Standard Operating Procedure

Subject Triglycerides – Cobas c501

Index Number Lab-4455
Section Laboratory
Subsection Chemistry
Category Departmental
Contact Amy Van Lin
Last Revised 4/4/2018

References
Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicare and Medicaid Services (CMS) and/or COLA.

Applicable To
Employees of the Gundersen Health System clinical laboratory.

Detail
INTENDED USE:
In vitro test for the quantitative determination of triglycerides in human serum and plasma on
Roche/Hitachi cobas c systems.

PRINCIPLE:
Enzymatic colorimetric test. This method uses a lipoprotein lipase for the rapid and complete hydrolysis
of triglycerides to glycerol followed by oxidation to dihydroxyacetone phosphate and hydrogen
peroxide. The hydrogen peroxide produced then reacts with 4-aminophenazone and 4-chlorophenol
under the catalytic action of peroxidase to form a red dye. The color intensity of the red dye formed is
directly proportional to the triglyceride concentration and can be measured photometrically.

CLINICAL SIGNIFICANCE:
Triglycerides are the main lipids present in human plasma; the others are cholesterol, phospholipids,
and nonesterified fatty acids. They are formed in the intestinal mucosa by the esterification of glycerol
and free fatty acids. They are then released into the mesenteric lymphatics and distributed to most
tissues for storage. Triglycerides are the main storage lipids in humans, where they constitute about
95% of adipose tissue lipids.

Elevated levels of triglycerides have been associated with high risk in severe atherosclerosis. High
triglyceride levels and hyper-lipidemia in general can be an inherited trait or can be secondary to
disorders including diabetes mellitus, nephrosis, biliary obstruction, and metabolic disorders associated
with endocrine disturbances.

SPECIMEN:
Serum or heparinized plasma collected using standard sampling tubes or tubes containing separating
gel. Patients should refrain from eating for at least 9 hours before blood is drawn. Samples must be
drawn in a soap or glycerol free collection device.

La Crosse Lab Only

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 Standard Operating Procedure

Peritoneal fluid and pleural fluid can also be tested.

Stability:
Serum, plasma, 5 days at 2-8°C, 3 months at -20°C.

La Crosse Lab Only –

body fluid: 5 days at 2-8°C
Body fluid specimens must be nonviscous and clear. Only light colored fluids will be accepted.
Viscous and colored fluids cannot be assayed. Blood cells or other material must be removed by
centrifugation.

Centrifuge samples containing precipitates before performing the assay. Universal precautions apply.

REAGENTS / MATERIALS:
Triglycerides, 250 tests – the reagent cassette is labeled as TRIGL. R1 is in position B.
R1 - PIPES buffer: 50 mmol/L, pH 6.8; Mg2+: 40 mmol/L; sodium cholate; 0.20 mmol/L; ATP: ≥1.4
mmol/L; 4-aminophenazone: ≥0.13 mmol/L; 4-chlorophenol: 4.7 mmol/L; lipoprotein lipase
(Pseudomonas spec.): ≥83 ukat/L; glycerokinase (Bacillus stearothermophilus): ≥3 ukat/L;
glycerol phosphate oxidase (E. coli): ≥41 ukat/L; peroxidase (horseradish): ≥1.6 ukat/L;
preservative, stabilizers
Diluent NaCl 9%, 50 mL – the diluent cassette is labeled as NACL.

Precautions and warnings:

For in vitro diagnostic use. Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.

Storage and Stability:

Triglycerides – Unopened at 2-8°C – up to the stated expiration date. On-board in use and refrigerated
on the c501 – 8 weeks.
Diluent NaCl 9% - Unopened at 2-8°C - up to the stated expiration date. On-board in use and
refrigerated on the c501 – 12 weeks.

EQUIPMENT / INSTRUMENTATION:
Roche c501 analyzer - Refer to the Operator’s Manual for operating instructions, maintenance, and
troubleshooting.

Calibration: This method has been standardized against the ID/MS method. Calibration mode: Linear, 2-
point calibration.

Calibrators: C.f.a.s. Use deionized water as zero calibrator.

Preparation: Add 3.0 mL of DI water and let stand for 30 minutes. Mix carefully, avoiding foam
formation.
Stability: 2 days at 2-8°C for all analytes except total bilirubin.

