Quantitative determination of creatine kinase MB (CK-MB)
IVD
Store at 2-8ºC
PRINCIPLE OF THE METHOD
An antibody to the anti CK-M inhibits completely CK-MM and subunit (M) of the
CK-MB. The activity of the non-inhibited CK-B subunit is then assayed by the
following series of reactions:
Phosphocreatine + ADP
CK
Creatine + ATP
ATP + Glucose
HK
ADP + Glucose-6-phosphate
G6P + NADP+
G6PDH
6-Phosphogluconate + NADPH + H+
The rate of NADPH formation, measured photometrically, is proportional to the
catalytic concentration of CK-B present in the sample1,2
.
CLINICAL SIGNIFICANCE
CK-MB is an enzyme formed by the association of two subunits from muscle (M)
and nerve cells (B). CK-MB is usually present in serum at low concentration; it is
increases after an acute infarct of myocardium and later descends at normal
levels. Also is increased, rarely, in skeletal muscle damage5,6
.
Clinical diagnosis should not be made on a single test result; it should integrate
clinical and other laboratory data.
REAGENTS
R 1
Buffer
Imidazol pH 6.7
Glucose
Magnesium acetate
EDTA
100 mmol/L
20 mmol/L
10 mmol/L
2 mmol/L
R 2
Anti CK-M
*Anti CK-M
ADP
AMP
di-Adenosine-5- pentaphosphate
NADP+
Hexoquinase (HK)
Glucosa-6-phosphate deshydrogenase
N-acetylcysteine
Creatinine phosphate
2000 U/L
2 mmol/L
5 mmol/L
10 mmol/L
2 mmol/L
2500 U/L
1500 U/L
20 mmol/L
30 mmol/L
*Anti CK-M sufficient to inhibit up to 2000 U/L of CK-MM.
Optional
CK-Nac / CK-MB CONTROL Lyophilized human
serum
Ref: 1002260
PRECAUTIONS
R1: H360- May damage fertility or the unborn child.
Follow the precautionary statements given in MSDS and label of the product.
CK-Nac / CK-MB CONTROL, Components from human origin have been tested
and found to be negative for the presence of HBsAg, HCV, and antibody to HIV
(1/2). However handle cautiously as potentially infectious.
PREPARATION
- Working reagent (WR)
Dissolve (
) one tablet of R2 in one vial of R1.
Cap vial and mix gently to dissolve contents.
Stability: 8 days at 2-8ºC or 24 hours at 15-25ºC.
- CK-Nac / CK-MB CONTROL
Dissolve (
) the contents in 2 mL of distilled water.
Cap vial and mix gently to dissolve contents.
Stability: 5 days at 2-8ºC or 4 weeks at -25ºC to -15ºC.
STORAGE AND STABILITY
All the components of the kit are stable until the expiration date on the label when
stored tightly closed at 2-8ºC, protected from light and contaminations prevented.
Do not use the tablets if appears broken.
Do not use reagents over the expiration date.
Signs of reagent deterioration:
- Presence of particles and turbidity.
- Blank absorbance (A) at 340 nm 1,60.
ADDITIONAL EQUIPMENT
- Spectrophotometer or colorimeter measuring at 340 nm.
- Thermostatic bath at 25ºC, 30ºC ó 37º C ( 0,1ºC).
- Matched cuvettes 1,0 cm light path.
- General laboratory equipment.
SAMPLES
Serum or plasma1
: Stability 7 days at 2-8ºC, protected from light.
CK-MB activity decreases a 10% after 24 hours at 4ºC or 1 hour at 25ºC.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 nm
Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . . 1 cm light path
Constant temperature . . . . . . . . .. . . . . .25ºC / 30ºC / 37ºC
2. Adjust the instrument to zero with distilled water or air.
3. Pipette into a cuvette:
4. Mix. Incubate for 10 minute.
5. Read initial absorbance (A) of the sample, start the stopwatch and read again after
5 minutes (A2).
6. Calculate the difference between absorbances: A= A2 – A1.
CALCULATIONS
A x 825 = U/L de CK-B A x 1651 = U/L de CK-MB
Calculating factor in automatic analyzers by kinetic method (A/min.) is 8255 of CK-MB.
Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of
substrate per minute, in standard conditions. The concentration is expressed in units per
litre of sample (U/L).
Percentage of CK-MB activity in sample:
100
CK Total Activity
CK MB Activity
= % CK-MB Activity
Temperature conversion factors
To correct results to other temperatures multiply by:
Assay
temperature
Conversion factor to
25ºC 30ºC 37ºC
25ºC
30ºC
37ºC
1,00
0,65
0,42
1,53
1,00
0,64
2,38
1,56
1,00
QUALITY CONTROL
CK-Nac / CK-MB specific sera controls (Ref. 1002260) are recommended to
monitor the performance of assay procedures.
If control values are found outside the defined range, check the instrument, reagents and
technique for problems.
Each laboratory should establish its own Quality Control scheme and corrective actions
if controls do not meet the acceptable tolerances.
REFERENCE VALUES
Heart infarct probability is high at the following conditions:
25ºC 30ºC 37ºC
CK-MB 10 U/L 15 U/L 24 U/L
CK-MB activity is between 6 and 25% of total CK activity.
These values are for orientation purpose; each laboratory should establish its own
reference range.
PERFORMANCE CHARACTERISTICS
Measuring range: Detection limit: 3,11 U/L.
Linearity: The total CK activity must be determined by the CK-NAC activated method
prior to the CK-MB assay. If the CK activity exceeds 1000 U/L, dilute the sample 1/2 with
NaCl 9 g/L and multiply the result by 2.
Precision:
Intra-assay (n=20) Inter-assay (n=20)
Mean (U/L) 54,2 138,3 55,7 141,6
SD 1,45 1,33 1.62 1.39
CV (%) 2,67 0,96 2,92 0,98
Sensitivity: 1 U/L = 0,00029 A / min.
Accuracy: Results obtained using SPINREACT reagents (y) did not show systematic
differences when compared with other commercial reagents (x).
The results obtained using 50 samples were the following:
Regression coefficient (r)2
: 0,996.
Equation of the regression line: y = 0,9919x - 0,1042.
The results of the performance characteristics depend on the analyzer used.
INTERFERENCES
Hemolysis interferes with the assay2
.
A list of drugs and other interfering substances with CK-MB determination has been
reported by Young et. al3,4
.
NOTES
SPINREACT has instruction sheets for several automatic analyzers.
BIBLIOGRAPHY
1. Abbot B et al. Creatinine kinase. Kaplan A et al. Clin Chem The C.V. Mosby Co.
St Louis. Toronto. Princeton 1984: 1112-116.
2. Gerhardt W. et al. Creatine kinase B-Subunit activity in serum after
immunohinhibition of M-Subunit activity. Clin Chem 1979;(25/7): 1274-1280.
3. Young DS. Effects of drugs on Clinical Lab. Tests, 4th ed AACC Press, 1995.
4. Young DS. Effects of disease on Clinical Lab. Tests, 4th ed AACC 2001.
5. Burtis A et al. Tietz Textbook of Clinical Chemistry, 3rd ed AACC 1999.
6. Tietz N W et al. Clinical Guide to Laboratory Tests, 3rd ed AACC 1995.
