The presence of memory B cells may contribute to graft damage. In fact, pre-transplant B
cell sensitization in the absence of circulating antibody represents a risk factor, since
grafts transplanted into recipients with historical positive but current negative direct
crossmatch do less well than patients with no evidence of B cell sensitisation. This
definition of ‘negative’ is based on conventional complement-dependent cytotoxicity
(CDC) assays and, more recently, on flow cytometry. In this study, I have analysed the
significance of antibodies undetectable by these conventional techniques but found to be
present using the more sensitive novel Luminex technology which has now become
available to detect class I and class II HLA-specific antibodies present in pre-transplant
patients’ sera at the time of transplantation. Even in the absence of low level antibodies,
there may still be undetected B cell sensitization. An ELISpot assay has previously been
developed to quantitate B cell responses. In this study, I have developed the ELISpot
assay in an attempt to determine B cell allosensitization.
This project aims were:
1. To evaluate the relationship between renal transplant outcome and the presence of
low grade HLA-specific IgG-antibodies pre-transplant, both non-donor-specific as
well as donor-specific antibodies detected using Luminex technology.
2. To develop ELISpot assays that detect the potential responses of the various cells of
the B cell compartment, reflecting naive allo-reactivity as well as that which has
been induced by historical sensitisation events.
3. To detect the presence of post-transplant HLA-specific IgG antibodies (both donorspecific
and non-donor specific) in tolerant drug-free patient and determine their
significance in defining the state of tolerance.
Summary of the results:
1. Luminex technology is a more sensitive technique than flow cytometry and CDC in
detecting low-level donor-HLA-specific antibodies which were present in pre-transplant patients’ sera at the time of transplantation and is associated with a higher
incidence of rejection episodes.
2. IgM- and IgG-detecting ELISpot assays were developed to determine the frequency
of antibody-secreting cells. I have begun to apply these methods to determine the
humoral reactivity against alloantigen in sensitised patients.
3. Tolerant drug-free patients had no detectable donor-specific antibodies (DSA). In the
non-tolerant patients, DSA-positive patients had worse graft function than DSAnegative
patients