25OH Vitamin D Total Assay - University of California, San Francisco

UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
Procedure Title: 25OH Vitamin D Total Assay – DiaSorin LIAISON
Document Section: CHEMISTRY Author: Kirk Sujishi, Linda Post Date Authored: NOVEMBER 2009
Laboratory Director: TED KURTZ, MD (PARNASSUS & CHINA BASIN)
Distribution: China Basin: CHEMISTRY
In Use Date: December 7, 2009 Discontinued Date: ___________
Revised By : ________________________ Reason:______________________________________Date: ____________
Revised By : ________________________ Reason:______________________________________Date: ____________
Revised By : ________________________ Reason:______________________________________Date: ____________
Revised By : ________________________ Reason:______________________________________Date: ____________
Revised By : ________________________ Reason:______________________________________Date: ____________
Annual Director’s Approval (P/CB): ___________________________ Date: ________________
Annual Director’s Approval (P/CB): ___________________________ Date: ________________
Annual Director’s Approval (P/CB): ___________________________ Date_________________
Annual Director’s Approval (P/CB): ___________________________ Date_________________
Annual Director’s Approval (P/CB): ___________________________ Date_________________
Annual Director’s Approval (P/CB): ___________________________ Date: ________________
Annual Director’s Approval (P/CB): ___________________________ Date: ________________
Annual Director’s Approval (P/CB): ___________________________ Date_________________
Annual Director’s Approval (P/CB): ___________________________ Date_________________
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I. PURPOSE
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
The LIAISON 25 OH Vitamin D TOTAL Assay uses chemiluminescent immunoassay (CLIA)
technology for the quantitative determination of 25-hydroxyvitiamin D and other hydroxylated vitamin
D metabolites in human serum to be used in the assessment of vitamin D sufficiency. Assay results
should be used in conjunction with other clinical or laboratory data to assist the clinician in making
individual patient management decisions in an adult population.
SUMMARY AND EXPLANATION OF TEST
The role of vitamin D in bone and mineral metabolism was recognized from its first identification as a
factor that could cure rickets. However, vitamin D is now recognized as a prohormone, which has
multiple roles in maintaining optimal health.
Vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) are the most abundant forms of vitamin D
in the body. Vitamin D is synthesized in the skin from 7-dehydrocholesterol in response to sunlight.
The best nutritional sources of D3 are oily fish, primarily salmon and mackerel. Vitamin D2‘s nutritional
sources are some vegetables, yeast and fungi. The vegetarian diet is abundant in vitamin D2.
Vitamin D (D3, D2 and metabolites) is converted to 25-hydroxyvitamin D in the liver. The
measurement of 25-OH vitamin D concentration in the serum or plasma is the best indicator of
vitamin D nutritional status.
The optimal level of 25-OH vitamin D is subject to some debate, but >32ng/mL (>80nmol/L) is
accepted as sufficient for bone health. Vitamin D toxicity is a recognized problem, but a rare
occurrence. Instead, a recent growing public health problem is vitamin D insufficiency. Several
research studies have identified widespread vitamin D insufficiency in apparent healthy populations
world-wide.
Vitamin D deficiency is commonly treated with vitamin D2 or D3 medications in doses ranging from
50,000 IU/month to 50,000 IU/week. Fortified foods and nutrition supplements may contain either
form. To ensure accurate assessment of the total vitamin D all forms, including D3, D2 and
metabolites, must be measured.
Recent studies have identified an inactive 3-epimer of 25-OH vitamin D, which may be present in the
serum of infants under the age of one year. Thus it is important that an assay avoid measuring the
inactive 3-epi form and only measure the active D3 and D2 forms equally.
BIOLOGICAL PRINCIPLES OF THE PROCEDURE
The LIAISON 25 OH Vitamin D assay is a direct competitive chemiluminescence immunoassay
(CLIA) for quantitative determination of total 25 OH vitamin D in serum. During the first incubation, 25
OH vitamin D is dissociated from its binding protein and binds to the specific antibody on the solid
phase. After 10 minutes, the tracer (vitamin D linked to an isoluminol derivative) is added. After a
second 10 minute incubation, the unbound material is removed with a wash cycle. Subsequently, the
starter reagents are added to initiate a flash chemiluminescent reaction. The light signal is measured
by a photomultiplier as relative light units (RLU) and is inversely proportional to the concentration of
25 OH vitamin D present in calibrators, controls or samples.
II. POLICY/SCOPE
This procedure is intended for use by licensed CLS staff in the China Basin Chemistry section.
