Detection of HLA class I- and class II-specific antibodies by flow cytometry and PRA-STAT screening in renal transplant recipients.

BACKGROUND: Screening for HLA-specific antibodies has been performed by complement-dependent lymphocytotoxicity for many years. In recent years, methods involving the use of flow cytometry or ELISA have been developed. METHODS: This study has compared a flow cytometric screening technique for the detection of HLA class I- and class II-specific antibodies with a commercially available ELISA technique, PRA-STAT. RESULTS: A significant correlation was found between the two methods for the detection of antibodies in patients after transplantation (P<0.001). Specificity analysis confirmed that the PRA-STAT technique detected both HLA class I- and class II-specific antibodies. Screening of serum samples from patients who experienced graft loss by cytotoxic, flow cytometric, and PRA-STAT techniques showed that there was a significant correlation between all three methods for the detection of antibody, but that the best correlation for the panel-reactive antibody level was that between the flow cytometric and PRA-STAT techniques (r=0.86). This was principally due to the detection of both HLA class I- and class II-specific antibodies by these methods, whereas cytotoxic screening detected only class I-specific antibodies. CONCLUSIONS: These results suggest that PRA-STAT is a useful technique for the detection of both HLA class I- and class II-specific antibodies, rather than only class I-specific antibodies as previously described.

The clinical importance of variation within the HLA-Bw4 complex.

BACKGROUND: Antibody screening of a patient with a failed renal transplant showed positive reactions with most, but not all HLA-Bw4-associated B-locus antigens. However, the patient's serological HLA class I type suggested the presence of HLA-Bw4. METHODS: Standard molecular techniques were used to re-type the patient and donor. ELISA antibody screening helped determine the patient's antibody specificity. RESULTS: The patient's type was HLA-B*1402,4703;Bw6 and the donor HLA-B*4703,51011;Bw4,6. Analysis of ELISA results identified three amino acids (positions 77,80,81) as the most likely epitope recognised by the patient's serum. These corresponded to HLA-B*51011 amino acid mismatches, explaining the lymphocytotoxic reactivity pattern. This epitope is located on a subgroup of the HLA-Bw4 antigen suggesting anti-Bw4 was not a sufficient description of this antibody. CONCLUSIONS: This report identifies an antibody to a sub-group of the Bw4 public specificity and also confirms the need for sequence-level analysis in the tissue-typing laboratory to determine future unacceptable mismatches.

Detection of HLA-specific IGG antibodies using single recombinant HLA alleles: the MonoLISA assay.

BACKGROUND: Because of the presence of confounding antigens, the assignment of HLA antibody specificity is difficult in highly sensitized patients, and the definition of an acceptable HLA mismatch requires a significant workload per patient. We describe a new ELISA method, monoLISA, for detection of immunoglobulin (Ig)G HLA antibody using single recombinant HLA class I monomers bound to microtiter plates. METHODS: HLA-A2 and -B8 monomers were synthesized and used as screening targets for 85 sera from renal patients. The sera contained various IgG and IgM HLA-specific antibodies, including anti-A2 and anti-B8,defined in a conventional complement-dependent cytotoxicity test (CDC). Investigations were performed to determine possible effects on antibody binding of differential monomer peptide presentation as well as lack of glycosylation. RESULTS: A good correlation was found between CDC-defined specificities and the reactivity observed with HLA monomers. MonoLISA attained means of 100% sensitivity and 92.5% specificity compared with CDC. Neither the presence of different peptides, nor the absence of glycosylation of the monomer affected the ability of monoLISA to detect antibody. CONCLUSION: This study demonstrates that the mono-LISA method for HLA antibody detection is valid. Because this has the potential to reduce the work involved in screening sensitized patients awaiting transplantation for HLA antibodies, resources aimed at increasing the number of constructed monomers would be well targeted.

Evaluation of the flow cytometric crossmatch. Preliminary results of a multicenter study.

The flow cytometric crossmatch is a technique that is increasingly being used by clinical transplant laboratories. In this multicenter study by the British Society for Histocompatibility and Immunogenetics Flow Cytometry Group, a series of crossmatches were carried out to determine whether different centers obtained same results when performing the same crossmatch. There was greater than 80% agreement among participating laboratories on the results of 35/54 tests. There was no clear agreement in the remaining 20 cases. Quantitative analysis, estimating the number of cell-bound fluorescein molecules, demonstrated that differences in the criteria used by each center to define a positive crossmatch were responsible for some discordant results. When applied, definition of positivity based on the molecules of fluorescein increased concordance from 57.5% to 81.4%.l. These results suggest that a criterion for the interpretation of results based on quantitative analysis of bound antibody may be more reliable than methods in current routine use.

Renal disease, epidermal necrosis, and epithelial cell antibodies.

