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Methotrexate Assay

Accurately screen for MTX with low-end sensitivity.

Precision
Precision was determined as described in CLSI/NCCLS Protocol EP5-A2. The six-level ARK Methotrexate Control and pooled human specimens containing methotrexate were used in the study. Each level was assayed in quadruplicate twice a day for 20 days. Each of the runs per day was separated by at least two hours. The within run, between day, total SD, and percent CVs were calculated.

   Within RunBetween DayTotal
SampleNMean (µg/mL)SDCV (%)SDCV (%)SDCV (%)
ARK Methotrexate Control
LOW1600.060.0058.20.0057.30.00710.7
MID1600.370.0113.00.0082.10.0143.8
HIGH1600.760.0324.30.0304.00.0455.9
5*1604.80.153.10.132.80.204.2
50*160491.362.82.324.82.725.6
500*16047615.173.230.756.534.667.3
Patient Pool
LOW1600.070.0069.10.0057.50.00811.7
MID1600.410.0133.30.0266.40.0297.2
HIGH1600.820.0374.50.0435.20.0577.0
5*1604.60.143.10.1834.00.245.3
50*160451.333.02.635.92.936.6
500*16046111.842.627.045.929.606.4

 

*Samples were diluted in ARK Methotrexate Dilution Bufer.
Mean result and SD were multiplied by the dilution factor.

Specificity
Cross-reactivity to 7-Hydroxymethotrexate, the major metabolite
The ARK Methotrexate Assay did not crossreact with the major metabolite 7-OH-MTX.

Reagent Lot7-OH MTX (µmol/L)Serum Control (µmol/L)MTX (µmol/L)Cross Reactivity (%)
Cross Reactivity 7-OH MTX in the presence of 0.05 µmol/L MTX
D25.00.060.060.01
D35.00.040.050.02
Cross Reactivity 7-OH MTX in the presence of 0.05 µmol/L MTX
D250.00.440.470.07
D350.00.460.480.05

Cross-reactivity to the minor, inactive metabolite 2,4-diam ino-N10-methylpteroic acid (DAMPA)
The ARK Methotrexate Assay crossreacts substantially with the minor metabolite DAMPA. Tests were performed in the absence of the parent drug methotrexate. Crossreactivity to DAMPA ranged 64.3 to 100%. The assay should not be used during possible compassionate therapy with glucarpidase (carboxypeptidase G2) that rapidly converts circulating methotrexate to DAMPA.

Drugs that crossreact
The ARK Methotrexate Assay crossreacts slightly with triamterene and trimethoprim, however these drugs may be contraindicated for MTX cancer treatment due to additional adverse effects if co-administered. The structures of these compounds closely match the pteridine ring moiety of methotrexate.

CompoundTested (µmol/L)Apparent MTX (µmol/L)Cross Reactivity (%)
MTX Absent
Triamterene250.461.85
Trimethoprim1000.170.17
MTX Present 0.05 (µmol/L)
Triamterene250.893.32
Trimethoprim1000.160.12
MTX Present 0.05 (µmol/L)
Triamterene251.042.31
Trimethoprim1000.990.54

 

Cross-reactivity to folate analogs and other compounds
The ARK Methotrexate Assay did not cross-react (≤ 0.01%) with folate analogs or other compounds at ≥ 1000 μmol/L as tested.

CompoundTexted (μmol/L)
Adriamycin1000
Cyclophosphamide1500
Cytosine1000
Dihydrofolic Acid1000
DL-6-Methyl-5,6,7,8-Tetrahydropterine1000
Folic Acid1000
Folinic Acid (leucovorin)1000
5-Fluorouracil3000
6-Mercaptopurine1000
5-Methyltetrahydrofolic acid1000
Prednisolone1000
Pyrimethamine1000
Sulfamethoxazole1600
Tetrahydrofolic Acid1000
Vinblastine1000
Vincristine1000

The products/features (mentioned herein) are not commercially available in all countries. Due to regulatory reasons, their future availability cannot be guaranteed. Please contact your local Siemens organization for further details.

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