Total Mycophenolic Acid

Catalog Number: 04357213190

SYSTEM INFORMATION

Test TMPA, test ID 0-623

INTENDED USE

In vitro test for the quantitative determination of total mycophenolic acid in serum or plasma as an aid in the management of mycophenolic acid therapy in renal and cardiac transplant patients on COBAS INTEGRA systems.

STORAGE AND STABILITY

Shelf life at 2-8 °C See expiration date on cobas c pack label
COBAS INTEGRA 400 plus system
On-board in use at 10-15 °C 12 weeks
COBAS INTEGRA 800 system
On-board in use at 8 °C 12 weeks

The on-board in use stability period begins at the time of cobas c pack puncture.

APPLICATION FOR SERUM AND PLASMA

COBAS INTEGRA 400 PLUS TEST DEFINITION

Measuring mode Absorbance
Abs. calculation mode Kinetic
Reaction mode R1/R2-S
Reaction direction Increase
Wavelength 340/409 nm
Reading cycle blank/test 52/65
Unit µg/mL

PIPETTING PARAMETERS

Diluent (H2O)
R1 185 µL 4 µL
R2 19 µL 4 µL
Sample 3 µL 7 µL
Total volume 222 µL

Diluent (H2O)
R1 185 µL 4 µL
R2 19 µL 4 µL
Sample 3 µL 7 µL
Total volume 222 µL

COBAS INTEGRA 800 TEST DEFINITION

Measuring mode Absorbance
Abs. calculation mode Kinetic
Reaction mode R1/R2-S
Reaction direction Increase
Wavelength 340/409 nm
Reading cycle blank/test 175/197
Unit µg/mL

PIPETTING PARAMETERS

Diluent (H2O)
R1 185 µL 4 µL
R2 19 µL 4 µL
Sample 3 µL 7 µL
Total volume 222 µL

CALIBRATION

A calibration curve must be prepared using the Total MPA Calibrators. Calibrators must be placed from the highest concentration (F) first, to the lowest (A) last, on the CAL/QC rack. This curve is retained in memory by the COBAS INTEGRA systems and recalled for later use.

Traceability: The Total MPA Calibrators are prepared to contain known quantities of mycophenolic acid in normal human serum and are traceable to a primary reference method (HPLC).

Calibrators Total MPA CalibratorsBottles A-F
MPA conc. 0, 1, 3, 5, 10, 15 µg/mL
Calibration mode Logit/log 4
Calibration replicate Duplicate recommended
Calibration interval Each lot, every 9 days and as required following quality control procedures

LIMITS AND RANGES

Measuring range

0.4-15 µg/mL (1.2-46.8 µmol/L)

Extended measuring range

Postdilution factor: 5 recommended

0.4-50 µg/mL (1.2-156.1 µmol/L)

Manually dilute samples above the measuring range with the diluent (equivalent to the 0 µg/mL calibrator) from the Roche Total MPA Calibrators (1 part sample + 4 parts diluent) and reassay. Multiply the result by 5 to obtain the specimen value.

Lower limits of measurement

Lower detection limit of the test:

0.3 µg/mL (0.9 µmol/L)

The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying 2 standard deviations above that of the 0 µg/mL calibrator (standard A + 2 SD, repeatability, n = 21).

Functional sensitivity:

0.4 µg/mL (1.2 µmol/L)

The functional sensitivity is calculated as the lowest concentration from clinical samples with a CV of

≤ 20 %, tested in triplicate over 10 days (n = 30).

SPECIFIC PERFORMANCE DATA

Representative performance data on the COBAS INTEGRA analyzers are given below. Results obtained in individual laboratories may differ.

PRECISION

Precision was determined using Roche Total MPA Controls and human plasma samples according to CLSI (Clinical Laboratory Standards Institute) guidelines.

The following results were obtained on a COBAS INTEGRA 700 analyzer with repeatability (n = 63) and intermediate precision (3 aliquots per run, 1 run per day, 21 days):

*HP 1 and HP 2 are non-spiked pooled clinical samples.

