CREATINE KINASE -MB (CK-MB) – Beckman Coulter

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OSR61155

CREATINE KINASE -MB (CK-MB)
2 x 22 mL
2 x 4 mL
2 x 6 mL

R1-1
R1-2
R2

CK

Intended Use
System reagent for the quantitative determination of Creatine Kinase-MB isoenzyme in human serum and plasma on Beckman Coulter AU
analyzers.

Summary
Measurements of Creatine Kinase (EC 2.7.3.2) are used in the diagnosis and treatment of myocardial infarction and muscle disease, such as
progressive Duchenne-type muscular dystrophy.

Creatine Kinase is a dimeric enzyme composed of M and/or B subunits which associate to form the CK-MM, CK-MB and CK-BB isoenzymes.1,2
Following myocardial infarction the CK-MM level rises and reaches a peak between 18-30 hrs, the increase being similar to that of the total CK
activity. CK-MB also rises following Ml, however, it reaches a peak up to 12 hrs earlier than CK-MM making it an important early indicator of MI. The
use of Total CK and CK-MB in the diagnosis of MI is the most important single application of CK measurements in clinical chemistry.1,3

Methodology
This CK procedure is a modification of the IFCC method 4,5 The R1 reagent contains an antibody which binds to the M subunit of CK in the serum
sample thereby inhibiting the activity of the M subunit. The B subunit of the enzyme remains free to act on the substrate present in the R2 reagent.
CK reversibly catalyzes the transfer of a phosphate group from creatine phosphate to adenosine diphosphate (ADP) to give creatine and adenosine
triphosphate (ATP) as products. The ATP formed is used to produce glucose-6-phosphate and ADP from glucose. This reaction is catalyzed by
hexokinase (HK) which requires magnesium ions for maximum activity. The glucose-6-phosphate is oxidized by the action of the enzyme glucose-6-
phosphate dehydrogenase (G6P-DH) with simultaneous reduction of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) to give
NADPH and 6-phosphogluconate. The rate of increase of absorbance at 340/660 nm due to the formation of NADPH is directly proportional to the
activity of CK-MB in the sample.

Creatine Phosphate + ADP

ATP + Glucose

G-6-P + NADP+

System Information
For AU400/400e/480, AU600/640/640e/680 and AU2700/5400/AU5800 Beckman Coulter Analyzers.

Reagents
Final concentration of reactive ingredients:
Imidazole (pH 6.7)
Hexokinase (HK)
NADP
G6P-DH
ADP
Mg-Acetate
AMP
Diadenosine pentaphosphate
EDTA
Glucose
Creatine Phosphate
N-Acetylcysteine
Activator
Antibody to CK-M subunit
Also contains preservatives.

Precautions
1. For in vitro diagnostic use.
2. Do not ingest. Harmful if swallowed.
3. Contains sodium azide as a preservative which may react with lead joints in copper plumbing to form explosive compounds. Even though the

2.0 mmol/L
10 mmol/L
5.0 mmol/L
10 µmol/L

2.0 mmol/L
20 mmol/L
30 mmol/L
0.2 mmol/L
26 mmol/L
Variable

100 mmol/L
≥ 4 KU/L
2.0 mmol/L

(41.67 µkat/L)

6-phosphogluconate + NADPH + H+

ADP + Glucose-6-phosphate (G-6-P)

Creatine + ATP

≥ 2.8 KU/L

(25 µkat/L)

HK, Mg2+

G6P-DH

reagent contains minute quantities of sodium azide, drains should be well flushed with water when discarding the reagent.

4. Dispose of all waste material in accordance with local guidelines.

Preparation of Reagents
R1: Ensure complete transfer of R1-2 into R1-1 by pouring an aliquot of R1-1 buffer into R1-2, mix gently, then transfer entire contents back into

R2: The reagent is ready for use and can be placed directly on board the instrument. No preparation is required.

R1-1. Mix by gentle inversion before placing on board the instrument.

