One of the most disturbing side effects of CYA therapy is gingival hyperplasia. Although many studies were performed to determine the cause of this hyperplasia however, the mechanism of this effect is not established. The Dental school of the Lebanese University is performing a study to determine such a Mechanism. The study envisaged the determination of CYA levels in the blood, lymphocytes, saliva, dental plaque and gum biopsy. We here describe the method that we have developed and patented (MERI drug extraction solution, Beirut Lebanon) to measure the CYA levels in the saliva, dental plaque and gum tissue. 2 mls of saliva were collected from the transplant patients, 2 hours post the dose, and centrifuged for 10 minutes 4000 RPM. The supernatant was discarded and the pallet was dissolved in 200 mls of MERI solution. The plaque and the gum tissue was washed twice with saline and weighed. The gum tissue and the plaque were then homogenized with 200 ml of the MERI solution. The saliva as well as the homogenate was centrifuge at 100000 RPM for 10 minutes. The CYA concentration was determined in the supernatant using the Abbott TDx assay. Briefly 150 ml of the supernatant was added to 350 ml of lysing /precipitation reagent and centrifuged for 10 minutes at 10000 RPM. The CYA concentration in the supernatant was then measured using the TDx instrument.
The average CYA level was 93.7 ng/ml of saliva, 5.1 ng/mg of gum and 7.1 ng/mg of plaque respectively. The corresponding C2 blood level was 1123 ng/ml. The assay is sensitive up to 2 ng/ml and reproducible inter assay CV of 3.4% and intra assay CV of 6.7 %. There were many attempts in the past to measure CYA in saliva, most of which were not successful. The published data indicate that only one assay that was reproducible required as much as 40 ml of saliva. As for gum tissue or plaque CYA levels we believe that this is the only assay available.