Quantitative determination of creatine kinase MB (CK-MB)
IVD
Store at 2-8ºC
PRINCIPLE OF THE METHOD
The procedure involves measurement of CK activity in the presence of an
antibody to CK-M monomer. This antibody completely inhibits the activity of CKMM and half of the activity of CK-MB while not affecting the B subunit activity of
CK-MB and CK-BB. Then it’s used the CK method to quantitatively determine CKB activity1,2. The CK-MB activity is obtained by multiplying the CK-B activity by
two.
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
increased after an acute infarct of myocardium and later descends at normal
levels.
Also is increased, rarely, in skeletal muscle damage5,6,7,8.
Clinical diagnosis should not be made on a single test result; it should integrate
clinical and other laboratory data.
PREPARATION
Working reagent (WR): Mix 4 volumes of reagent 1 with 1 volume of reagent 2.
Stability: 7 days at 2-8ºC or 12 hours at room temperature (20-25ºC).
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 reagents over the expiration date.
Signs of reagent deterioration:
- Presence of particles and turbidity.
- Blank absorbance (A) at 340 nm 1,2.
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 free of hemolysis or heparin plasma: 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:
WR (mL) 1,0
Sample (L) 40
4. Mix and incubate 10 minutes.
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 of CK-B A x 1651 = U/L of CK-MB
Calculating factor in automatic analyzers by kinetic method (A/min.) is 8255.
Units: One international unit (IU) is the amount of enzyme that transforms 1mol
of substrate per minute, in standard conditions. The concentration is expressed
in units per litre of sample (U/L).
QUALITY CONTROL
CK-NAC/CK-MB specific control sera (Ref. 1002260) is 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.
PERFORMANCE CHARACTERISTICS
Measuring range: From detection limit of 1,9 U/L to linearity limit of 1000 U/L.
If the results obtained were greater than linearity limit, dilute the sample 1/1 with NaCl 9
g/L and multiply the result by 2.
Precision:
Sensitivity: 1 U/L = 0,000134 (A).
Accuracy: Results obtained using SPINREACT reagents (y) did not show systematic
differences when compared with other commercial reagents (x).
The results obtained were the following:
Correlation coefficient (r)2: 0,999.
Regression equation: y= 0,976 x - 0,269.
The results of the performance characteristics depend on the analyzer used.
INTERFERENCES
Bilirubin (mixed isomers): Less than 10% interference up to 600 µmol/L Bilirubin.
Hemolysis: Less than 10% interference up to 1,25 g/L Hemoglobin.
Lipemia: Les than 10% interference up to 2,5 g/L Intralipid.
A list of drugs and other interfering substances with CK determination has been
reported3,4.
LIMITATION OF THE PROCEDURE
The method will also measure any CK-BB isoenzyme present in serum. The activity of
the isoenzyme is usually negligible, however, if a significant amount of CK-BB activity is
present the CK-MB activity will be overestimated.
A macro form of BB (immunoglobulin complexed) has been observed wich will be
measured as B in the assay. If the measured CK-B activity exceeds 20% of the total CK
activity, the presence of macro BB should be suspected.
NOTES
SPINREACT has instruction sheets for several automatic analyzers. Instructions
for many of them are available on request.