The following results were obtained on a COBAS INTEGRA 400 analyzer with repeatability (n = 30) and intermediate precision (3 aliquots per run, 1 run per day, 10 days):

**HP 1 and HP 2 are spiked MPA plasma pools.

Repeatability Meanµg/mL (µmol/L) SDµg/mL (µmol/L) CV%
Level 1 0.93 (2.90) 0.01 (0.03) 1.1
Level 2 3.54 (11.05) 0.01 (0.03) 0.4
Level 3 12.37 (38.62) 0.06 (0.19) 0.5
HP 1* 1.61 (5.03) 0.01 (0.03) 0.8
HP 2* 6.36 (19.86) 0.04 (0.12) 0.7

Intermediate precision Meanµg/mL (µmol/L) SDµg/mL (µmol/L) CV%
Level 1 0.93 (2.90) 0.04 (0.12) 4.5
Level 2 3.54 (11.05) 0.06 (0.19) 1.8
Level 3 12.37 (38.62) 0.16 (0.50) 1.3
HP 1* 1.61 (5.03) 0.04 (0.12) 2.2
HP 2* 6.36 (19.86) 0.11 (0.34) 1.8

Repeatability Meanµg/mL (µmol/L) SDµg/mL (µmol/L) CV%
Level 1 0.91 (2.84) 0.02 (0.06) 2.0
Level 2 3.48 (10.86) 0.04 (0.12) 1.1
Level 3 12.22 (38.15) 0.03 (0.09) 0.3
HP 1** 1.55 (4.84) 0.03 (0.09) 1.7
HP 2** 9.11 (28.44) 0.07 (0.21) 0.8

Intermediate precision Meanµg/mL (µmol/L) SDµg/mL (µmol/L) CV%
Level 1 0.91 (2.84) 0.04 (0.12) 4.5
Level 2 3.48 (10.86) 0.06 (0.19) 1.8
Level 3 12.22 (38.15) 0.09 (0.28) 0.7
HP 1** 1.55 (4.84) 0.05 (0.16) 3.0
HP 2** 9.11 (28.44) 0.08 (0.25) 0.9

METHOD COMPARISON

Mycophenolic Acid values for human plasma samples obtained with the Roche Total Mycophenolic Acid assay were compared to those determined with three independent validated MPA HPLC methods. Samples from renal and cardiac transplant patients were tested with the Roche Total Mycophenolic Acid assay at two external clinical sites and also internally, with concurrent HPLC testing at each site.

The trial included a total of 571 samples collected from post-transplant patients. The Passing-Bablok statistics of the correlations are shown in the table below.20 Samples tested on the COBAS INTEGRA 800 analyzer were obtained from an international trial of renal transplant recipients (147 samples from 86 adult patients). Demographics for the other sites are subsequently described with the representative regression plots that follow the table.

The following graph shows the correlation testing of the Roche Total Mycophenolic Acid assay on the COBAS INTEGRA 700 analyzer vs HPLC. The same data sets are depicted in the Bland-Altman difference plot shown below the regression plot. The sample population for this internal study included 89 renal and 70 cardiac patients. Other demographics for this sample population are unknown. Passing-Bablok statistics for the correlation are included in the method comparison table above.

The following graph shows the correlation testing of the Roche Total Mycophenolic Acid assay on the COBAS INTEGRA 400 plus analyzer vs HPLC. The same data sets are depicted in the Bland-Altman difference plot shown below the regression plot. The sample population for this external study included 265 samples (148 renal and 117 cardiac) from a total of 209 “routine” adult transplant recipients. Passing-Bablok statistics for the correlation are included in the method comparison table above.

Methodology vs. HPLC
Slope(95 % CI) Intercept(95 % CI) Correlation Coefficient Sample Size Sample Range(µg/mL)
COBAS INTEGRA 700 analyzer
Renal 1.062(1.044-1.079) 0.019 (-0.021-0.073) 0.997 89 0.46-13.6
Cardiac 1.088(1.050-1.125) -0.028(-0.110-0.045) 0.993 70 0.57-14.2
Combined 1.068(1.052-1.085) 0.005(-0.029-0.050) 0.995 159 0.46-14.2
COBAS INTEGRA 400 plus analyzer
Renal 1.010 (0.988-1.035) 0.068(0.034-0.105) 0.995 148 0.4-14.8
Cardiac 1.014(0.996-1.031) 0.057 (-0.004-0.103) 0.992 117 0.4-13.6
Combined 1.011(1.000-1.025) 0.064(0.038-0.090) 0.993 265 0.4-14.8
COBAS INTEGRA 800 analyzer
Renal 1.100(1.073-1.120) -0.120(-0.192-(-0.066)) 0.994 147 0.5-14.7

