Gordon D. MacFarlane

Clinical Utility of Monitoring Tacrolimus Blood Concentrations in Liver Transplant Patients

The relationship between the dose of tacrolimus, trough tacrolimus blood concentration, and selected clinical endpoints (acute rejection, nephrotoxicity, and other toxicities) were examined in a prospective, multicenter clinical trial to validate the use of an enzyme‐linked immunosorbent assay (ELISA) for monitoring whole‐blood concentrations of tacrolimus in liver transplant patients. A total of 111 subjects from six transplant centers were evaluated over 12 weeks posttransplantation. In addition to trough tacrolimus blood concentrations, hematocrit, ALT, AST, GGTP, alkaline phosphatase, total bilirubin, serum creatinine, BUN, serum potassium, serum magnesium, blood glucose, and serum albumin were also measured. The relationship between trough tacrolimus blood concentrations and clinical endpoints was analyzed using both a logistic regression model and a Cox proportional hazard model. By logistic regression analysis, a statistically significant (p = 0.0465) relationship between increasing trough tacrolimus blood concentrations and decreasing risk of acute rejection was demonstrated over a 7‐day time window. Nephrotoxicity and other toxicities also demonstrated statistically significant relationships with trough tacrolimus blood concentrations. The results of the Cox analysis were consistent with the logistic regression analysis. Using receiver operator characteristic curves, trough tacrolimus concentrations as measured by the ELISA method were able to differentiate the occurrence of nephrotoxicity and toxicity from nonevents. To minimize nephrotoxicity of tacrolimus, it is necessary to maintain trough blood concentrations below 15 ng/ml. This study demonstrates that the ELISA method used to measure tacrolimus blood concentrations in this study provides information of predictive value for managing the risk of nephrotoxicity, other toxicity, and rejection in liver transplant patients.

Hypovitaminosis D in a normal, apparently healthy urban European population

Serum 25 OH Vitamin D (25 OH D) concentrations generally vary with latitude, season, and the composition of the population studied. There is a growing recognition that rather than a seasonal specific decline in serum 25 OH Vitamin D, a significant proportion of the population may exhibit asymtomatic subclinical Vitamin D insufficiency. Vitamin D insufficiency has been described in populations at risk, such as nursing home residents and the homebound elderly. We assessed a population of normal, apparently healthy volunteers at a single European urban center for 25 OH Vitamin D sufficiency. Serum 25 OH D concentrations were determined using an automated LIAISON((R)) 25 OH Vitamin D assay. For the purposes of this study, Vitamin D insufficiency was defined as a serum 25 OH Vitamin D concentration of <15 ng/mL. Of the total population (n = 126) 34% exhibited 25 OH Vitamin D concentrations of <15 ng/ml. The mean +/- S.D. serum 25 OH Vitamin D concentration among the total, sufficient, and insufficient populations was 19.4 +/- 7.7, 23.6 +/- 6.4, and 12.1 +/- 2.3 ng/mL. From these data, we conclude that 25 OH Vitamin D insufficiency is more common than previously thought, and is not restricted to high-risk groups.

A simplified whole blood enzyme-linked immunosorbent assay (ProTrac II) for tacrolimus (FK506) using proteolytic extraction in place of organic solvents

Tacrolimus (FK506) is a macrolide antibiotic with potent immunosuppressive properties. FK506 is 10- to 100-fold more potent than cyclosporin A in preventing organ rejection and in toxicity. Extreme inter- and intrapatient variability and lack of correlation between drug dosage and drug blood levels necessitate consistent and reliable therapeutic drug monitoring. Previous methods for monitoring drug levels have been lengthy and labor intensive and have required organic solvents for sample extraction. We have developed a manual enzyme-linked immunosorbent assay (ProTrac II ELISA) that employs standardized reagents and a nonorganic solvent extraction and yields good assay sensitivity and precision. The calculated mean sensitivity of the assay was 0.18 ng/ml. Interassay precision ranged from 6.3% to 13.1% (coefficient of variation) for FK506 in whole blood (concentration range, 1.0-25.0 ng/ml). Recovery of the drug from spiked EDTA whole blood was 91-98% over the same concentration range. Mean recovery of the drug from clinical samples spiked with kit standards was 99.5%. Dilutions of high-concentration spiked EDTA whole blood samples and high-concentration samples from patients exhibited linearity of observed versus expected values (y = 0.86x - 1.15; r = 0.99). Comparison of ProTrac II with the INCSTAR ProTrac FK506 ELISA by paired Students t test showed no statistical difference between the methods (p = 0.46). Comparison of ProTrac II ELISA to the IMx microparticle enzyme immunoassay (MEIA) by linear regression resulted in a line with a slope of 1.22 (r = 0.91). Analysis by t test resulted in a p value < 0.001, indicating that the absolute values obtained in these assays are significantly different. The mean (+/-SD) difference in the absolute values was 4.2 +/- 2.6 ng/ml, with the MEIA values consistently higher. These results indicate that ProTrac II is a sensitive, precise ELISA for the determination of tacrolimus in whole blood; it can be performed in < 4 h.

