ck mb assay

1. CK-MB FS*
Diagnostic reagent for quantitative in vitro determination of CK-MB in serum and plasma on
photometric systems
Order Information Warnings and Precautions
Cat. No. Kit size 1. The reagents contain sodium azide (0.95 g/l) as preservative.
1 1681 99 10 021 R1 5x 20 ml + R2 1 x 25 ml Do not swallow! Avoid contact with skin and mucous
1 1681 99 10 026 R1 5x 80 ml + R2 1 x 100 ml membranes.
1 1681 99 10 730 R1 4x 20 ml + R2 2 x 10 ml 2. Take the necessary precautions for the use of laboratory
1 1681 99 10 930 R1 4x 20 ml + R2 2 x 10 ml reagents.
1 1681 99 10 191 R1 4x 36 ml + R2 4 x 9 ml Waste Management
For determination with CK-MB DS additionally required:
Please refer to local legal requirements.
1 1690 99 10 065 3x 3 ml
Reagent Preparation
Summary [1,2]
Creatine kinase (CK) is an enzyme which consists of isoenzymes Substrate Start
mainly of the muscles (CK-M) and the brain (CK-B). CK exists in The reagents are ready-to-use.
serum in dimeric forms as CK-MM, CK-MB, CK-BB and as macro- For the determination with CK-MB DS:
enzymes. Measurement of CK-MB is a quite specific test for Mix 1 part of CK-MB DS with 20 parts of Reagent 1.
detection of cardiac muscle damage and is therefore used for Use mixture as described for reagent R1.
diagnosis and monitoring of myocardial infarction. Stability in premixed R1:
6 days at 2 - 8 °C
Method 24 hours at 15 - 25 °C
Optimized UV test according to DGKC (German Society of Clinical Sample Start
Chemistry) and IFCC (International Federation of Clinical
Chemistry and Laboratory Medicine) for CK with inhibition of CK-M (without CK-MB DS)
isoenzymes by polyclonal antibodies. Mix 4 parts of R1 + 1 part of R2
(e.g. 20 ml R1 + 5 ml R2) = monoreagent
Principle Stability: 2 weeks at 2 – 8 °C
24 hours at 15 – 25 °C
Inhibition of CK-M subunits The monoreagent must be protected from light.
CK-MB consists of the subunits CK-M and CK-B. Specific polyclonal Materials required but not provided
antibodies against CK-M inhibit the complete CK-MM activity (main NaCl solution 9 g/l. General laboratory equipment
part of the total CK activity) and the CK-M- subunit of CK-MB.
Only CK-B activity is measured, which is half of the CK-MB Specimen
activity.
Serum and heparin plasma
Reaction principle Loss of activity:
CK after 24 h at 2 – 8 °C < 10 %
Creatine phosphate + ADP < > Creatine + ATP
after 1 h at 15 – 25 °C < 10 %
HK
Glucose + ATP < > Glucose-6-phosphate + ADP Stability at – 20 °C: 4 weeks (in the dark)
G6P-DH Discard contaminated specimens!
Glucose-6-phosphate + NADP+ < >
6-Phospho-glucono lactone + NADPH + H+ Assay Procedure
CK-MB DS
Application sheets for automated systems are available on
In samples with low CK-MB concentrations the measuring signals request.
