and compared regarding CDC- and ELISA-based crossmatch outcomes. In all cases, it became evident that the classical CDC-based crossmatch was completely unfeasible for the detection of donor-specific anti-HLA antibodies, whereas ELISA-based cross-matching not requiring vital cells was not artificially affected. We conclude that ELISA-based cross-matching is a valuable tool to methodically circumvent false positive CDC-based crossmatch results in the presence of therapeutically applied antibodies.
It has been known for more than forty years that antibodies which are directed against HLA antigens of a given donor represent the dominating reason for hyperacute or acute rejections of renal allografts and allografts of other organs. These donor-specific anti-HLA antibodies (DSA) are thus regarded as a contraindication for grafting according to the transplantation guidelines of most countries and supranational societies (e.g., Eurotransplant) which are responsible for the supervision of the allocation of organs. In order to detect and exclude these harmful DSA, the so-called crossmatch (CM) procedure was developed in the late sixties of the last century. As standard technique to detect DSA, the complement-dependent cytotoxicity (CDC) assay was first established [
All of the patients (
All of the patients were initially investigated for DSA by the standard CDC-CM procedure in detail described elsewhere [
As procedure of ELISA-based cross-matching, the Antibody Monitoring System (AMS) class I/II ELISA (GTI, Waukesha, USA, FDA-number BK 060038 from June 26, 2006) was first implemented by us until its discontinuation in 2013 when it had to be replaced by the alternative AbCross HLA class I/II ELISA (Biotest/BioRad, Dreieich, Germany). In order to lead to a higher sensitivity and to considerably faster results, the laborious procedure presented in the manual of the manufacturer was completely modified by us resulting in a workflow which was very similar to that of the former AMS ELISA. Both assays, however, reliably allowed the direct detection of DSA by immobilizing extracted HLA molecules from donor cells/tissues onto which, in a consecutive step, only donor-specific but not anti-HLA antibodies in general out of a given recipient’s serum bind. The principle of work is demonstrated in the flow scheme (Figure
Flow diagram of the crossmatch-ELISA for the detection of donor-specific anti-HLA class I antibodies. (a) Binding of the donor’s solubilized HLA class I molecules by monoclonal capture antibodies recognizing a monomorphic epitope on HLA class I molecules. (b) Binding of the donor-specific anti-HLA antibodies out of the recipient’s serum to the HLA molecules of the donor. (c) Binding of enzyme-conjugated secondary anti-human IgG (anti-human IgG/M/A) antibodies to the bound recipient’s donor-specific anti-HLA class I antibodies and subsequent color reaction. (d) Lysate control using an enzyme-conjugated monoclonal antibody directed against a second monomorphic epitope (AMS-ELISA) or the
The recipients’ sera were generally screened for anti-HLA class I antibodies using the QuikScreen ELISA (BioRad) and for anti-HLA class II antibodies using the B-Screen ELISA (BioRad). Serum samples positive in this first screening step were afterwards investigated for antibody specification using the DynaChip HLA antibody analysis technique (Invitrogen/Dynal, Bromborough, UK) until 2011 when this system was discontinued by the manufacturer. This miniaturized chip-based technique was the only completely automated system available for the detection and specification of anti-HLA antibodies. In its second generation design 106 positions of each microchip were covered with HLA class I and 48 positions with HLA class II molecules of different single donors, respectively. Thus, apart from a number of HLA class II DQ-antigens immobilized on some positions, this assay did not provide a resolution at the single antigen level. However, the combination of the single donors’ immobilized HLA class I or II antigens, respectively, allowed the identification of the patients’ antibody specificities in most cases (70–80%) especially if the immunization level/PRA-level (see below) was not too high. The system, however, was discontinued by the manufacturer in 2011 for commercial reasons leading to the implementation of the Luminex technique in our laboratory. This technique currently represents the dominating tool for anti-HLA antibody specification. Its technical aspects and drawbacks for antibody specification have in detail been reviewed and discussed elsewhere [
During the last decade, humanized monoclonal antibodies have increasingly been used for preconditioning AB0 blood group-incompatible recipients of living kidney donations (anti-CD20/Rituximab) or for the therapy of acute rejection episodes (anti-CD25/Basiliximab). Rituximab which was originally used to administer a therapy against B-cell non-Hodgkin’s lymphoma [
Comparison of the outcome of CDC-based cross-matching with ELISA-based cross-matching (AMS- or AbCross-ELISA, resp.) as shown for twenty-seven patients treated with anti-CD20 mAb Rituximab and four patients treated with anti-CD25 mAb Basiliximab (Simulect).
