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UW Health SMPH
American Family Children's Hospital

UW Hospital and Clinics Lab Test Directory

Test Name: Methotrexate

Test Code(s): MTX / HCMTX

CPT Code(s): 80299

Methodology: Homogenous Enzyme Immunoassay

Clinical Significance: Antineoplastic

Days Performed: Daily.

Turnaround Time: Stat: 1 hour.
Routine: 24 hours.

Specimen Requirements

Specimen: Blood

Collection Container: Preferred: Red top

Collection Volume: Preferred: 4 mL
Pediatrics: 0.6 mL

Sample Analyzed: Serum

Volume Required: Preferred: 1 mL
Pediatrics: 0.2 mL

Specimen Processing: Centrifuge. Transfer cell-free serum to plastic vial. Refrigerate.

Specimen Transport: Transport specimen to laboratory. Transport with coolant pack if coming from outreach location.

Unacceptable Criteria: Specimens collected in red cap with yellow ring (SST) tubes are not acceptable.

Stability: Ambient: 8 hours
Refrigerated: 2 weeks
Frozen: 3 months at -20C or below


The laboratory does not interpret methotrexate levels.

Test Limitations:

The manufacturer has not determined the efficacy of this test when performed on Siemens Vista.  The performance characteristics of this test were determined by UWHC Clinical Laboratory.


Patients who have received glucarpidase (carboxypeptidase G2) as a high dose methotrexate rescue therapy should not be tested with the ARK Methotrexate Assay.  These specimens have increased serum levels of 4-[[2,4-diamino-6-(pteridinyl)methyl]-methylamino]-benzoic acid (DAMPA) that result from metabolism of methotrexate by glucarpidase.  DAMPA cross reacts with the methotrexate antibody used in this assay, and may continue to circulate for at least five to seven days before accurate measurement of serum methotrexate may return.

Additional Information:

Result of "BELOW" indicates that the result is below the method detection limit of 0.04 umol/L. Dependent on test results as applied to dose and interval, adjustments may need to be made in the leucovorin rescue dose, interval, and/or duration. Levels and rescue should be continued on a 24 hour basis until methotrexate level is less than 0.1 umol/L.


This test was performed using a modification of an FDA approved method.  The performance characteristics of this test were determined by the UWHC Clinical Laboratories and meet the standards for clinical testing.

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