are rather low. The supplementary reagent CK-MB DS produces an
additional reaction step which duplicates the measuring signal and Wavelength 340 nm, Hg 334 nm
Optical path 1 cm
therefore leads to an improvement of the precision and sensitivity:
Temperature 37 °C
6-Phospho-glucono lactone PGL > 6-Phospho-gluconate Measurement Against reagent blank
6-Phospho-gluconate + NADP+ 6-PGDH > Substrate Start
Ribulose-5-phosphate + CO2 + NADPH + H+
Blank Sample
Sample - 50 µl
Reagents
Dist. water 50 µl -
Components and Concentrations Reagent 1 1000 µl 1000 µl
N.B. Concentrations are those in the final test mixture. Mix, incubate for approx. 5 min., then add:
Reagent 2 250 µl 250 µl
R1: Imidazole buffer pH 6.7 100 mmol/l Mix, read absorbance after 3 min. and start the stopwatch. Read
N-Acetylcysteine (NAC) 20 mmol/l absorbance again after 1, 2 and 3 min
Glucose 20 mmol/l
ADP 2 mmol/l A/min = [A/min Sample] – [A/min Blank]
NADP 2 mmol/l
Magnesium acetate 10 mmol/l Sample start
EDTA-Na2 2 mmol/l Blank Sample
Hexokinase (HK)  4 kU/l Sample - 40 µl
Glucose-6-phosphate dehydrogenase (G6P-DH)  2.8 kU/l Dist. water 40 µl -
AMP 5 mmol/l Monoreagent 1000 µl 1000 µl
Diadenosine pentaphosphate 10 µmol/l Mix, read absorbance after 5 min. and start the stopwatch. Read
Polyclonal antibodies (goat) against human absorbance again after 1, 2 and 3 min.
CK-M; inhibiting capacity  2 kU/l
Stabilizers and preservatives A/min = [A/min Sample] – [A/min Blank]
R2: Creatine phosphate 30 mmol/l
Glucose-6-phosphate dehydrogenase (G6P-DH)  2.8 kU/l Calculation
Stabilizers and preservatives From absorbance readings calculate A/min and multiply by the
corresponding factor from table below:
Storage Instructions and Reagent Stability
The reagents are stable up to the end of the indicated month of A/min x factor = CK-MB activity [U/l]
expiry, if stored at 2 – 8 °C, protected from light and without CK-MB DS with CK-MB DS
contamination is avoided. Do not freeze the reagents! 334 nm 8414 4207
340 nm 8254 4127
CK-MB FS – Page 1 * fluid stable

2. Controls Method Comparison
Control material containing CK-MB of non-human origin is not without CK-MB DS
appropriate for quality control of CK-MB tests (independent of the
A comparison between DiaSys CK-MB FS (y) and a commercially
reagent manufacturer). Controls with human CK-MB should be
available test (x) using 41 samples gave following results:
used. DiaSys TruLab N and TruLab P controls are recommended. y = 0.949 x – 0.057 U/l; r= 1.000
Cat. No. Kit size with CK-MB DS
TruLab N 5 9000 99 10 062 20 x 5 ml
5 9000 99 10 061 6 x 5 ml A comparison between DiaSys CK-MB FS with CK-MB DS (y) and a
commercially available test (x) using 49 samples gave following
TruLab P 5 9050 99 10 062 20 x 5 ml
results: y = 0.992 x + 0.498 U/l; r= 0.998
5 9050 99 10 061 6 x 5 ml
Reference Range
Performance Characteristics
Myocardial infarction: the risk of myocardial infarction is high if
Measuring range following three conditions are fulfilled [6]:
In samples with total CK activities up to 2000 U/l the CK-M 1. CK (Men) > 190 U/l (3.12 µkat/l)*
components are completely inhibited by the present antibodies. If CK (Women) > 167 U/l (2.87 µkat/l)*
that value is exceeded, samples should be diluted with NaCl 2. CK-MB > 24 U/l (0.40 µkat/l)*
solution (9 g/l) to activities of less than 2000 U/l. 3. CK-MB activity is between 6 and 25 % of total CK activity.
* calculated using temperature conversion factor 2.38 (25 °C  37 °C)
Specificity / Interferences
If myocardial infarction is suspected and the conditions are not
No interference was observed by ascorbic acid up to 30 mg/dl, fulfilled, the infarction may be fresh. In this case the
unconjugated bilirubin up to 50 mg/dl, conjugated bilirubin up to measurements should be repeated after 4 hours with fresh
60 mg/dl and lipemia up to 600 mg/dl triglycerides. samples.