III. TEST AVAILABILITY
Test performed Monday, Wednesday and Fridays. Results will be available by 16:00.
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IV. SPECIMEN REQUIRMENTS
SPECIMEN TYPES
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
1. Human serum or SST tubes.
2. Fasting samples are recommended, but not required.
3. Grossly hemolyzed or lipemic samples as well as samples containing particulate matter or
exhibiting obvious microbial contamination should not be tested.
4. The Diasorin LIAISON does not provide the capability to verify specimen type. It is the
responsibility of the operator to verify that the correct specimen type is used in the 25OH Vitamin
D TOTAL assay.
PREPARATION FOR ANALYSIS
STORAGE
STABILITY
1. The minimum volume required is 250 µL of specimen for the first test, and 25 µL more for each
additional test.
2. Mix thawed specimens thoroughly by low speed vortexing or by inverting 10 times. Visually
inspect the specimens. If layering or stratification is observed, continue mixing until specimens
are visibly homogeneous.
3. Samples having particulate matter, turbidity, lipemia or erythrocyte debris may require clarification
by filtration or centrifugation before testing. Check for and remove air bubbles before assaying.
1. Frozen (-20˚C or below). Mix thawed samples well before testing.
2. Specimens may be stored in glass or plastic vials. No additives or preservatives are required to
maintain integrity of the samples.
3. Avoid multiple freeze/thaw cycles. Samples should not be tested if exposed to more than three
freeze/thaw cycles.
After separation from cells:
• Ambient: 3 days
• Refrigerated: 1 week
• Frozen: 6 months
V. EQUIPMENT, REAGENTS AND SUPPLIES
TEST INSTRUMENT: DiaSorin LIAISON System
MATERIALS PROVIDED
DiaSorin LIAISON Reagent Integral (Cat. No. 310600)
MATERIALS REQUIRED BUT NOT PROVIDED
1. DiaSorin LIAISON System (including sample racks).
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REAGENTS
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
2. LIAISON Module (Cat. No. 319130)
3. LIAISON Starter Kit (Cat. No. 319102)
4. LIAISON Light Check (Cat. No. 319101)
5. LIAISON Wash/System Fluid (Cat No. 319100)
6. LIAISON Waste Bags (Cat. No. 9450003)
7. LIAISON 25 OH Vitamin D TOTAL Control Set (Cat. No. 310601)
8. LIAISON 25 OH Vitamin D TOTAL Specimen Diluent (Cat. No. 310602)
9. LIAISON 25 OH Vitamin D TOTAL Calibration Verifiers (Cat. No. 310603)
10. LIAISON Cleaning Kit (Cat. No. 310990)
11. LIAISON Xcelerator (Cat. No. A0090)
12. Centrifuge (IEC Centra-7, International Equipment Company)
13. 12 x 75 glass or plastic tubes
14. Transfer pipettes
15. Calibrated pipettes for delivering volumes of 75 µL and 225 µL
16. For information on materials required for maintenance procedures, refer to the DiaSorin Liaison
User Manual, Chapter 18.
DIASORIN LIAISON REAGENT INTEGRAL, 100 TESTS, the following components are all contained
within the Reagent Integral:
1. Magnetic Particles (2.4 mL) Magnetic particles coated with antibody against 25 OH Vitamin D,
protein, phosphate buffer, < 0.1% sodium azide.
2. Assay Buffer (28.0 mL) Buffer with 10% ethanol, surfactants and preservatives.
3. Conjugate (4.5 mL) 25 OH Vitamin D conjugated to an isoluminol derivative, in phosphate buffer
with 10% ethanol, EDTA, surfactant and preservatives.
4. Calibrator 1 (1.0 mL) Human serum, Tris Buffer, < 0.1% sodium azide and 25 OH Vitamin D.
The calibrator concentrations (ng/mL) are referenced to standard preparations containing highly
purified 25 OH Vitamin D.
