1.0     INTENDED USE

This reagent is intended for the quantitative determination of the activity of the isoenzyme CK-MB in serum.

 

2.0     BACKGROUND

2.1     METHOD AND HISTORY

Conventional methods for the separation and quantitation of Creatine Kinase (CK) isoenzymes have been based on electrophoresis and ionexchange chromatography (10.6.)  While these procedures can be useful, newer methods have recently been introduced by Wuerzburg et al. (10.7) and Gerhardt et al. (10.8.)  Their procedure employs a polyclonal antibody to the CK-M monomer to completely inhibit the activity of CK-MM and one-half the activity of CK-MB.  The activity of the non-inhibited CK-B monomer can then be measured.  The present reagent includes the polyclonal antibody in a reagent which measures CK activity.  Inhibition of CK-M occurs during the lag phase of the CK assay system, leaving only CK-B activity to be measured.  The CK measuring reagent is based on a modification of the IFCC recommended formulation.

2.2     TEST PRINCIPLE

The sample is incubated in the CK-MB reagent which includes the anti-CK-M antibody.  The activity of the noninhibited CK-B is then determined using the following series of reactions:

                                                CK

          Creatinine phosphate +ADP -----‑‑‑‑> creatine + ATP

                                   HK

          ATP + Glucose ‑‑‑----‑> ADP + Glucose‑6‑phosphate G‑6‑PDH

 

          G-6-P + NAD⁺ ‑‑‑‑‑‑‑---‑-> 6‑Phosphogluconate + NADH + H⁺

 

The remaining CK-B catalyzes the reversible phosphorylation of ADP in the presence of creatine phosphate to form ATP and creatine. The auxiliary enzyme hexokinase (HK) catalyzes the phosphorylation of glucose by the ATP formed, to produce ADP and glucose-6-phosphate (G-6-P).  The G-6-P is oxidized to 6-phosphogluconate with the concomitant production of NADH.  The rate of NADH formation, measured at 340nm, is directly  proportional to serum CK-B activity.

2.3     CLINICAL SIGNIFICANCE (10.5)

Creatinine Kinase exists as dimers formed by two subunits which derive primarily from either muscle (M) or nerve cells (B).  The combination of these subunits produces three isoenzymes of CK (10.1, 10.2.)  CK-MM and CK-MB are found primarily in skeletal and heart muscle, respectively, while CK-BB is found mainly in the brain and smooth muscle tissue.  Most CK activity in normal serum is due to the presence of the CK-MM isoenzyme.  CK-MM activity increases following exercise, muscle trauma, shock and major surgery.  The CK-BB isoenzyme is usually present in serum at very low activities and has been found to be rather unstable.  Its activity increases in brain damage, malignant neoplasms, liver metastases and damage to the pregnant uterus (10.3).  CK-MB is also present in low concentration in normal human serum but is increased as a result of heart injury and rarely, skeletal muscle damage.  CK-MB is widely used as an indicator of acute myocardial infarction as the detection of elevated activities is considered highly specific for this condition (10.4.)

3.0     SPECIMEN COLLECTION AND HANDLING

3.1     SPECIMEN COLLECTION.

Fresh, clear, unhemolyzed serum is the preferred specimen.  Avoid exposures of samples to strong light.

Hemolyzed samples should not be used since erythrocytes contain contaminants and enzymes that interfere with the assay.

The amount of sample required will depend on the analyzer used.  The amount of sample required is in the range of 5-50 µl.  Call Biotron's technical service department at 1-800-595 8766 for the recommended sample volume for your analyzer.

3.2     SPECIMEN STORAGE

The specimen can be stored refrigerated (2°-8°C) but not longer than 1 week.  Freezing of samples results in minimal loss of activity.

It is recommended that testing be done as soon as possible after sample collection and preparation.  If testing cannot occur immediately, store the sample properly using the guidelines above.

 

4.0     MATERIALS                                                            (4 X 6.5 ml)

Reagents necessary for the determination of CK-MB are included in the kit.

4.1     REAGENT

4.1.1  CK-MB Reagent contains, after reconstitution with CK-MB diluent:

          creatine phosphate disodium                                      ³ 32 mmol/L

          ADP                                                                         ³ 2.0 mmol/L

          glucose                                                                       ³ 20 mmol/L

          NAD                                                                         ³ 2.2 mmol/L

          hexokinase                                                                  ³ 3,000 U/L

          glucose-6-phosphate dehydrogenase                            ³ 2,500 U/L

          magnesium acetate                                                     ³ 12 mmol/L

          EDTA                                                                         ³ 2 mmol/L

          NAC                                                                          ³ 20 mmol/L

          buffers, stabilizers

4.1.2  CK-MB Diluent contains buffer 100 mmol/L, anti-human polyclonal CK-M antibody (from goat) sufficient to inhibit up to 2000 U/L CK-MM at 37° C, surfactant and sodium azide (0.1%) preservative.

