"ELISA DETERMINATION OF ANTI-HLA SPECIFICITY FOR IMMUNIZED PATIENTS: A STRATEGY TO MAKE GRAFTING POSSIBLE"

One of the still unsolved questions of renal transplantation is immunized candidates, generally defined as patients with serum antibodies (IgG and IgM) reacting against HLA class I and II antigens in a percentage ranging from 30 to 70% (immunized), or from 50 to 100% (hyperimmunized). The degree of immunization is usually assessed by antibody screening against a panel of separate lymphocytes, harvested from donor blood (Panel Reactive Antibody, PRA), via complement-dependent lympho-cytotoxicity testing. The presence of immunization lowers the chances of receiving a graft, so that such patients tend to accumulate on waiting lists.

The best strategy to make grafting possible (as drawn up by the HIT project conducted by the Collaborative Transplant Study between 1986 and 1996), consists in seeking high donor-recipient compatibility after an extensive pre-transplant analysis of antibody specificities, to establish the so-called acceptable incompatibilities. Patient sera are routinely tested against panels of HLA-typed donor blood, establishing the individual HLA antigens against which the patient has not formed antibodies. Knowledge of these acceptable antigens (or epitopes shared by different antigens) comes in useful whenever organ donors are available. By centrally coordinated organ allocation one is thus able to select all the HLA-compatible kidney donors who will presumably give a negative cross-match to all the sera of a given immunized patient.

In the light of the foregoing experience, the strategy illustrated in our study showed that a programme, which use an Elisa method to identify anti-HLA class I and II antibody specificities in the transplant candidate, makes it possible, first to perform transplants, and second, to have an acceptable clinical course.

For this purpose 387 patients, who underwent to cadaver kidney transplantation at the Nephrology Department of St. Orsola University Hospital between 1995 and 2001, were assessed by the following methods:

Before transplantation: 1) Genome typing of HLA antigens (DNA typing), an almost 100% reliable way of establishing even those antigens that serological methods fail to identify. 2) Search for antibody specificities against class I and II HLA antigens, via an ELISA (Sangstat or OneLambda) test (besides standard screening with the complement-dependent cytotoxicity technique (CDC). 3) Obtaining the HLA typing of the first donor in the case of a second- or third-time transplantee, that of the husband of multiparous women, and the potential donor’s typing wherever candidates had had a positive cross-match at a previous opportunity for transplantation.

At transplantation: after ruling out the existence of previously identified HLA antigens, we set about ensuring maximum HLA compatibility: typing was by the genome technique, even on the donor; donor-recipient cross-match was assessed by the standard NIH method on several sera: the two most recent and the highest historical (so-called peak) sera.

Immediately post-transplant - 90 immunised patients underwent renal transplantation from cadaver donor by this programme: 52 had a medium degree of immunisation (at least one pre-transplant PRA above 50%); 16 were hyperimmunized (more than 3 PRA above 50%); 22 patients were undergoing a second transplant.

For patients hyperimmunized and second transplants the standard therapy regimen (steroids and calcineurin inibitor) was boosted for the first days by induction therapy, following which a third immunosuppressant was added. In the other group the enhancement therapy was only added if steroid resistant rejection was present.

To check for group homogeneity and gather statistical data, we compared the patients personal and clinical details to a control group of 297 patients, matched by sex, age, blood group, number of pregnancies, age-gap from donor, cold ischemia and post-transplant function recovery times according to the commonest statistical methods.

Although it is no surprise that the mean and peak percentage of cytotoxic antibodies against HLA (PRA) antigens is decidedly higher than controls in all three groups (immune, hyperimmune, and retransplanted), the Elisa tests sharply distinguish which patients are at most risk (hyperimmune and retransplanted: respectively 13.4 ± 16.1 % and 22.5 ± 28.8 %) as against controls (1.5 ± 3.6 %, p<0.001) and - more to the point - patients with a low level of immunization (2.2 ± 7.3 %, p < 0.001) (Table I). Antigen specificity (both against Class I and II) were identified with high frequency only in retransplant and hyperimmune patients (68% and 50% respectively) (Table I). The personal details of the 4 groups differ only in the time of awaiting transplantation which is clearly more than double that of non-immunized patients. The clinical details of transplantation and the early outcome pattern do not differ significantly among the four groups.

* p<0.001

Analysis of the compatibility achieved at the moment of choosing the recipient (the only true strategy for ensuring graft success) showed an overall average of 1 mismatch on the DR locus and slightly more than 1 mismatch for Class I antigens. The immune patient group had slightly greater compatibility on being transplanted, but where the difference is most marked is in the hyperimmune and retransplant groups.

There the values are almost identical for the DR locus (0.44 ± 0.53 mismatches for the hyperimmune; 0.54 ± 0.5 for the retransplanted, as against 0.96 ± 0.62 for controls), and around 2 mismatches for class I loci, with a preference for locus B (0.83 ± 0.40 mismatches for the hyperimmune; 0.73 ± 0.98 for the retransplanted, as against 1.47 ± 0.5 for controls). The difference is obvious and also statistically significant, thus confirming the importance of greater antigen exposure, the predictive value of pre-transplant anti-HLA antigen determination and the need for careful selection to exclude the presence of antigen against which pre-formed antibodies have been identified.

Renal function, seen as serum creatinine is virtually the same in all 4 groups one year after surgery (1.56 ± 0.5 mg/dl for the hyperimmune; 1.47 ± 0.5 mg/dl for the retransplanted; 1.52 ± 0.6 mg/dl for the immune and 1.41 ± 0.4 mg/dl for controls). The number of rejection episodes in the first three months is 0.53 ± 0.7 in the immune and hyperimmune (0.44 ± 0.64 among the controls). Only the retransplant group has a higher incidence (0.61 ± 0.52), though not statistically so.

The really outstanding finding, however, comes from analysing the long-term actuarial graft survival curves (follow-up over six years). Three months from surgery, good graft function (graft survival) accounts for 91% of the hyperimmune group, 89% of the re-transplantees, 94% of the moderately immunized and 93% of controls. The same proportions are kept over the five years of follow-up (81%, 83%, 87%, 88% respectively).

In conclusion this study suggests that even hyperimmunized patients may be transplanted successfully. Careful and technologically advanced pre-transplant immune screening is required, as well as high compatibility with the donor. Some more specific points also emerge from the experience: patients with a medium-low degree of immunization (only one PRA peak above 50%) may have double the mean PRA values (vis-à-vis controls) but have a low pre-transplant Elisa and good organ function expectancy even in the long term. In this case, by eliminating the antigens the patient will react to at the moment of choosing the donor, one can transplant without any additional immunological risk. For these patients there are no reasons in favour of supplementary induction immunosuppressive therapy.

With patients undergoing retrasplantation and with those where sensitization is high (more than three PRA determinations above 50%). the method we advocate gave best results. ELISA test higher positivity works as a marker of immunological risk but knowing the anti-HLA specificities opens the door that makes grafting possible. Such patients have a good graft function expectancy, which is only slightly inferior to controls, although they do for preventive induction immuno-suppression, and it is wise to take extra care in post-transplant immunological monitoring, in view of the more aggressive immunosuppressive therapy.