Roche Total Mycophenolic Acid (COBAS INTEGRA 700 analyzer) vs HPLC
COBAS INTEGRA 700 analyzerµg/mL
HPLCµg/mL

N = 159Mean (Y-X) = 0.38SD (Y-X) = 0.521.96 SD = 1.02Mean + 1.96 SD = 1.41Mean – 1.96 SD = -0.64

Roche Total Mycophenolic Acid (COBAS INTEGRA 400 plus analyzer) vs HPLC
COBAS INTEGRA 400 plus analyzer µg/mL
HPLC µg/mL

N = 265Mean (Y-X) = 0.13SD (Y-X) = 0.381.96 SD = 0.75Mean + 1.96 SD = 0.881Mean – 1.96 SD = -0.62

ANALYTICAL SPECIFICITY

The following cross-reactive substances were evaluated on the COBAS INTEGRA systems in normal human serum spiked with mycophenolic acid at 1.7 µg/mL (5.3 µmol/L) and 8.0 µg/mL (25.0 µmol/L). Cross-reactivity was designated as “not detectable” (ND) if the obtained value was less than the sensitivity of the assay.

The following compounds were tested at the concentrations listed for interference in normal human serum spiked with mycophenolic acid at approximately 1.5 µg/mL (4.7 µmol/L) and 9.0 µg/mL (28.1 µmol/L). No significant interference with the assay was found.

In addition, tests were performed on 9 drugs. No significant interference with the assay was found.

Cross-reactivity (%) = 100 × (analytical result - analyte concentration)
concentration of interferent

Drug Level tested(µg/mL) Cross-reactivity%
Mycophenolic acid glucuronide (MPAG) 1000 ND
Mycophenolic acid acyl glucuronide (AcMPAG) 10 5.6

Compound µg/mL Compound µg/mL
Acetaminophen 60 Isoproterenol 0.18
Acyclovir 45 Itraconazole 6
Albuterol 1.2 Kanamycin 180
Allopurinol 120 Ketoconazole 10.5
Alprazolam 6 Labetalol 0.573
Amikacin 105 Lidocaine 36
Amphotericin B 240 Lovastatin 0.0357
Ascorbic Acid 120 Methylprednisolone 36
Atenolol 30 Metoclopramide 1.35
Azathioprene 9 Minoxidil 0.921
Bromocriptine 0.75 Misoprostol 0.018
Caffeine 180 Morphine sulfate 1.5
Captopril 15 N-Acetylprocainamide 120
Carbamazepine 90 Nadolol 3.6
Cefaclor 225 Naproxen 500
Ceftriaxone 2430 Niacin 2400
Cephalosporine 0.3 Nicardipine 0.564
Chloramphenicol 150 Nifedipine 1.2
Chloroquine 2.5 Omeprazole 18
Cimetidine 60 Penicillin G 36
Ciprofloxacin 30 Phenobarbital 300
Clonidine 0.03 Phenytoin 150
Colchicine 0.033 Piperacillin 120
Cyclophosphamide 1125 Prazosin 0.057
Cyclosporine A 1.2 Prednisolone 1.173
Digitoxin 0.075 Prednisone 0.9
Digoxin 0.015 Primidone 120
Diltiazem 0.12 Probucol 2400
Dipyridamole 7.5 Procainamide 72
Disopyramide 30 Promethazine 3.6
Erythromycin 180 Propanolol 6
Ethanol 12000 Quinidine 36
Everolimus 0.12 Ranitidine 18
Fluconazole 225 Rifampicin 192
Flucytosine 240 Salicylic Acid 1800
Folic Acid 0.060 Sirolimus 0.084
Furosemide 180 Spectinomycin 480
Ganciclovir 48 Sulfamethoxazole 1200
Gentamicin 36 Tacrolimus 0.12
Glipzide 6 Theophylline 120
Glyburide 6 Ticlopidine 4.26
Heparin 8000 U/L Tobramycin 36
Hydralazine 1.62 Triamterene 18
Hydrochlorothiazide 18 Trimethoprim 120
Ibuprofen 500 Valproic Acid 1500
Insulin 1320 mU/L Vancomycin 300
Isoniazid 120 Verapamil 6

Acetyl Cysteine Ampicillin-Na
Ca-Dobesilate Cefoxitin
Doxycycline (Tetracycline) Levodopa
Methyldopa + 1.5 H20 Metronidazole
Phenylbutazone