5. Calibrator 2 (1.0 mL) Human serum, Tris Buffer, < 0.1% sodium azide and 25 OH Vitamin D.
The calibrator concentrations (ng/mL) are referenced to standard preparations containing highly
purified 25 OH Vitamin D.
OTHER REAGENTS
1. LIAISON STARTERS
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UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
a. LIAISON STARTER 1: Catalyst in 4% NaOH
b. LIAISON STARTER 2: 0.12% peroxide solution
2. LIAISON WASH/SYSTEM LIQUID: Phosphate buffer solution (10x concentrate). Preservative:
sodium azide.
3. LIAISON LIGHT CHECK
4. LIAISON 25 OH VITAMIN D TOTAL CONTROL SET (with Bar Code Labels for Controls 1 and 2)
a. CONTROL LEVEL 1: Human serum with buffer salts and < 0.1% sodium azide as a
preservative.
b. CONTROL LEVEL 2: Human serum with buffer salts and < 0.1% sodium azide as a
preservative.
5. LIAISON 25 OH VITAMIN D TOTAL SPECIMEN DILUENT: Human serum with buffer salts and

UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
6. LIAISON 25 OH VITAMIN D TOTAL SPECIMEN DILUENT is stored at 2 – 8˚C upon receipt.
When stored and handled as directed, the reagent is stable until the expiration date.
7. LIAISON 25 OH VITAMIN D TOTAL CALIBRATION VERIFIERS are stored at 2 – 8˚C upon
receipt. When stored and handled as directed, the reagent is stable until the expiration date.
Once the vials are opened, the reagents are stable for 4 weeks when properly stored.
INDICATIONS OF REAGENT DETERIORATION
When a control value is out of the specified range, it may indicate deterioration of the reagents or
errors in technique. Associated test results are invalid and samples must be retested. Assay
recalibration may be necessary.
VI. WARNINGS AND PRECAUTIONS
CAUTION
For In Vitro Diagnostic Use.
1. This product requires the handling of human specimens. It is recommended that all human
sourced materials be considered potentially infectious and be handled in accordance with the
OSHA Standard on Bloodborne Pathogens. Biosafety Level 2 or other appropriate biosafety
practices should be used for materials.
2. Some reagents used in this assay contain human source material and should be treated as
potentially infectious. Each unit used in the preparation of this product has been tested by an
FDA-approved method and found non-reactive for the presence of HBsAg, antibody to HCV and
antibody to HIV1/2. While these methods are highly accurate, they do not guarantee that all
infected units will be detected. This product may also contain other human source material for
which there is no approved test. Because no known test method can offer complete assurance
that hepatitis B virus, hepatitis C virus (HCV), Human Immunodeficiency Virus (HIV) or other
infectious agents are absent, all products containing human source material should be handled in
accordance with good laboratory practices using appropriate precautions as described in the
Centers for Disease Control and Prevention / National Institutes of Health Manual, “Biosafety in
Microbiological and Biomedical Laboratories.” 4th ed., May 1999.
3. Some reagents contain sodium azide as a preservative. Because sodium azide may form
explosive lead azide or copper azide in plumbing, it is recommended that drains be thoroughly
flushed with water after disposal of solutions containing sodium azide.
4. For a detailed discussion of safety precautions during system operation, refer to the LIAISON
User Manual.
HANDLING PRECAUTIONS
Do not use reagent kits beyond the expiration date.
1. Contents of starter reagent bottles should never be pooled when replacing new containers with
those in use, regardless of lot number.
2. Before placing an integral on the instrument, suspend the magnetic particles in the first
compartment by placing the integral in the Xcelerator for 30 seconds, then rotate the
compartment for an additional minute. Check that all particles have been resuspended. Place
the integral into the reagent bay and allow the instrument mixing to proceed for at least 30
minutes prior to starting a test run.
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3. Before loading an integral onto the instrument, check reagent compartments for bubbles and
liquid adherence to the covers; remove if present.
4. Newly prepared Wash/System Liquid must sit for 6 hours to de-gas prior to use on the instrument.
5. Wash/System Liquid tanks must be bleached monthly according to manufacturer’s
recommendations and rinsed thoroughly to prevent build up of residue from Wash/System Liquid,
which could affect background readings. See maintenance procedures.