Interference by Adenylate Kinase In healthy individuals different values are found depending on race
Hemolysis interferes strongly as adenylate kinase is released from and age [6,7].
the erythrocytes leading to following side reaction: Each laboratory should check if the reference ranges are
ADP + ADP Adenylate kinase > ATP + AMP transferable to its own patient population and determine own
The side reaction of adenylate kinase can be measured before reference ranges if necessary. For diagnostic purposes CK values
adding R2 and can be used for a correction of the result according should always be assessed in conjunction with the anamnesis, the
to following procedure: clinical examination and other findings
Substrate start with correction of adenylate kinase Literature
Measurement of blank see above 1. Stein W. Creatine kinase (total activity), creatine kinase
isoenzymes and variants. In: Thomas L, ed. Clinical
Sample 50 µl
laboratory diagnostics. Frankfurt: TH-Books
Reagent 1 1000 µl
Verlagsgesellschaft;1998.p.71-80.
Mix, read absorbance after 2 min. and start stopwatch. Read
2. Moss DW, Henderson AR. Clinical enzymology. In: Burtis CA,
absorbance again after 1, 2 and 3 min. = (ΔA/min)pre
Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd
Reagent 2 250 µl
ed. Philadelphia: W.B Saunders Company; 1999. p. 617-721.
Mix, read absorbance after 3 min. and start stopwatch. Read
3. Würzburg U, Hennrich N, Orth HD, Lang H. Quantitative
absorbance again after 1, 2 and 3 min. = (ΔA/min)total
determination of creatine kinase isoenzyme catalytic
A/mincorr = (A/min)total - 0,81(A/min)pre concentrations in serum using immunological methods. J Clin
A/min = [(A/min)corr Sample] – [(A/min) Blank] Chem Clin Biochem 1977;15:131-7.
4. Recommendations of the German Society for Clinical
With the correction of the adenylate kinase no interference was Chemistry. Standardization of methods for the estimation of
observed by hemoglobin up to 150 mg/dl. enzyme activities in biological fluids: Standard method for the
Sensitivity / Limit of Detection determination of creatine kinase activity. J Clin Chem Clin
Biochem 1977;15:255-60.
The lower limit of detection is 5 U/l. Using the supplementary
5. Schumann G, Bonora R, Ceriotti F, Férard G et al. IFCC
reagent CK-MB DS the lower limit of detection is 2 U/l.
primary reference procedure for the measurement of catalytic
Precision without CK-MB DS activity concentrations of enzymes at 37 °C. Part 5:
Intra assay Mean SD CV Reference procedure for the measurement of catalytic
n = 20 [U/l] [U/l] [%] concentration of creatine kinase. Clin Chem Lab Med
Sample 1 24.0 0.98 4.07 2002;40:635-42.
Sample 2 31.8 0.97 3.06 6. Stein W. Strategie der klinisch-chemischen Diagnostik des
Sample 3 101 0.98 0.97 frischen Myokardinfarkts. Med Welt 1985:36:572-7.
7. Myocardial infarction redefined – a consensus document of
the Joint European society of Cardiology / America College of
Inter assay Mean SD CV
Cardiology Committee fort he redefinition of myocardial
n = 20 [U/l] [U/l] [%]
Infarction. Eur Heart J 2000;21:1502-13.
Sample 1 32.9 1.08 3.29
Sample 2 63.9 1.06 1.65 Manufacturer
Sample 3 83.4 0.99 1.18
DiaSys Diagnostic Systems GmbH
Precision with CK-MB DS Alte Strasse 9 65558 Holzheim Germany
Intra assay Mean SD CV
n = 20 [U/l] [U/l] [%]
Sample 1 24.2 0.53 2.17
Sample 2 32.2 0.66 2.05
Sample 3 100 0.90 0.89
Inter assay Mean SD CV
n = 20 [U/l] [U/l] [%]
Sample 1 34.0 1.01 2.97
Sample 2 63.7 0.56 0.87
Sample 3 83.1 0.90 1.09
CK-MB FS – Page 2 844 1681 10 02 00 July 2007/4