Donor-Specific Antibodies and Lymphocytotoxic Crossmatch

Pre-formed or de novo antibodies against donor HLA antigens cause hyperacute and antibody mediated rejection and graft loss in solid organ transplantation, and engraftment failure in haematopoietic stem cell transplantation with mismatched donors. Three families of tests are used.

Antibody screening (panel reactive antibody) uses single antigen bead assays on a Luminex platform or ELISA to identify HLA class I and II antibodies, with mean fluorescence intensity (MFI) as a semi-quantitative reading. Complement dependent cytotoxic crossmatch mixes recipient serum with donor T or B lymphocytes and complement, and lysis indicates antibody.

Flow cytometric crossmatch uses fluorescent anti-human IgG to detect antibody binding to donor cells and is about 10 to 50 times more sensitive. Virtual crossmatch compares the recipient's antibody specificities with the donor's typed HLA, without cells.

Sensitisation occurs through pregnancy, transfusion or previous transplant. Around 30% of kidney transplant waiting list patients are sensitised, and highly sensitised patients (calculated panel reactive antibody over 80% or 98%) wait years for a compatible organ. A positive cytotoxic crossmatch with T cells is a contraindication to kidney transplantation because of hyperacute rejection.

De novo donor specific antibodies develop in 15 to 25% of kidney recipients within 5 years and are the main cause of late graft loss.

Indications. Kidney, heart, lung and pancreas transplant candidates (periodic screening on the waiting list, and after sensitising events). Pre-transplant crossmatch with the actual donor (before the organ is implanted). Post-transplant surveillance (at 1, 3, 6 and 12 months and yearly) and when graft function deteriorates. Haploidentical or mismatched stem cell transplant.

Refractoriness to platelet transfusion. Acute or chronic antibody mediated rejection. Desensitisation therapy monitoring. Non-HLA antibody testing in selected cases.

Specimen. Serum 2 to 5 mL (clotted blood) from the recipient, with no anticoagulant. Recent sera are used, taken within 3 months (and after any sensitising event). Donor sample: heparinised or EDTA blood, spleen or lymph node cells, 10 to 30 mL, for lymphocyte isolation (donor DNA typing is used for virtual crossmatch). Samples are shipped at room temperature, and results are time critical.

Reference values.

• Panel reactive antibody (calculated, based on unacceptable antigens): 0% (no sensitisation) to 100%. Under 20% is low, 20 to 80% moderate, over 80% high, and over 98% very high sensitisation.
• Single antigen bead MFI (laboratory defined thresholds): under 500 negative, 500 to 1000 weak (usually not clinically important), 1000 to 3000 moderate, over 3000 to 5000 positive, over 10 000 strong. Thresholds depend on centre and assay.
• Cytotoxic crossmatch: negative if lysis is under 10 to 20%, positive when lysis exceeds the threshold. T-cell positive suggests anti-class I antibody, B-cell positive suggests class I or II.
• Flow crossmatch: reported as median channel shift or ratio of fluorescence, with a laboratory cut off.

Interpretation.

• Negative DSA and negative crossmatch: immunologically compatible, proceed with transplant (usual immunological risk).
• Positive cytotoxic T-cell crossmatch (IgG, confirmed): hyperacute rejection risk, so the transplant does not proceed unless desensitisation is done or an alternative donor is used.
• Positive flow crossmatch with negative cytotoxic crossmatch: elevated risk of antibody mediated rejection, handled with a risk based approach, desensitisation and intensified immunosuppression.
• DSA with high MFI, particularly against HLA-DR, DQ or complement binding (C1q positive) antibodies: higher risk of rejection and graft loss. DQ antibodies are the commonest de novo antibodies.
• Pre-transplant DSA: avoided by listing unacceptable antigens and virtual crossmatch.
• Post-transplant new DSA with stable graft function: increased surveillance and optimisation of immunosuppression. DSA with rising creatinine, proteinuria or biopsy features (microvascular inflammation, C4d staining, transplant glomerulopathy): antibody mediated rejection, treated by plasma exchange, intravenous immunoglobulin, rituximab, or bortezomib or eculizumab in selected cases.
• A rising MFI or falling MFI on repeat tests tracks response, though absolute levels are not comparable between laboratories.

Critical values and findings needing immediate action.

• Positive cytotoxic crossmatch with donor T cells just before implantation: do not proceed, since hyperacute rejection leads to graft thrombosis within minutes to hours.
• Sudden anuria, fever, pain at the graft and rising creatinine within hours to days of transplant: hyperacute or accelerated rejection, urgent biopsy, plasma exchange and nephrology review.
• New strong DSA with rising creatinine after transplant: urgent biopsy and treatment of antibody mediated rejection.
• Missed immunosuppression doses in a transplant recipient: drug levels and DSA measured, since non-adherence is a major cause of de novo DSA.

Interferences and pitfalls.

• Single antigen bead assays are not fully quantitative. MFI is affected by antibody concentration, bead antigen density, the prozone effect (falsely low MFI in high titre serum, corrected by dilution or EDTA treatment) and interference from IgM, denatured antigens and non-HLA antibodies.
• Cutoffs for MFI are centre specific, and a low MFI does not guarantee absence of risk (memory B-cell response after re-exposure).
• Autoantibodies (IgM, cold reactive) cause false positive cytotoxic crossmatch, and treating the serum with dithiothreitol removes IgM and distinguishes it from clinically relevant IgG.
• Rituximab and other B-cell therapy, pronase, and drugs such as anti-thymocyte globulin can distort flow crossmatch results.
• Antibodies against denatured HLA on beads can be present without clinical effect.
• Complement fixing assays (C1q, C3d) refine risk but are not routine in all centres.
• Non-HLA antibodies (anti-angiotensin II type 1 receptor, anti-MICA, anti-endothelial) can cause rejection when HLA antibody is negative.
• Sera used for crossmatch must be recent, since sensitising events (transfusion, pregnancy, infection, vaccination) change antibody profile.
• Positive DSA is not itself an indication to alter therapy, and decisions combine antibody, graft function and biopsy.

Special groups.

• Children: avoid unnecessary transfusion of leucoreduced and irradiated products to reduce sensitisation, and young recipients' parents as donors raise issues of prior pregnancy (mother as donor sensitisation to father's antigens).
• Pregnancy: multiparous women are often sensitised against paternal HLA, so spouses are poor donors.
• Older adults: lower sensitisation rates, and risk benefit favours accepting older donor organs.
• Regions: limited access to luminex based antibody testing. Where the cytotoxic method is the only one, a negative crossmatch is the minimum, and sharing of tissue typing resources between centres helps.

Follow up tests. HLA typing of recipient and donor (high resolution), single antigen bead antibody identification with MFI, C1q or C3d binding assay, calculated panel reactive antibody, repeat crossmatch on current serum, ABO compatibility, creatinine and urine protein to creatinine ratio, drug levels (tacrolimus), BK and cytomegalovirus PCR, donor derived cell free DNA where available, and graft biopsy with C4d staining and molecular rejection scoring.