The possibility of using the specific RIA method for the area-under-time-concentration curve sparse sampling calculation of cyclosporin A despite a large post-dose overestimation.

OBJECTIVE: To find limited sampling strategies (LSS) for prediction of the real AUC using the RIA analytical method. METHOD: Blood samples of 40 male renal transplant patients taken pre-dose and after 0.5, 1, 1.5, 2, 3, 5, 8, and 12 h in the steady-state were analyzed with HPLC and the specific RIA method. I. Eight equations for AUC0-12 and one for AUC0-8 obtained from the literature, that produced the mean percentage prediction error (%PE) < ± 15% and absolute %PE < 30% in 95% of predictions, were analyzed for possibility to predict the real AUC of CsA. II. Multiple regression analysis (MRA) was provided for the AUC equation proposal. Patients were divided into two groups according to the AUC0-12. Group I was used for LSS : s proposals while Group II for validation. The bias and precision were expressed as %PE, r2 and RMSE. The relationship of %PE interassay and with LSS:s was expressed as Pearson correlation r. GraphPad InStatt Software was used for MRA and Pearson r calculation. RESULTS: None of the equations described in the literature predicts AUC of CsA proprietarily. Seven equations for AUC0-12 and five for AUC0-8 were proposed with MRA for prediction of real AUC from RIA values. CONCLUSIONS: LSS:s can moderate the interassay %PE in AUC of CsA. New patients should be tested with both RIA and HPLC for the level of overestimation. The conversion factors should be calculated for patients with an overestimation higher than 90%. Our equation 251.09 + 0.5195 × C1h + 4.926 × C3h or 196.13 + 4.526 Â× C0h + 2.089 × C1.5h for AUC0-12, and 171.80 + 0.4759 × C1h + 4.132 × C3h for AUC0-8 may be used in patients with medium or low RIA and HPLC differences. Repeated analysis with HPLC is thus suggested in cases with AUC:s results close to the lower or upper margin of the therapeutic window.

Significant discrepancy in cyclosporin A post-dose concentrations when analyzed with specific RIA and HPLC method.

C(2) or AUC sparse sampling methods are widely recommended for therapeutic monitoring of cyclosporin A (CsA). One additional reason for promoting the C(2) sampling time in place of commonly used C(0) is that the C(2) level may actually provide more accurate measurement of parent drug concentration by immunoassays, as lower portion of metabolites has been formed 2 hours post-dose than at the steady-state trough time point. HPLC and RIA whole blood levels of CsA and its main metabolites AM1, AM9 and AM4N were compared during 12 hours profile after chronic administration. 40 stable renal transplant male patients (age 49 +/- 6 years, body weight 76 +/- 7 kg) were treated with CsA (Sandimmun Neoral, Novartis s.r.o, Prague, Czech Republic) in doses 198 +/- 56 mg twice daily. Samples were collected in steady state (after 2 weeks of regular treatment regimen) as follows: pre-dose, 0.5, 1, 1.5, 2, 3, 5, 8 and 12 hours after dose. CsA concentrations were determined both specific RIA assay (Cyclo-Trac SP Whole, Dia Sorin) and HPLC method, where concentrations of metabolites AM1, AM9 and AM4N were simultaneously analyzed. The AUC(0-12) was calculated by the linear trapezoidal rule. The percentage prediction error defined as [(RIA value-HPLC value)/HPLC value] x 100 was used for estimation of differences. C(max), t(max), and C(avg) were compared using Student's t-test. RIA produced significantly higher CsA levels than HPLC method in the period of 0.5 - 5 hours after application. The greatest differences (43 - 56%) occurred between 1 and 3 hours after dose. AUC(0-12), C(max) a C(avg) calculated from RIA results were consequently significantly higher. Only t(max) remained unchanged. The ratio of metabolites/parent drug after CsA intake is decreasing but their absolute concentrations are significantly increasing. Mean levels at C(0)/C(2) were CsA-RIA 82/612, CsA-HPLC 89/425, AM1 121/179, AM9 4.1/81.4, AM4N 9.5/21.0 ng/ml. TDM using C(2) and AUC sparse sampling may cause misleading interpretation using both methods alternately for the same patient.

[Therapeutic monitoring of cyclosporine A]. / Terapeutické monitorování cyklosporinu A.