VII. CALIBRATION / CALIBRATION VERIFICATION
1. The predefined Master Curve is adjusted to a new instrument-specific curve using the two
calibrators supplied in the Reagent Integral via the bar codes on the reagent integral label.
2. Renewed calibration is required:
a. With each new lot of reagents (Reagent Intergral or Starter Reagents).
b. Every 7 days.
c. After each servicing of the LIAISON Analyzer.
d. If quality controls are out of acceptable range.
3. Refer to the LIAISON Daily Quick Start Procedure for detailed information on how to perform an
assay calibration.
4. Refer to Appedix II for detailed information on how to perform a Calibration Verification.
VIII. QUALITY CONTROL
LIAISON 25 OH VITAMIN D TOTAL CONTROL SET (with Bar Code Labels for Controls 1 and 2)
1. Controls are received in liquid form and require no further dilution prior to use.
2. The controls are stored at 2 – 8˚C upon receipt. When stored and handled as directed, the
reagent is stable until the expiration date.
3. LIAISON 25 OH VITAMIN D TOTAL controls levels 1 and 2 are run each day of testing.
IX. PROCEDURE
ASSAY PROCEDURE
For detailed instructions on the following steps, refer to LIAISON Daily Quick Start Procedure.
For additional information on principles of operation, refer to the LIAISON User Manual.
1. Prepare the Analyzer.
a. Prepare Reagent Intergals.
b. Check Instrument Status.
c. Check Status of Data from Prior Run.
d. Check Printer Status.
2. Perform System Test.
3. Perform Calibration if required.
4. Run LIAISON 25 OH VITAMIN D TOTAL controls levels 1 and 2.
5. Program, place and run patient samples.
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6. Validate and print control and patient results.
7. Save the database to the designated flash drive.
8. Perform Daily Shut-Down procedure.
9. For optimal performance, it is important to perform routine maintenance as described in the
LIAISON User Manual. Detailed instructions may be found in the DisSorin LIAISON Maintenance
procedure.
SPECIMEN DILUTION PROCEDURES
Specimens with a 25 OH Vitamin D Total result of >150 ng/mL will be flagged as “>>”. Samples
should be diluted with LIAISON 25 OH Vitamin D TOTAL Specimen Diluent. Note that all lots of
specimen diluent are interchangeable between LIAISON 25 OH Vitamin D TOTAL Assay kit lots.
Manual dilutions should be done as follows:
a. A dilution of one part patient to 3 parts diluent is recommended, resulting in a 1:4 dilution.
b. Mix specimen diluent thoroughly by gentle inversion prior to use.
c. Add 75 µL of patient sample to 225 µL of specimen diluent.
d. Mix well or vortex gently.
X. RESULTING/REPORTABLE RANGE
The predefined Master Curve is adjusted to a new instrument-specific curve using the two calibrators
supplied in the reagent integral via the bar codes on the reagent integral label.
Some results may contain information in the flags column of the report. For a description of the flags
that may appear refer to the posted reference table or to the LIAISON Quick Guide Appendix, page
25.
ANALYTICAL MEASUREMENT RANGE & REPORTABLE RANGE
The analytical measurement range for the DiaSorin LIAISON 25 OH Vitamin D Total Assay is 4
ng/mL to 150 ng/mL.
The lowest reportable value is 4 ng/mL, based on an inter-assay precision that approximates 20% CV
(functional sensitivity). Values below 4 ng/mL should be reported as

XII LIMITATIONS OF PROCEDURE
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
1. Assay results should be utilized in conjunction with other clinical and laboratory data to assist the
clinician in making individual patient management decisions in an adult population.
2. The antibody utilized in this assay will demonstrate cross reactivity to many dihydroxylated
metabolites of vitamin D. However, in humans, these compounds are naturally present in
picomolar concentrations.
3. Bacterial contamination or repeated freeze-thaw cycles (>3) may affect the test results.
4. A skillful technique and strict adherence to the instructions are necessary to obtain reliable
results.
5. The concentration of vitamin D in a given specimen determined with assays from different
manufacturers may vary due to differences in methods and reagent specificity.
