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UW Health SMPH
American Family Children's Hospital

UW Hospital and Clinics Lab Test Directory

Test Name: Vitamin D, 25-Hydroxy by HPLC

Test Code(s): LCD25 / HCLCD25

CPT Code(s): 82306

Test Description: Test Component:

Total 25-Hydroxy Vitamin D, 25-Hydroxy Vitamin D2 and 25-Hydroxy Vitamin D3

Methodology: Liquid Chromatography

Days Performed: Mon-Fri, dayshift.

Turnaround Time: Routine: 4 - 7 days.

Specimen Requirements

Specimen: Blood

Collection Container: Preferred: Lavender top
Also Acceptable: Red top

Collection Volume: Preferred: 4 mL
Pediatrics: 1 mL

Sample Analyzed: Plasma or serum

Volume Required: Preferred: 1 mL
Pediatrics: 0.5 mL

Specimen Processing: Centrifuge. Transfer plasma 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: 7 days
Refrigerated: 2 weeks
Frozen: 6 months


Reference Interval:


Vitamin D status in a patient is judged by

Total 25-0H Vitamin D


 Total 25-hydroxy Vitamin D clinical reference values: 
 < 10 ng/mL  Severe deficiency
 10-29 ng/mL  Mild to moderate deficiency
 30-80 ng/mL  Optimum levels
 > 80 ng/mL  Toxicity possible

Test Limitations:

Patients treated with Atovaquone may exhibit a falsely elevated 25-OH Vitamin D2 level regardless of whether the patient is receiving Vitamin D2 (ergocalciferol) therapy.


Vitamin D requests more frequent than 60 days will automatically be cancelled by the laboratory as a duplicate request.

Additional Information:

A patient's vitamin D status should be judged by the total 25-Hydroxy vitamin D which is equal to the combined 25-Hydroxy D2 and 25-Hydroxy D3 levels. Vitamin D by HPLC (UV) quantitates both the D2 and D3 forms of the hydroxylated vitamin. D3 is the endogenous form of the vitamin that is derived from sunlight and the conversion of cholestane to cholecalciferol which is subsequently converted by the liver to 25-Hydroxy D3. Occasionally, D3 (cholecalciferol) is found in multi-day vitamin preparations but not in concentrations that achieve therapeutic doses required for treating osteoporosis. Whereas, D2 (ergocalciferol) is an exogenous form derived from plant sources and generally reflects supplementation. Levels of 25-Hydroxy vitamin D3 vary seasonally, especially in the northern climate of Wisconsin.


This test was developed and its performance characteristics determined by the UWHC Clinical Laboratories.  This test meets the standards for clinical testing.  This test should not be regarded as investigational or for research use.  The US Food and Drug Administration (FDA) has not approved or cleared this test; however, the FDA has determined that such clearance or approval is not necessary.

Vitamin D2,
Vitamin D3

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