Cyclosporine A (CyA) is a drug with a specific influence on the immune system and it is used both to prevent tissue rejection of transplanted organs and to treat autoimmune diseases. The properties, metabolism, and methods of therapeutic drug monitoring (TDM) in patients under immunosuppressive therapy are described. TDM of CyA reflects the clinical condition of the patient during immunosuppression and may include pharmacokinetic and pharmacodynamic data obtained either from measuring CyA levels in blood, or from determining some other parameters, which are modulated by CyA (II-2). Although TDM is mainly based on analyzing trough levels of CyA, the determination of the whole biological exposition calculated as AUC enables better correlation with the clinical state of patients. Pharmacodynamic parameters have not been measured routinely yet.

[Determination of cyclosporine A using high-performance liquid chromatography]. / Stanovení cyklosporinu A metodou vysoce úcinné kapalinové chromatografie (HPLC).

Recent analytical possibilities of therapeutic drug monitoring (TDM) of cyclosporine A (CyA) both in solid organ transplanted patients and in patients with autoimmune diseases are described. The standard method for determination of CyA in blood is a validated HPLC method. HPLC methods were developed which make it possible to determine not only the parent drug, but also the main metabolites of CyA: AMI (M17), AM9 (M1), and AM4N (M21). Preparation of blood samples, their extractions and purifications are discussed. Chromatography is usually carried on C18 or CN columns by isocratic elution under high temperature (70 degrees C). CyD or CyC is used as the internal standard HPLC-MS method enables unambiguous identification of CyA metabolites after their separation on a chromatographic column and it is used mostly for research purposes only.

[Immunoanalytical methods of cyclosporine A determination]. / Imunoanalytické metody stanovení cyklosporinu A.

The standard method for determination of CyA in blood is a good validated HPLC method. HPLC methods were developed which make it possible to determine not only the parent drug, but also the main metabolites of CyA: AMI (M17), AM9 (M1), and AM4N (M21). HPLC methods for determination of CyA are very laborious and expensive. Most transplantation centres in the world use immunoanalytical methods for TDM of CyA. In recent years there has been an increase in the number of users of nonisotopic automated methods (Abbott TDx mono, AxSYM, Dade-Behring EMIT or Microgenic Cedia), which render instantaneous analysis possible. The analysis of the results of CyA in the blood in patients after transplantation shows that all immunoanalytical methods overestimate the concentration of CyA against HPLC results. The usually presented series of results is as follows: HPLC < RIA = EMIT < CEDIA < AxSYM < TDx. The differences are difficult to explain only by a different degree of cross reactivity for particular metabolites of CyA with the antibody used in the immunoanalytical method. The criterion of IFCC, which should be met by each immunoanalytical method in order to be used for TDM of CyA, should be redrawn, because it is not able to ensure sufficient specificity of the method. A suitable, cheap, and quick analytical method for TDM of CyA is still wanted. Some transplantation centres, including ours, carry out not only one method for effective TDM of CyA.

Evaluation and comparison of therapeutic monitoring of whole-blood levels of cyclosporin A and its metabolites in renal transplantation by HPLC and RIA methods.

BACKGROUND: The aim of the work was to evaluate the possibility to estimate the level of cyclosporin A (CyA) metabolites as the difference of radioimmunoassay (RIA) non-specific and RIA specific methods. METHODS: Blood samples of renal transplant patients were analyzed by three different methods: RIA specific method (CYCLO-Trac, DiaSorin, USA) (RIA(SP)), RIA non-specific method (Immunotech, Czech Republic) (RIA(NS)), and high performance liquid chromatography (HPLC) method. RESULTS: Although values obtained by RIA(SP) correlated well those obtained by HPLC (RIA(SP)=0.995.HPLC+9.68; r(2)=0.962, n=448), the results of HPLC methods were lower by 8%. The values obtained by RIA(NS) were 2.57 times higher than the values obtained by RIA(SP) (RIA(SP)=0.356RIA(NS); r(2)=0.713, n=448). The ratio (CyA+CyA metabolites)/(CyA) calculated as the ratio RIA(NS)/RIA(SP) values for 42 renal transplant patients was relatively stable for each particular patient. The sum of selected CyA metabolites (M1+M17+M21) measured by HPLC correlated well with that estimated from the difference of RIA(NS)-RIA(SP): HPLC(metab)=0.921.(RIA(NS)-RIA(SP))+21.3; (r(2)=0.746, n=448). CONCLUSION: The combination of both the specific and non-specific methods for the determination of CyA presents an improved means for the TDM of CyA and CyA metabolites in renal transplant patients. Moreover, a combination of both methods can help to elucidate some unexpected events, such as the persistence of high cyclosporin blood levels.