Therapeutic drug monitoring of tacrolimus in pediatric liver transplant patients

Abstract: The clinical utility of tacrolimus monitoring in adults has been well documented. The present study compared tacrolimus monitoring in a pediatric population of 34 liver transplant patients in four US centers with an adult population of 111 patients in six US centers. Subjects (adult and pediatric) were evaluated, at defined intervals over 12 weeks post‐transplantation (Tx), for tacrolimus trough concentrations and 12 additional laboratory chemistries. Pediatric patient and graft survival for the 12 weeks were 91% and 88%, respectively, as compared to 97% and 93%, respectively, for the adult population. The mean oral dosage of tacrolimus for pediatric patients was 0.13 ± 0.1 mg/kg/day at week 1, increased to 0.30 ± 0.3 mg/kg/day by week 3 and remained constant for the remainder of the study. These dosages were two‐ to three‐fold higher than the dosage used in the adult population. In contrast, the mean whole‐blood trough concentration, as determined by PRO‐Trac™ II enzyme‐linked immunosorbent assay (ELISA), decreased from 11.3 ± 5.1 ng/mL at week 1 to 6.3 ± 3.7 ng/mL by week 12 and was not significantly different from the trough concentration in adults. The incidence and distribution of the clinical end‐points for the pediatric subjects (rejection, nephrotoxicity, death, re‐Tx) were different from those observed in adults. The total percentage of pediatric subjects reaching any end‐point was 74%, as compared to 54% in the adult population. These data indicate several differences between the adult and pediatric populations in their response to tacrolimus.

Analysis of whole blood tacrolimus concentrations in liver transplant patients exhibiting impaired liver function.

In transplant patients with impaired liver function, HPLC methodologies have been suggested for monitoring whole blood tacrolimus concentrations because of the reported inaccuracy of immunoassay for whole blood tacrolimus concentrations. One hundred fifty whole blood samples from 50 subjects enrolled in a multicenter liver transplant trial were chosen for HPLC/MS/MS analysis without consideration of the clinical status of the patient at the time of sampling. These samples were chosen to represent the sampling intervals during the 12-week posttransplantation period. Retrospectively, the authors identified a subset of 39 samples from 27 subjects exhibiting impaired liver function as demonstrated by bilirubin concentrations > 3.0 mg/dL (mean +/-SD = 7.5 +/- 5.6 mg/dL). The authors compared the agreement of concentrations obtained from the PRO-Trac II ELISA and HPLC/MS/MS by least squares linear regression analysis and Bland/Altman analysis, in this subset against the agreement of concentrations for 76 samples with normal bilirubin. In the samples obtained from patients with impaired liver function the resulting regression equation was: ELISA = 1.19(HPLC) + 0.7; r = 0.9. The mean difference (HPLC/MS/MS - ELISA) was -2.5 ng/mL +/- 2.9 ng/mL (mean +/- SD). While 71% of samples agreed within 3 ng/mL, 3% (n = 1) exhibited a difference >10 ng/ml. The corresponding evaluation of the samples with normal bilirubin concentrations resulted in the regression equation ELISA = 0.96(HPLC) + 0.9; r = 0.9, and a mean difference of -0.6 ng/mL +/- 2.3 ng/mL. The authors conclude that while a small subset of patients with cholestasis may require closer evaluation with a more specific methodology, the majority of the patients may be satisfactorily monitored with the PRO-Trac II ELISA.

Evaluation of 25 OH Vitamin D in chronic renal failure and end stage renal disease subjects

Analysis of laboratory samples from chronic renal failure (CRF) and end stage renal disease (ESRD) patients can be problematic. Current HPLC and RIA methods for the determination of 25 OH Vitamin D involve sample extraction. However, the differences between a normal and CRF or ESRD matrix can lead to interference or inaccuracy in non-extracted, automated methods now available. The objective of this study was to assess the accuracy of the non-extracted LIAISON 25 OH Vitamin D assay in the analysis of CRF and ESRD samples as compared against RIA as reference. Samples were collected from regional reference laboratories and analyzed in both the LIAISON 25 OH Vitamin D assay and the DiaSorin 25 OH Vitamin D RIA. By Students t test, no significant difference was observed between the RIA values and the LIAISON values (P = 0.07 CRF; P = 0.28 ESRD). The linear regression analysis resulted in the equations: CRF: LIAISON = 0.91 (RIA) + 0.6; r = 0.82 and ESRD: LIAISON = 0.93 (RIA) - 0.6; r = 0.78. From these data we conclude that the LIAISON 25 OH Vitamin D assay correctly assesses the 25 OH Vitamin D status of CRF and ESRD patients.