Standard Operating Procedure

Subject Beta-HCG – Cobas e601 and e411

Index Number Lab-4005
Section Laboratory
Subsection Chemistry
Category Departmental
Contact Amy VanLin
Last Revised 8/23/2018

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 laboratories and Gundersen Palmer Lutheran
Hospital and Clinic Laboratories.

Detail
INTENDED USE:
Immunoassay for the in vitro quantitative determination of the sum of human chorionic gonadotropin
(hCG) plus the hcG β-subunit in human serum and plasma. This assay is intended for the early detection
of pregnancy.

PRINCIPLE:
The Elecsys HCG + β assay uses a sandwich principle. Total duration of assay: 18 minutes.
1. 1st incubation: 10 μL of sample, a biotinylated monoclonal hCG-specific antibody and a
monoclonal hCG-specific antibody labeled with a ruthenium complex react to form a sandwich
complex.
2. 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes
bound to the solid phase via interaction of biotin and streptavidin.
3. The reaction mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are then removed
with ProCell M. Application of a voltage to the electrode then includes chemiluminescent
emission which is measured by a photomultiplier.
4. The results are determined via a calibration curve which is instrument-specifically generated by
2-point calibration and a master curve provided via the reagent barcode.

CLINICAL SIGNIFICANCE:
Similarly to LH, FSH and TSH, human chorionic gonadotropin (hCG) is a member of the glycoprotein
family and consists of 2 subunits (α- and β-chains) which are associated to the intact hormone. The α-
chains in all four of these glycoprotein hormones are virtually identical, whereas the β-chains have
greatly differing structures and are responsible for the respective specific hormonal functions.

HCG is produced in the placenta during pregnancy. In non-pregnant women, it can also be produced by
tumors of the trophoblast, germ cell tumors with trophoblastic components and some non-
trophoblastic tumors. Human chorionic gonadotropin consists of a number of isohormones having the

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 Standard Operating Procedure

same biological activity but differing molecular size. The biological action of hCG serves to maintain the
corpus luteum during pregnancy. It also influences steroid production. The serum of pregnant women
contains mainly intact hCG. Measurement of the hCG concentration permits the diagnosis of pregnancy
just one week after conception. The determination of hCG in the 1st trimester of pregnancy is of
particular importance. Elevated values here serve as an indication of chorionic carcinoma, hydatiform
mole or multiple pregnancy. Depressed values indicate threatening or missed abortion, ectopic
pregnancy, gestosis or intra-uterine death. Elevated hCG concentrations not associated with pregnancy
are found in patients with other diseases such as tumors of the germ cells, ovaries, bladder, pancreas,
stomach, lungs and liver.

The specific monoclonal antibodies used in the Elecsys HCG + β assay recognize the holo-hormone,
“nicked” forms of hCG, the β-core fragment and the free β-subunit. The ruthenium-labeled and
biotinylated antibodies used are directed against different epitopes of the hCG molecule.

SPECIMEN:
Serum or lithium-heparin plasma collected using standard sampling tubes or tubes containing separating
gel.

Stability: 3 days at 2-8°C, 12 months at -20°C. To avoid carryover issues, this test cannot be added on to
any previously tested specimen. Only one freeze/thaw cycle is allowed. Samples containing precipitate
must be centrifuged before performing the assay. Samples and controls stabilized with azide cannot be
used. Ensure that patient samples, calibrators and controls are at ambient temperature (20-25°C) before
measurement. Universal precautions apply.

La Crosse Only: This test cannot be added on to samples that have run through the Cobas c501.
Due to possible evaporation effects, samples, calibrators and controls on the analyzer should be
analyzed/measured within 2 hours.

REAGENTS/MATERIALS:
Intact human chorionic gonadotropin + the β-subunit, 100 tests – the reagent rackpack is labeled as
HCG-BETA.
M - Streptavidin-coated microparticles (transparent cap), 6.5 mL: Streptavidin-coated
microparticles 0.72 mg/mL; preservative
R1 - Anti-hCG-Ab~biotin (gray cap), 9 mL: Biotinylated monoclonal anti-hCG antibodies (mouse)
2.6 mg/L; phosphate buffer 40 mmol/L, pH 7.5; preservative
R2 - Anti-hCG-Ab~Ru(bpy)23+ (black cap), 10 mL: Monoclonal anti-hCG antibody (mouse) labeled
with ruthenium complex 4.6 mg/L; phosphate buffer 40 mmol/L, pH 6.5: preservative
Diluent Universal, 36 mL – the diluent rackpack is labeled as Dil. Uni.

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:

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 Standard Operating Procedure

Intact human chorionic gonadotropin + the β-subunit – Store at 2-8°C. Do Not Freeze. Store the reagent
kit upright in order to ensure complete availability of the microparticles during the automatic mixing
prior to use.