High-performance liquid chromatographic method for therapeutic drug monitoring of cyclosporine A and its two metabolites in renal transplant patients.

A novel fast HPLC method was developed for the determination of cyclosporine A (CyA) and its two metabolites M17 (AM1) and M21 (AM4N) in blood. Whole blood was precipitated with zinc sulphate, extracted with diethyl ether, evaporated, dissolved in aqueous methanol and partitioned twice with n-hexane. Chromatography was carried out using a microbore RP-column under isocratic elution with acetonitrile-methanol-water (200:80:140, v/v/v) at 70 degrees C and a detector set at 205 nm. Linearity for all three compounds was tested in the range of 1-1000 ng/ml. Recovery was 97-109%, and a coefficient of variation was 1.6-8.8% depending on the particular compound and its concentration. The method was used for a group of renal transplant patients having an inadequate response to CyA therapy in order to evaluate the possible role of CyA and its metabolites on the occurrence of hypertension and other toxicological events.

Quality control program of steroid receptor assays: an international study.

Lyophilized calf uterine cytosol standards were prepared for quality control of estrogen receptor (ER) determination, and lyophilized cytosols and tissue powders were used for quality control of progesterone receptor (PR) analysis. Two series of four samples were analyzed either for ER or PR contents, twice within one month, by 7 laboratories in 5 countries. Coefficient of variation (CV) of the between-laboratory averages assayed in a single run of ER-positive (ER+) and PR-positive (PR+) standards varied from 29.6 to 61.8% and from 32.4 to 76.2%, respectively. All laboratories, with the exception of a single value, could recognize samples of low, medium, an high ER level, as well as a negative sample. Most laboratories evaluated properly also the level of PR samples. The average between-laboratory CV values of protein determination in the relevant standards were 23%.

[Antibodies against thyroxine, triiodothyronine and thyroglobulin as a possible cause of error in the diagnosis of disorders of thyroid gland function]. / Protilátky proti tyroxinu, trijodtyroninu a tyreoglobulinu jako mozná prícina omylu pri diagnostice poruch funkce stítné zlázy.

The authors examined in a 82-year-old female patient T4, T3, free T4, TSH, thyroglobulin and made the TRH-TSH functional test because of suspected thyroid disease. For thyroid hormones they obtained controversial resulting levels: in T4 signalizing an unreasonable negative value, in T3 a very high level, in TSH and the TRH-TSH test levels suggesting normal thyroid function which was consistent with the clinical picture. A more detailed laboratory analysis revealed the presence of antibodies binding 125I-T4 and 125I-T3 (radioindicators used for assessment of T4 and T3). The authors identified antibodies against T4, T3 and thyroglobulin. Their presence influenced the radioimmunoassay of T4 and T3 and also the immunoradiometric estimation of thyroglobulin (artificially raised level). The reliable result of the eufunctional condition was not influenced in the estimation of the TSH level.

[Effects of venous compression for blood sampling on the results of in vitro tests of thyroid function (author's transl)]. / Einfluss der Venenkompression bei der Blutentnahme auf die Ergebnisse der in vitro-Teste der Schilddrüsenfunktion.

In a group of subjects with a normal as well as pathological thyroid function there was a significant increase in RT3U, serum-thyroxine-concentrations and TSH-levels but there were no significant changes in the T4-RT3U-indices and ETR-tests after 5 min of venous compression. The discordant results were obtained in 21 cases (26.9%) with the RT3U-test and in 4 cases (5,1%) with the T4-serum-level-determination, which may cause diagnostic errors. The non-standardized venous compression time causes a widening of the euthyroid zone which reduces the discrimination capabilities of these function tests. With TSH-determinations these deviations may be of diagnostic importance in the evaluation of TRH-TSH-test results. A standardized and minimum compression time when drawing blood samples is recommended in order to eliminate this potential source of diagnostic error.