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No significant interference up to 40 mg/dL Bilirubin

No significant interference up to 400 mg/dL Intralipid**

Intralipid, manufactured by KabiVitrium Inc., is a 20% IV fat emulsion used to emulate extremely turbid samples.

Creatine Kinase – MB (CK-MB)

Storage and Stability
1. The unopened reagents are stable until the expiration date printed on the label when stored at 2 – 8°C.
2. Opened reagents are stable for 30 days when stored in the refrigerated compartment of the analyzer.

Indications of Deterioration
Visible signs of microbial growth, gross turbidity or precipitate, or any change in reagent color may indicate degradation and warrant discontinuance
of use.

Specimen Collection and Preparation
Serum and heparinized plasma samples free from hemolysis are the recommended specimens. Allow specimen to clot. Remove serum from cells
promptly to minimize hemolysis and contamination by adenylate kinase from the red cells. Plasma samples may occasionally produce unpredictable
rate reactions resulting in false low results.4 Plasma with EDTA, oxalate or citrate is not recommended.

Sample Storage and Stability
Protect samples from light for maximum stability. CK is stable in serum for 4 hours at 15 – 25°C, 8 – 12 hours at 2 – 8°C or 1 month at ≤ -20°C.1

Interfering Substances
Results of studies6 show that the following substances interfere with this Creatine Kinase-MB assay.

The criteria for no significant interference is recovery within 10% of the initial value

Bilirubin:
Hemolysis:*
Lipemia:

* Adenylate Kinase from red blood cells may react with the reagent to produce spurious results and such specimens should not be used.
**

In very rare cases gammopathy, especially monoclonal IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

The information presented is based on results from Beckman Coulter studies and is current at the date of publication. Beckman Coulter Inc. makes
no representation about the completeness or accuracy of results generated by future studies. For further information on interfering substances, refer
to Young7 for a compilation of reported interferences with this test.

Procedure
A complete list of test parameters and operational procedure can be found in the User’s Guide appropriate to the analyzer.

Materials Provided
CK-MB Reagent.

Stability of Final Reaction Mixture
The Beckman Coulter AU analyzer automatically computes every determination at the same time interval.

Calibration
Calibration of this CK-MB procedure is based upon the theoretical extinction coefficient for NADPH, which has a molar absorptivity of. 6300 at
340/660 nm.

Quality Control
During operation of the Beckman Coulter AU analyzer at least two levels of appropriate quality control material, should be tested a minimum of once
a day. In addition, controls should be performed with each new lot of reagent and after specific maintenance or troubleshooting steps described in
the appropriate Beckman Coulter User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and each
laboratory’s standard procedure. Please note that recovery of non-Beckman Coulter controls may vary with reagent lots, due to changes of antisera.

Results
Results are automatically printed out for each sample in U/L at 37°C.

Dynamic Range
The CK-MB procedure is linear from 10 to 2000 U/L. Samples exceeding the upper limit of linearity should be diluted and repeated. The sample may
be diluted, repeated and multiplied by the dilution factor automatically utilizing the AUTO REPEAT RUN.

Note: For inhibition of adenylate kinase the recommended inhibitors AMP/Ap5A are included, but as the inhibition can never be completely 100 % a
residual activity could affect low CK-MB activity results.
Inhibition capacity of the anti-CK-M antibody is > 99 % at a CK-MM level of 8000 U/L. In samples where the total CK activity exceeds 8000 U/L,
CK-MB should be measured using a pre-diluted sample to ensure adequate inhibition of CK-M.

Expected Values
Serum:8

Expected values may vary with age, sex, diet and geographical location. Each laboratory should determine its own expected values as dictated by
good laboratory practice.

Specific Performance Characteristics
The following data was obtained using the CK-MB reagent on Beckman Coulter AU analyzers according to established procedures. Results obtained
in individual laboratories may differ.