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 Standard Operating Procedure

Calibration Frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not
more than twenty-four hours since the reagent pack was registered on the analyzer). Renewed
calibration is recommended as follows:
1. If necessary after instrument service or repair
2. If dictated by quality control results

QUALITY CONTROL:
BioRad Unassayed Chemistry controls, levels 1 and 2

Refer to Lab-4405 Quality Control Criteria for Chemistry for interpretation of QC. Each level of Quality
Control should be performed at a minimum:
1. once every twenty-four hours
2. if a new pack of reagent is put in use
3. if a calibration is performed

Implementation
Refer to the Cobas 6000 Operator’s Manual Located in the chemistry department.

PROCEDURE NOTES:
Results are reported to the nearest whole number in mg/dL.

AMR (Analytical Measurement Range): 9-885 mg/dL. The extended measuring range with Decrease
mode (1:5) is 9-4425 mg/dL.
Values below 9 are reported as <9 mg/dL. In serum/plasma specimens, triglyceride levels that low
are biologically unlikely.
Values above 4425 are reported as >4425 mg/dL. If a data flag is generated, i.e. >Kin, use 0.9% NaCl
to manually dilute the specimen. The concentration of a manually diluted specimen must fall within
the extended measuring range before applying the dilution factor.

Specimens which have a lipoprotein analysis (LPA1), HDL cholesterol (HDL), Non-HDL Cholesterol Lipid
Panel (NHDLPNL), or comprehensive chemistry panel (COMP) ordered and have a triglyceride value of
>900 mg/dL must have an aliquot of the sample ultracentrifuged for analysis of the HDL cholesterol.
Gundersen Tri-County Hospital laboratory must send sample to La Crosse laboratory for
ultracentrifugation if triglyceride value is >900.

La Crosse Laboratory Only -

Create an aliquot label in the LIS. Manually program the new specimen ID to run the HDL and TRIG on
the cleared specimen. If the TRIG result on the cleared sample is <900 mg/dL, you can report the
cleared HDL result. Report the original uncleared TRIG result.

When an LPA is ordered on a regional clinic or outreach specimen and no indication is given of the
number of hours fasting, hold results until the number of hours fasting can be verified.

CALCULATIONS:
The COBAS 6000 system automatically calculates the triglyceride concentration of each sample.

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 Standard Operating Procedure

INTERPRETATION:
Expected Range:
Age Male Female
0–9 < 75 mg/dL < 75 mg/dL
10 – 19 < 90 mg/dL < 90 mg/dL
20 – 29 40 – 141 mg/dL 33 – 137 mg/dL
30 – 39 45 – 150 mg/dL 38 – 150 mg/dL
> 40 50 – 150 mg/dL 45 – 150 mg/dL

LIMITATIONS:
Hemolysis: No significant interference up to an H index of 700 (approximate hemoglobin concentration:
0.7 g/dL).
Icterus: No significant interference up to an I index of 10. If the I index is greater than 10, test direct and
total bilirubin. Indirect bilirubin can be determined by subtracting the direct bilirubin from the total
bilirubin. There is no significant interference up to a direct bilirubin of 10 mg/dL or an indirect
bililrubin of 35 mg/dL.
Lipemia: The L index correlates with sample turbidity but not with triglycerides level. Extremely Lipemic
samples (triglycerides greater than 3000 mg/dL) can produce a normal result. Perform a manual
dilution if necessary - do not clear.
See package insert for additional interference and cross-reactivity studies. The results should always be
assessed in conjunction with the patient’s medical history, clinical examination and other findings.

Special Wash Requirements: The determination of certain analytes interferes with this assay requiring a
special wash step. Refer to the NaOHD/SMS/SmpCln1+2/SCCS method sheet and the operator manual
for further instructions.

REVIEW AND CHANGES:

This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical
Director, or designated person. Changes require retyping document or form and review by the Medical
Director.

REFERENCES:
1. Roche Triglycerides package insert
2. Roche C.f.a.s. package insert
3. Roche Diluent NaCl 9% package insert
4. Roche Cobas 6000 Operator’s Manual

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