Fiona Hudson

Review article: Luminex technology for HLA antibody detection in organ transplantation.

Since its inception in the early 1960s, the serologically based complement‐dependent cytotoxicity (CDC) assay has been the cornerstone technique for the detection of human leucocyte antigen (HLA) antibodies, not only in pre‐transplant renal patients, but also in other forms of organ transplantation. Recently, solid phase assays have been developed and introduced for this purpose, and in particular the Flow‐based bead assays such as the Luminex system. This latter assay has proved to be far more sensitive than the CDC assay and has revealed pre‐sensitization in potential transplant recipients not detected by other methods of HLA antibody detection. However, the clinical implications of this increased sensitivity have not been convincingly demonstrated until recently. This technology for HLA antibody detection permits the evaluation of the clinical importance of antibodies directed at, for example, HLA‐DPB1 and HLA‐DQA1, which has not been possible to date. There are Luminex issues, however, requiring resolution such as the ability to distinguish between complement fixing and non‐complement fixing antibodies and determination of their relative clinical significance. Luminex technology will permit a re‐evaluation of the role of HLA antibodies in both early and late antibody‐mediated rejection.

A single low-fixed dose of rituximab to salvage renal transplants from refractory antibody-mediated rejection.

Rituximab may improve graft survival in renal acute antibody-mediated rejection (AMR), but data confirming efficacy and optimal dosing is lacking. High-dose regimens may be associated with significant rates of infective complications. We therefore conducted a pilot study of a single low-fixed dose (500 mg) of rituximab in seven consecutive patients with AMR resistant to standard therapy. After a mean follow-up of 21 months (range, 9.5–33 months), graft and patient survival were 100% with serum creatinine levels significantly lower than peak rejection levels (171±73 &mgr;mol/L vs. 559±358 &mgr;mol/L, P=0.028). B cells were undetectable in all patients for more than or equal to 6 months and in six of seven patients for more than or equal to 12 months after rituximab. Three patients encountered a significant infective complication including cytomegalovirus reactivation, viral pneumonia, and polyoma viral nephropathy. All have since resolved. A single low-fixed dose of rituximab may help improve graft survival in AMR and offers the potential advantage of reduced infective complications.

Solid phase HLA antibody detection technology – challenges in interpretation

The introduction into routine diagnostic laboratories of solid phase assays for human leukocyte antigen (HLA) antibody detection has resulted in the application of new laboratory matching algorithms in clinical organ transplantation which have improved pre-transplant detection of immunization, in turn resulting in avoidance of rejection in many cases which until their introduction would not have been possible using the historical complement dependent serological techniques. There have been two generations of solid phase assays introduced into routine practice, namely, the enzyme-linked immunosorbent assay (ELISA) technique and the use of fluorescent beads with HLA molecules bound to their surface which can either be used in conventional flow cytometry or in conjunction with Luminex instrumentation, the latter having become the most popular approach. The use of the fluorescent bead techniques has raised interesting questions both with respect to technical performance and the interpretation of the results obtained. The advantages of bead technology for HLA antibody determination and the technical issues requiring resolution are the subject of this review.

Rapid screening for the detection of HLA-B57 and HLA-B58 in prevention of drug hypersensitivity

HLA-B57 and HLA-B58 are major histocompatibility class (MHC)-I allotypes that are potentially predictive of important clinical immune phenotypes. HLA-B*5701 is strongly associated with hypersensitivity to the HIV drug abacavir, liver toxicity from the antibiotic flucloxacillin and is a marker for slow progression of HIV AIDS. HLA-B*5801 is associated with hypersensitivity to allopurinol used to treat hyperuricaemia and recurrent gout. Here we describe a monoclonal antibody (mAb) specific for HLA-B57 and HLA-B58 that provides an inexpensive and sensitive screen for these MHC-I allotypes. The usefulness of HLA-B57 screening for prediction of abacavir hypersensitivity was shown in three independent laboratories, including confirmation of the mAb sensitivity and specificity in a cohort of patients enrolled in the PREDICT-1 trial. Our data show that patients who test negative by mAb screening comprise 90%-95% of all individuals in most human populations and require no further human leukocyte antigen (HLA) typing. Patients who test positive by mAb screening should proceed to high-resolution typing to ascertain the presence of HLA-B*5701 or HLA-B*5801. Hence, mAb screening provides a low-cost alternative to high-resolution typing of all patients and lends itself to point-of-care diagnostics and rapid ascertainment of low-risk patients who can begin immediate therapy with abacavir, flucloxacillin or allopurinol.

Human Leukocyte Antigen Mismatch and Other Factors Affecting Cryopreserved Allograft Valve Function

The causes of cryopreserved allograft heart valve degeneration are poorly understood. We investigated HLA mismatch and other factors implicated in allograft valve degeneration. For this study we recruited 110 adult recipients of allograft heart valves who underwent surgery between June 1998 and March 2003 in the state of Victoria, Australia. Recipients and donors were HLA typed using serological and molecular methods. Valve function at most recent echocardiographic follow-up was examined for an association with the following variables using univariate and multivariate methods: HLA-A,-B, and -DR donor-recipient mismatch; HLA class I mismatch; total HLA mismatch; valve ischemic time; recipient age; donor age; ABO blood group donor-recipient match; and allograft size. Mean recipient age was 45 years (18-75 years), 75% were men. Seventy-four pulmonary (62 Ross procedure) and 36 aortic allografts were examined. Median valve ischemic time was 31 hours, range 20-48 hours. Echocardiographic follow-up was complete at a mean of 41 (+/-18) months, range 6-85 months. At univariate analysis longer ischemic time and younger recipient age were associated with valve dysfunction. HLA-A, -B, or DR mismatch, HLA class I mismatch, total HLA mismatch, donor age, ABO mismatch, and allograft size were not associated with valve dysfunction. Only younger recipient age remained significant at multivariate analysis. In conclusion, longer ischemic times and younger patient age predicted valve dysfunction at a mean of 3 years follow-up. Recipient age remained the strongest predictor of valve dysfunction. These results indicate that allograft ischemic times should be minimized.