0 – 10 U/L

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Precision11
Estimates of precision, based on CLSI recommendations,9 are consistent with typical performance. The within run precision is less than 5% CV or
SD ≤ 2.3 U/L and total precision is less than 6.5% CV or SD ≤ 3 U/L. Assays of control sera were carried out and data reduced following CLSI
guidelines above.

Creatine Kinase – MB (CK-MB)

N = 80
Mean, U/L
17
86
194

Within run

Total

SD
0.69
0.65
1.04

CV%
4.03
0.75
0.54

SD
0.86
0.99
1.76

CV%
5.05
1.15
0.90

Method Comparison11
Patient samples were used to compare this CK-MB Reagent on the AU640 to another Beckman Coulter AU analyzer CK-MB method (Method 2).
The table below demonstrates representative performance on the AU analyzers.:

Y Method
X Method
Slope
Intercept
Correlation Coeff. (r)
No. of Samples (n)
Range (U/L)

AU640
Method 2
1.061
2.207
1.000
103
12 – 1860

Sensitivity
Typical change in absorbance per minute for 1 U/L of CK-MB is approximately 0.12 mAbsorbance.

Limit of Quantitation
The Limit of Quantitiation (LOQ) using serum settings for the CK-MB reagent was determined to be 4 U/L. The Limit of Quantitiation (LOQ) using
serum settings for the CK-MB reagent on the AU5800 was determined to be 9.42 U/L. This was determined according to CLSI protocol EP17-A12 and
represents the lowest concentration of CK-MB that can be measured with a total imprecision of 20%.

Note
Macro CK is an atypical form of CK that is composed of immunoglobulin complexes of normal isoenzymes. It migrates electrophoretically between
MM & MB and is found mainly in elderly women. It is of no clinical significance, but its presence may cause falsely elevated results. If Macro-CK
contribution is suspected its presence should be confirmed by electrophoresis.10
In very rare cases gammopathy, especially monoclonal IgM (Waldenström’s macroglobulinemia) can cause unreliable results.

References

Tietz, N.W., Fundamentals of Clinical Chemistry. 3rd Edition, (1987), 383pp.

1.
2. Christenson, R.H., Ohman, E.M., Topol, E.J., Hanesian, M.A., et al. Creatine Kinase MM and MB Isoforms in Patients Receiving Thrombolytic

Therapy and Acute Angiography. Clin Chem 1995; 41(6) 844-852.

3. Adams, J.E., Abendschein, D.R., Jaffe A.S., Biochemical markers of myocardial injury: Is MB creatine kinase the choice for the 1990s.

Circulation 1993; 88: 750-63.

4. Horder M., Elsner R., et al., Approved Recommendation of IFCC Methods for the Measurement of Catalytic Concentration of Enzymes, Part 7

IFCC Method for Creatine Kinase, Appendix A, J. Clin. Chem. Clin. Biochem. 29, 435, 1991.

5. Szasz, G., Gerhardt, W. and Gruber, W., Clin Chem, 23: 1888, 1977.
6. CLSI/NCCLS, Interference Testing in Clinical Chemistry EP7-P, 1986.
7. Young, D.S., Effects of Drugs on Clinical Laboratory Tests, 5th Edition, AACC Press, 2000.
Tietz, N.W., (ed), Clinical Guide to Laboratory Tests, 3rd Edition, W.B. Saunders, 1995.
8.
9. CLSI/NCCLS Evaluation Protocol, EP5-T2, 1992.
10. Jacobs, C., Kasten, B., Dermott, W., Wolfson, W. The Laboratory Test Handbook, 2nd Edition. Stow/Ohio, Lexi-comp/Mosby, 1988.
11. Data is on file for specific AU analyzers.
12. Tholen DW, Linnet K, Kondratovich M, Armbruster DA, Garrett PE, Jones RL, et al. Protocols for determination of limits of detection and limits

of quantitation; approved guideline. NCCLS Document EP17-A. NCCLS, Pennsylvania, USA, 2004

Manufactured by: Beckman Coulter, Inc., 250 S. Kraemer Blvd. Brea, CA 92821, USA

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2014-04

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