Standard Operating Procedure

Subject C-Reactive Protein (CRP) – Cobas c501 and c502

Index Number Lab-4232
Section Laboratory
Subsection Chemistry
Category Departmental
Contact Amy Vanlin
Last Revised 12/5/2019

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, Gundersen Moundview Hospital and Gundersen Tri-County
Hospital laboratories.

Detail
INTENDED USE:
Immunoturbidimetric assay for the in vitro quantitative determination of CRP in human serum and
plasma on Roche/Hitachi cobas c systems.

PRINCIPLE:
Particle enhanced immunoturbidimetric assay. Human CRP agglutinates with latex particles coated with
monoclonal anti-CRP antibodies. The aggregates are determined turbidimetrically.

CLINICAL SIGNIFICANCE:
C-reactive protein is the classic acute phase protein in inflammatory reactions. It is synthesized by the
liver and consists of five identical polypeptide chains that form a five-membered ring having a molecular
weight of 105000 daltons. CRP is the most sensitive of the acute phase reactants and its concentration
increases rapidly during inflammatory processes. Complexed CRP activates the classical complement
pathway. The CRP response frequently precedes clinical symptoms, including fever. In normal healthy
individuals CRP is a trace protein with a range up to 0.5 mg/dL. After onset of an acute phase response
the serum CRP concentration rises rapidly and extensively. The increase begins within 6 to 12 hours and
the peak value is reached within 24 to 48 hours. Levels above 100 mg/L are associated with severe
stimuli such as major trauma and severe infection (sepsis). CRP response may be less pronounced in
patients suffering from liver disease. CRP assays are used to detect systemic inflammatory processes; to
assess treatment of bacterial infections with antibiotics; to detect intrauterine infections with
concomitant premature amniorrhexis; to differentiate between active and inactive forms of disease with
concurrent infection, e.g. in patients suffering from SLE or Colitis ulcerosa; to therapeutically monitor
rheumatic disease and assess anti-inflammatory therapy; to determine the presence of post-operative
complications at an early stage, such as infected wounds, thrombosis and pneumonia; and to distinguish
between infection and bone marrow rejection. Postoperative monitoring of CRP levels of patients can
aid in the recognition of unexpected complications (persisting high or increasing levels). Measuring
changes in the concentration of CRP provides useful diagnostic information about how acute and how
serious a disease is. It also allows judgments about the disease genesis. Persistence of a high serum CRP

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 Standard Operating Procedure

concentration is usually a grave prognostic sign which generally indicates the presence of an
uncontrolled infection.

SPECIMEN:
Serum or heparinized plasma collected using standard sampling tubes or tubes containing separating
gel. Separate the serum or plasma from the clot or cells promptly.

Stability: 11 days at 15-25oC, 2 months at 2-8oC, 3 years at -15 to -20oC.

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

REAGENTS/MATERIALS:
C-Reactive Protein Gen.3, 250 tests – the reagent cassette is labeled as CRPL3. R1 is in position B and R2
is in position C. Before loading a cassette on the analyzer, invert gently 10 times to mix.
R1 - TRIS buffer with bovine serum albumin; preservatives
R2 - Latex particles coated with anti-CRP (mouse) in glycine buffer; immunoglobulins (mouse);
preservative
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:

C-Reactive Protein Gen.3 – Unopened at 2-8oC – up to the stated expiration date. On-board in use and
refrigerated on the c501 and c502 – 12 weeks.
Diluent NaCl 9% – Unopened at 2-8°C – up to the stated expiration date. On-board in use and
refrigerated on the c501 and c502 – 12 weeks.

EQUIPMENT/INSTRUMENTATION:
Roche Cobas c501 and c502 analyzer - Refer to the Operator’s Manual for operating instructions,
maintenance, and troubleshooting.

Calibration: This method has been standardized against an internal method traceable to CRM 470
(RPPHS – Reference Preparation for Proteins in Human Serum). Calibration mode: 6-point spline, full
calibration.

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

Calibration type: 2-point end
Calibration mode: 6-point spline
Preparation: Ready for use. Mix carefully, avoiding foam formation.
Stability: 4 weeks at 2-8°C.

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:

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 Standard Operating Procedure

1. If necessary after instrument service or repair

2. If dictated by quality control results

QUALITY CONTROL:
BioRad Liquichek Immunology levels 1 and 3

Storage and Stability:

This product will be stable until the expiration date when stored unopened at -20 to -70°C.
Thawed Unopened: When thawed and stored unopened at 2 - 8°C, this product will be stable for 45
days.
Thawed Opened: Once thawed, opened, and stored tightly capped at 2 - 8°C, this product will be stable
for 30 days.
Do not refreeze this product after opening the vials.

This product is shipped under frozen conditions.

Refer to Lab-4405 Quality Control Criteria for Chemistry for interpretation of QC. Two levels of quality
control should be performed at a minimum:
1. Once every twenty-four hours.
2. If a new pack of reagent is put into use.
3. If a calibration is performed.

Implementation
Refer to the Cobas 6000 or Cobas 8000 Operator’s Manual located in the Chemistry Department.

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

AMR (Analytical Measurement Range): 0.03-35.0 mg/dL.

Values below 0.1 are reported as <0.1 mg/dL.
Values above 35.0 are reported as >35 mg/dL.

CALCULATIONS:
The Cobas 6000 and the Cobas 8000 system automatically calculates the C-Reactive Protein
concentration of each sample.

INTERPRETATION:
Expected value: <0.8 mg/dL

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 60 (approximate total bilirubin concentration: 60
mg/dL).

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 Standard Operating Procedure

Lipemia: No significant interference up to an L index of 1000. There is poor correlation between the L
index (corresponds to turbidity) and triglycerides concentration.
Rheumatoid factors: No significant interference from rheumatoid factors up to a concentration of 1200
IU/ml.
Immunoglobulins: No significant interference from immunoglobulins up to a concentration of 5000
mg/dL. (Simulated by human immunoglobulin G)
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.
Cobas c502 analyzer: All special wash programming necessary for avoiding carry-over is available via the
cobas link, manual input is required in certain cases.

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 C-Reactive Protein Gen.3 package insert
2. Roche C.f.a.s. Proteins package insert
3. Roche Diluent NaCl 9% package insert
4. Roche Cobas 6000 and Cobas 8000 Operator’s Manual

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