XIII SPECIFICITY/INTERFERENCES
SPECIFICITY
Data on the cross-reactivity of the antiserum used in this assay were obtained by spiking up to 100
ng/mL of the potential cross-reactant and assaying. The cross-reactivity of each compound,
normalized to 25 OH Vitamin D3 is listed below.
INTERFERENCE
Steroid % Cross-reactivity
25 OH Vitamin D2 104%
25 OH Vitamin D3 100%
Vitamin D2

Plasma
METHOD COMPARISON
TRUENESS
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
Repeatability P1 P2 P3 P4
Number of determinations 80 80 80 80
Mean (ng/mL) 5.91 45.9 52.6 62.7
Standard Deviation (ng/mL) 0.48 1.51 1.71 3.25
Coefficient of Variation (%) 8.1 3.3 3.2 5.2
Reproducibility P1 P2 P3 P4
Number of determinations 80 80 80 80
Mean (ng/mL) 5.91 45.9 52.6 62.7
Standard Deviation (ng/mL) 0.75 3.19 3.65 4.92
Coefficient of Variation (%) 12.7 6.9 6.9 7.9
A total of 155 samples was tested by LIAISON 25 OH Vitamin D TOTAL and by DiaSorin 25 OH
Vitamin D RIA. The resulting regression equation was:
LIAISON = 0.99 (RIA) + 2.4; R = 0.97.
The assay trueness has been verified by the dilution and recovery tests.
1. Dilution Test:
Five samples were diluted with LIAISON 25 OH Vitamin D TOTAL Specimen Diluent and
analyzed. The results were analyzed as a linear regression of the Expected vs. Observed values.
The resulting regression equation is: Observed = Expected 0.99 + 0.1; R = 0.99.
2. Recovery Test
High concentration samples were mixed with low concentration samples in ratios of 1:2, 1:1, and
2:1. The observed values were then compared to the expected values to determine the %
recovery. The mean recovery is 102%.
Sample 1
High neat
2 H : 1 L
1 H : 1 L
1 H : 2 L
Low neat
Expected Concentration
(ng/mL)
35.1
30.6
26.0
Observed Concentration
(ng/mL)
44.0
34.1
32.1
25.7
17.2
% Recovery
97%
105%
99%
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Sample 2
High neat
2 H : 1 L
1 H : 1 L
1 H : 2 L
Low neat
Sample 3
High neat
2 H : 1 L
1 H : 1 L
1 H : 2 L
Low neat
Sample 4
High neat
2 H : 1 L
1 H : 1 L
1 H : 2 L
Low neat
Sample 5
High neat
2 H : 1 L
1 H : 1 L
1 H : 2 L
Low neat
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES
74.9
59.4
43.9
59.9
48.9
37.8
93.2
73.7
54.2
84.8
64.8
44.8
XV ALTERNATE METHODS
105.0
76.0
58.2
45.6
13.8
81.4
60.4
48.2
36.5
16.3
131.0
92.7
70.4
53.9
16.4
123.6
94.5
68.9
53.0
6.0
101%
98%
104%
101%
99%
97%
99%
96%
99%
111%
106%
118%
Mean Recovery 102%
No backup method is available in the laboratory. Hold samples until instrument is back up. If delay is
expected to be significant, consult with Supervisor or Director.
XVI REFERENCES
DiaSorin LIAISON 25 OH Vitamin D TOTAL Assay package insert
DiaSorin LIAISON Starter package insert
DiaSorin LIAISON Light Check package insert
DiaSorin LIAISON Wash/System Liquid package insert
DiaSorin LIAISON 25 OH Vitamin D TOTAL Control Set package insert
DiaSorin LIAISON 25 OH Vitamin D TOTAL Specimen Diluent Set package insert
DiaSorin LIAISON 25 OH Vitamin D TOTAL Calibration Verifiers package insert
DiaSorin LIAISON Cleaning Kit package insert
Holick MF, Vitamin D Status: Measurement, Interpretation, and Clinical Application, Annals of
Epidemiology, 2009 Feb; 19(2):73-8.
Bischoff-Ferrari HA et al., Estimation of optimal serum concentrations of 25-hydroxyvitamin D for
multiple health outcomes, American Journal of Clinical Nutrition, 2006; 84:18-28.
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