Diluent Universal – Store at 2-8°C.

Store the Elecsys reagent kit upright in order to ensure complete availability of the mircoparticles during
automatic mixing prior to use.

Stability:
Intact human chorionic gonadotropin + the β-subunit –Unopened at 2°-8°C: up to the stated expiration
date. Opened at 2-8°C: 12 weeks. On-board the analyzers: 4 weeks.

Diluent Universal – Unopened at 2-8°C: up to the stated expiration date. Opened at 20-25°C: 3 months.
On-board the e601: 3 months.

EQUIPMENT/INSTRUMENTATION:
La Crosse: Roche Cobas e601 analyzer.
Palmer: Roche cobas e411 analyzer.
Refer to the operator's manual for operating instructions, maintenance, and troubleshooting.

Calibration: This method has been calibrated against the 4th International Standard for Chorionic
Gonadotropin from the National Institute for Biological Standards and Control (NIBSC) code 75/589.
Every reagent set has a bar-coded label containing the specific information required for calibration of
the particular reagent lot. The predefined master curve is adapted to the analyzer by the use of the
relevant CalSet.

Calibrators: HCG+β CalSet. Store unopened at 2-8°C.

Preparation: Add 1.0 mL of DI water and let stand for 15 minutes. Mix carefully, avoiding foam
formation. Transfer reconstituted calibrators into two sets of labeled snap-cap bottles. One set
is used for immediate calibration and the second set is stored refrigerated.
Stability: 12 weeks at 2-8°C.

Calibration Frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e., not
more than 24 hours since the reagent pack was registered on the analyzer). Renewed calibration is
recommended as follows:
1. After 28 days when using the same reagent lot.
2. After 7 days when using the same reagent kit on the analyzer.
3. If dictated by quality control results.
4. If necessary after instrument service or repair.

QUALITY CONTROL:
BioRad Immunoassay Plus controls, levels 1 and 3
Storage and Stability:
This product will be stable until the expiration date when stored unopened at -20°C to -70°C.

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 Standard Operating Procedure

Thawed and Unopened: When thawed and stored unopened at 2°-8°C, this product will be stable as
follows: 30 days. Date of thaw should be noted on the bottle.

Thawed and Open: Once thawed, opened, and stored tightly capped at 2°-8°C, this product will be
stable as follows: 14 days.

Once thawed, do no refreeze this product. Discard the remaining material.

This product is shipped under frozen conditions.

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
Resuspension of the microparticles before use and the reading in of the test-specific parameters via the
reagent bar code take place automatically. No manual input is necessary. If in exceptional cases the bar
code cannot be read, enter the 15-digit sequence of numbers.

Bring the cooled reagents to approximately 20°C by allowing them to sit at room temperature for at
least 45 minutes. Place on the reagent disk of the analyzer. Avoid the formation of foam. The system
automatically regulates the temperature of the reagents and the opening/closing of the bottles.
PROCEDURE NOTES:
Results are reported to the nearest whole number in U/L.

AMR (Analytical Measurement Range): 0.5-10,000 U/L. The extended measuring range with Decrease
mode (1:100) is 0.5-1,000,000 U/L.
Values below 1 are reported as <1 U/L.
Values above 1,000,000 are reported as >1,000,000 U/L.

CALCULATIONS:
The La Crosse Cobas 6000 system automatically calculates the BHCG concentration of each sample.
When using Decrease mode, the analyzer automatically applies the onboard dilution factor before
displaying the result.

INTERPRETATION:
Reference Ranges:
Male:
0-59 years - less than 5 U/L
Over 60 years - less than 10 U/L
Female (not pregnant):
Premenopausal - less than 5 U/L
Postmenopausal - less than 10 U/L

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 Standard Operating Procedure

LIMITATIONS:
Hemolysis: No significant interference up to an H index of 1000 (approximate hemoglobin
concentration: 1.0 g/dL).
Icterus: No significant interference up to an I index of 24 (approximate total bilirubin concentration: 24
mg/dL).
Lipemia: No significant interference up to a triglyceride concentration of 1400 mg/dL. There is poor
correlation between the L index (corresponds to turbidity) and triglycerides concentration.
However, Gundersen Health System has established a L index of 90 as a lipemia screen for this test.
If the L index is above this number, test triglycerides to determine specimen suitability and then
ultracentrifuge specimen if necessary. The triglycerides value of the cleared specimen must be
below the limit in order to report results. It may be necessary to ultracentrifuge multiple times.
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.

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 Intact human chorionic gonadotropin + the β-subunit package insert.
2. Roche HCG+β CalSet package insert
3. Roche Diluent Universal package insert
4. Roche Cobas 6000 Operator’s Manual

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