OBJECTIVE: To describe the association between epithelial cell IgM, which has previously been associated with an increased incidence of loss of renal graft in children, with a novel cutaneous eruption and unexplained native renal disease. DESIGN: Observational study on children with epithelial cell antibody presenting with unexplained renal or skin disease. SETTING: General paediatric department and regional paediatric nephrology unit. PATIENTS: Six children (five girls, one boy), who presented to the unit in 1989-90. RESULTS: Three children, two of whom had a history of a hyperpigmented rash, presented with hypertension, proteinuria, and impaired renal function. Renal biopsy specimens from two of these children showed severe arteriolar endothelial cell swelling with arteriolar occlusion. These children fully recovered after treatment with antihypertensive drugs. The third child developed end stage renal failure and required dialysis. Three other children presented with an unusual cutaneous eruption but no evidence of renal disease. Histology of the skin lesions showed acute epidermal necrosis and features consistent with a viral infection. CONCLUSIONS: The aetiology and pathogenesis of the epithelial cell antibody are unknown. These cases indicate that it may have a role in native kidney disease and focal epidermal necrosis. Clinical and histological features suggest that the antibody may be associated with a viral infection.

Utilization of crossmatch techniques for renal transplantation.

The pre-transplant cytotoxic crossmatch test is a necessary requirement for renal transplantation to proceed. However, there is no universally accepted policy on the clinical significance of a positive flow-cytometric crossmatch despite the increasing use of this technique. Differences in clinical significance found by different groups may be due to the outcome measures used and differences in the technique. The definition of a positive crossmatch can affect the results and investigators may gain information of clinical importance by considering changes in antibody levels over a series of samples from individual patients. The use of sensitive screening methods may assist in the interpretation of crossmatch results. Pre- and post-transplant crossmatching may provide data which can be used to modify immunosuppression in patients at risk of graft dysfunction.

Influence of complement on the allospecific antibody response to a primary vascularized organ graft.

The induction of antibody responses against T cell-dependent antigens has been reported to be influenced by complement. We therefore asked if the primary induction of alloantibodies against transplantation antigens, an important determinant of transplant outcome, is complement sensitive and whether this has functional implications. We transplanted rat kidney allografts into fully major histocompatibility complex-mismatched recipients, in which complement activation was inhibited by daily injection of soluble recombinant human complement receptor type 1 (sCR1). Control allograft recipients were injected with saline. Animals in the control group showed a marked antibody response against donor-specific antigens and an increase in the proportion of activated B and T splenocytes by day 5 after transplantation. Complement-inhibited rats showed a reduced level of antibody binding on target cells sharing the same histocompatibility antigens as the donor strain (p < 0.001), and a reduced level of activated splenic B (p < 0.01) and T (p < 0.01) cells. In a functional assay, the plasma of complement-inhibited rats showed reduced cytotoxic activity against donor-specific cells, and their grafts contained less bound antibody than controls. Analysis beyond 6 days was obscured due to the development of antibodies against sCR1. We conclude that complement activation facilitates the induction of the alloantibody response. Sparing of vascular injury and prolongation of graft survival, previously reported in complement-inhibited rats (Pratt J. R. et al., Am. J. Path. 1996, 149: 2055), could therefore be due to down-regulation of the B cell response as well as reduced complement-dependent cytotoxicity. Inhibition of complement may provide an ancillary approach to the prevention of allospecific antibody formation and the prolongation of allograft survival in primary kidney grafting.

Preliminary report: dramatic rise in renal allograft failure rate.

In 1989 the 1-year renal allograft survival at the Guy's Hospital paediatric transplant unit fell from 80% to 48%. The children were negative for HLA antibodies but 10 of the 21 were positive for an IgM antibody against epithelial cells (AEC). The 10 AEC-positive children had received a total of 12 grafts; 9 were lost and 3 were saved by means of plasma exchange. The 11 children without AEC received 11 grafts; 3 were lost, 2 owing to thrombosis. The correlation between the presence of AEC and graft rejection was highly significant. AEC have since been found in adult transplant recipients who suffered clinically similar graft failure, both at Guy's Hospital and at another hospital. The apparent spread of the antibody suggests an infective agent, but until the cause is identified kidney transplants in the paediatric unit have been suspended.

Novel antibodies associated with unexplained loss of renal allografts.

Using an endothelial/epithelial hybrid cell line, three different non-HLA antibody types have been identified by flow cytometry in patients who have rapidly rejected multiple renal allografts. These antibodies may be classified as anti-endothelial-monocyte, anti-activated endothelial cell, or anti-epithelial cell.

A highly sensitive, rapid screening method for the detection of antibodies directed against HLA class I and II antigens.

Screening of potential transplant recipients for antibodies that can cause graft rejection is an essential part of the pre-transplant monitoring carried out by tissue typing laboratories. This is a time-consuming process and the rapid reporting of results is dependent on the maintenance of frozen cell panels. The usual procedure of screening against a panel of random cells takes up to 6 weeks. In this study we have used flow cytometric analysis of pooled chronic lymphatic leukaemia (CLL) cells to detect antibodies directed against HLA antigens. We show that FACS screening of pooled cells can accurately and rapidly detect these antibodies and that the method is suitable for routine use. An estimate of the degree of patient panel reactivity can be determined within a few hours. In addition, the technique is more sensitive than those conventionally used, an advantage that may be of importance in preventing graft damage.