Patient’s number | CDC-CM (NIH-score) | ELISA-CM | Antibody detection/specification (PRA max.) | |||
---|---|---|---|---|---|---|
PBL | T-cell | B-cell | Class I | Class II | ||
Rituximab (anti-CD20) [AB0-incompatible living kidney donations] | ||||||
(1) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(2) | 2 | 1 | 6 | neg. | neg. | PRA = 0% |
(3) | 2/4 | 1/2 | 8 | neg. | neg. | PRA = 0% |
(4) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(5) | 2/4 | 2 | 8 | neg. | neg. | PRA = 0% |
(6) | 2 | 1/2 | 8 | neg. | neg. | PRA = 0% |
(7) | 2/4 | 1/2 | 8 | neg. | neg. | PRA = 0% |
(8) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(9) | 2 | 1/2 | 8 | neg. | neg. | PRA = 0% |
(10) | 2/4 | 2 | 8 | neg. | neg. | PRA = 18%# |
(11) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(12) | 2 | 1/2 | 6 | neg. | neg. | PRA = 0% |
(13) | 2/4 | 2 | 8 | neg. | neg. | PRA = 0% |
(14) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(15) | 2 | 1/2 | 8 | neg. | neg. | PRA = 4%# |
(16) | 2 | 1 | 8 | neg. | neg. | PRA = 0% |
(17) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(18) | 2 | 1 | 8 | neg. | neg. | PRA = 0% |
(19) | 2/4 | 1/2 | 8 | neg. | neg. | PRA = 12%# |
(20) | 2/4 | 2 | 6/8 | neg. | neg. | PRA = 0% |
(21) | 2 | 1/2 | 8 | neg. | neg. | PRA = 0% |
(22) | 2/4 | 2 | 8 | neg. | neg. | PRA = 0% |
(23) | 2/4 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
(24) | 2 | 1 | 6/8 | neg. | neg. | PRA = 0% |
(25) | 2/4 | 2 | 8 | neg. | neg. | PRA = 0% |
(26) | 2 | 1/2 | 8 | neg. | neg. | PRA = 0% |
(27) | 2 | 1/2 | 6/8 | neg. | neg. | PRA = 0% |
|
||||||
Basiliximab (anti-CD25) | ||||||
(1) | 2/4 | 2/4 | 4 | neg. | neg. | PRA = 0% |
(2) | 2/4 | 2/4 | 4/6 | neg. | neg. | PRA = 86%# |
(3) | 4 | 4 | 6 | neg. | pos. | PRA = 12%& |
(4) | 6 | 4 | 6/8 | neg. | pos. | PRA = 20%& |
The outcomes of CDC-based and ELISA-based cross-matching are compared by showing the respective NIH-scores (introduced in Section
Interestingly, also the fraction of isolated T-cells in most cases appeared slightly positive (scores between 1/2 and 2) due to an apparent drawback of the RosetteSep cell separation system by Stemcell Technologies. This puzzling factor of residual B-cells in the fraction of isolated T-cells was presented and discussed a few years ago [
As part of these investigations, the other therapeutical moAb Basiliximab (Simulect) which is directed against the alpha-chain of the interleukin 2 receptor (CD25) [
In the same context, patient 1 was important as she/he did generally not exhibit any anti-HLA antibodies (
In spite of the limited number of only four cases presented in the context of posttransplant cross-matching under the immunosuppressive regimen of Basiliximab, the cases show that ELISA-based cross-matching represents a valid procedure to circumvent artificial outcomes induced by this therapeutical moAb which, for all these patients, has been applied to avoid the loss of kidney allografts due to clinically apparent rejection episodes. As a matter of course, fresh donor cells have to be available for any CDC-based cross-matching procedure. Thus, the comparative posttransplant investigations which comprised both crossmatch procedures were limited to living kidney donations for which a given donor’s material was again available for successive investigations after the kidney donation. Specifically due to this fact, only four cases were suitable for their observance in this group of patients.
Generally, it is noteworthy that the procedure of ELISA-based cross-matching does not require vital cells of the donor but advantageously uses detergent lysates of the respective donors (Figure
Interestingly, the aspect of medical treatment falsifying the outcome of CDC-based cross-matching has hitherto been described only a few times. This holds true for patients suffering from various forms of leukemia thus destined for a transfer of hematopoietic stem cells. Regularly, these patients do not have to fulfill the criterion of a negative crossmatch prior to the transfer since the recipient and her/his chosen donor have to be identical at the high resolution (four digits) level of HLA-typing. However, cases exist where the transfer is performed between two persons not completely HLA-identical in all genotypes or only identical for one HLA-haplotype if the donation is, for example, arranged between parents and their children (haploidentical donation). These configurations may lead to the requirement to exclude donor-specific anti-HLA antibodies. In this context, false-positive CDC-based crossmatch outcomes were reported to be the result of an unspecific cell death caused by the cytostatic agent 6-mercaptopurine which was used to administer a therapy against leukemia [
The diagnostic interference of the therapeutic humanized chimeric moAbs Rituximab and Basiliximab leading to false-positive results, mainly of the CDC-CM, but also of the flow cytometry-based crossmatch results, was first described about nine years ago [
In conclusion, the results of these investigations indicate the urgent need to implement ELISA-based cross-matching for the reliable exclusion of DSA (i) in the increasing field of AB0 blood group-incompatible living kidney donations and (ii) for any situation of cross-matching under the regimen of therapeutic antibodies not allowing the application of a cellular vitality assay such as CDC-based cross-matching.
The authors declare that there is no conflict of interests regarding the publication of this paper.