On-Cell Stability for Therapeutic Drug Monitoring

Abstract Introduction A common problem in many clinical laboratories and outpatient clinics is processing specimens rapidly to comply with specimen stability criteria. Labs receive thousands of samples a day and may face challenges with staffing, equipment malfunction, or workflow, which can prevent immediate processing of samples. Currently, for therapeutic drug monitoring, the lab is required to centrifuge specimens and remove the serum/plasma from cells within 2-hours (CLSI-GP44-A4). Many of these drugs are monitored in the outpatient setting, which may require longer transportation times to get specimens from the collection location to the laboratory. This creates challenges for the lab in processing samples within 2-hours and may result in unnecessary rejection of otherwise acceptable specimens. Not all clinical sites have the staffing or space to process the specimens on site. Our objective was to determine if the on-cell stability of therapeutic drugs can be extended to beyond two hours. Methods We performed spiking studies to determine the on-cell stability of phenytoin in whole blood that was collected from healthy donors in barrier-free red top tubes. Phenytoin was spiked into the whole blood at a final concentration of about 10 (therapeutic) or 40 (toxic) µg/mL. The spiked whole blood samples were mixed by inverting six times and then were left at ambient temperature for 30 minutes, 2-, 4-, 6-, or 12-hours, after which they were centrifuged, and the serum was removed for analysis of total phenytoin concentration on the Abbott Architect and free and total phenytoin on the Beckman AU (n=3 per time point and concentration). In vivo studies are ongoing for an extended list of therapeutic drugs, in which two extra red top tubes are collected from patients being monitored for phenytoin, valproic acid, digoxin, vancomycin, lithium, or carbamazepine therapy. The two study tubes are kept at ambient temperature for 6- or 12-hours after collection before being centrifuged and separated from cells. The 6- and 12-hour drug concentrations were compared to the patient’s standard of care tube that was processed within 2-hours, after clotting for 30 minutes. Results Spiking studies demonstrated phenytoin was stable on cells for at least 12-hours at both 10 and 40 µg/mL. The percent total phenytoin remaining at 12-hours relative to the 30-minute control was 104 ± 2% at 10 µg/mL and 99 ± 16% at 40 µg/mL. Furthermore, the fraction of free phenytoin did not change over the 12 hours (10 µg/mL: 8 ± 1% free; 40 µg/mL: 10 ± 1% free). Our preliminary in vivo studies have shown no change in vancomycin or lithium concentration in samples processed 6- and 12-hours post-collection. Conclusion We have demonstrated that phenytoin, vancomycin, and lithium collected in red-top tubes are stable on cells at ambient temperature for at least 12-hours.