Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.Background: Management of a transplant recipient involves the use of multiple
immunosuppressant drugs. Currently there is no test that reflects the overall immune
status of the patient. This results in under or over suppression of the immune system and
consequently increases in morbidity and mortality rates. Evaluation of the proliferative
response of PBMC's to a mitogen PHA by measurement of intracellular ATP was
evaluated as a tool to assess the immune response in kidney transplant patients.
Method: PBMC's were separated from the blood samples of healthy controls and kidney
transplant patients on cyclosporine, sirolimus, and tacrolimus based regimens by density
gradient centrifugation, cells were counted and incubated overnight with and without
PHA. The luciferin-Iuciferase enzyme reaction which induces bioluminescence and the
Turner Biosystem luminometer were used to measure intracellular ATP levels in relative
light units (RLU). An A TP standard curve was generated for each test.
Results: The ATP (nglml) levels measured in the transplant recipients were lower and
statistically significantly different (p< 0.0001) than the healthy controls. No statistically
significant difference was measured between the cycIosporine and sirolimus drug groups.
Patients on tacrolimus gave a statistically significant (p<O.0001) stronger immune
response than those receiving cyclosporine and sirolimus. Overall, the immune response
results of kidney transplant patients were statistically significantly lower than the healthy
control by 981 nglml. Linear regression analysis revealed no correlation between patient
A TP (nglml) levels and therapeutic drug blood levels, immunosuppressant drug dosages,
creatinine levels and white cell counts. The immune responses of patients who were diagnosed with infection or were clinically stable were characterised as low or moderate,
of interest, one patient who was diagnosed with rejection was found to have a strong
immune response (>501 nglml ATP).
Conclusion: Future studies to determine the predictive value of the A TP assay in
directing immunosuppressive therapy are required. The assay described in this study is
simple, sensitive and rapid and has possible application in immunological monitoring in a
variety of conditions that affects the immune system.
Keywords: kidney transplantation, immunosuppression, bioluminescence, lymphocyte,
Adenosine Triphosphate (A TP), Phytohemmagglutinin (PHA