4.2     WARNINGS AND PRECAUTIONS

For In Vitro Diagnostic Use.  Not for Internal use in Humans or Animals.  In Vitro Diagnostics reagents may be hazardous.  Avoid ingestion and skin or eye contact.

The reagent contains sodium azide.  The compound may react with lead and copper plumbing giving rise to explosive metal azides.  Flush with large volumes of water when disposing of reagent.

4.3     REAGENT PREPARATION

The working reagent is prepared by reconstituting each vial of CK-MB reagent with 6.5 ml of CK-MB diluent.  Replace rubber stopper and allow to sit for 5 minutes.  Swirl gently until dissolution is complete.  Then mix gently.  Record the date and time of reconstitution.

4.4     REAGENT STORAGE AND STABILITY

All reagents included in the kit are stable at 2‑8° C (refrigerated) until the expiration date stated on the labels.  The working reagent is stable when refrigerated (2‑8° C) for 6 days or at room temperature (18-26° C) for 24 hours.  Do not use if the working reagent is turbid.

4.5     ADDITIONAL MATERIALS REQUIRED

4.5.1  Spectrophotometer or colorimeter capable of reading absorbance accurately at 340 nm.

4.5.2  1 cm cuvettes or a flow cell capable of transmitting light at 340 nm.

4.5.3  Test tubes capable of holding 2 ml.

4.5.4  Pipettes capable of delivering 1 ml and 50 µl.

4.5.5  A timer with one minute increments.

4.5.6  A water bath which can be adjusted to 37° C.

4.5.7  Normal and abnormal control for quality control.

 

5.0     TEST PROCEDURE

          The following is a general procedure for use on a manual instrument.

5.1     PROCEDURE CONDITIONS

 Wavelength                                                                      340 nm

 Temperature                                                                        37° C

 Pathlength                                                                            1 cm

 Mode                                                                                 Kinetic

 Reaction time                                                                5 minutes

 Sample volume                                                                      50 µl

 Reagent volume                                                                  1.0 ml

 Total volume                                                                    1.05 ml

 Sample to reagent ratio                                                          1:20

5.2     INSTRUMENT

Any instrument capable of reading absorbance accurately with a sensitivity of 0.001 absorbance at 340 nm may be used.  The band width should be 10 nm or less, stray light 0.5% or less, and the wavelength accuracy within 2 nm.

5.3     CALIBRATION

No reagent calibration is necessary as the CK-MB activity is calculated by use of the molar absorptivity of NADH, which is taken as 6.22 at 340 nm.

5.4     PROCEDURE

Determine the total CK activity of the sample (with Biotron 's CK reagent kit.)  After determining the CK activity, dilute any sample with an activity higher than 2000 U/L at 37°C using physiological saline.  Multiply the activity by the dilution factor to compensate for the dilution.  Determine CK-MB as follows.

5.4.1  Prepare the required volume of CK-MB working reagent.  (See 4.3 Reagent Preparation section.)

5.4.2  Into separate cuvettes add 1.0 ml of CK-MB working reagent: one cuvette for test and one cuvette for blank.

5.4.3  Add 50 µl of sample to test cuvette.  Add 50 µl of distilled water to blank cuvette.

5.4.4  Mix, and incubate for 5 minutes at 37° C.

5.4.5  Record the change in absorbance at 340 nm at one minute intervals for the next 3 minutes.

5.5     CALCULATION AND RESULTS

5.5.1  Subtract DA/min of blank from DA/min of test.  Then use the corrected DA/min in the calculation below.

5.5.2  CK-B activity

                            DA/min x assay volume (ml) x 1000

          CK-B (U/L) = ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑------------------------

                            6.22 x light path (cm) x sample volume (ml)

                            = DA/min x 3376

          DA/min = (corrected) change in absorbance per minute

          Assay volume = total reaction volume expressed in ml

          1000 = converts U/ml to U/L

          6.22 = absorbance coefficient of NADH

          Lightpath= length of the light path expressed in cm (usually 1.0)

          Sample volume = sample volume expressed in ml

          3376 = factor derived from the constants in the equation

5.1.3  CK-MB activity

          CK-MB (U/L) = 2 times CK-B (U/L) activity.

5.1.4  Example

          Assume the corrected DA/min = 0.013 and total CK = 1200 U/L.

                      0.013 x 1.05 x 1000

          CK-B = ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑------ = 0.013 x 3376 = 43.9 U/L

                      6.22 x 1 x 0.05

          CK-MB = 2 x 43.9 = 87.8 U/L

                                         CK-MB                    87.8

          % CK-MB activity = -------------- x 100% = ---------- x 100% = 7.3%

                                         CK                          1200

 

6.0     INTERPRETATION OF RESULTS

6.1     EXPECTED VALUES (10.3)

          The range of expected values for the CK-MB is:

0-22 U/L (37° C)

A ratio between CK-MB and total CK activities above 4% should be considered suspicious, even though it could be caused by extensive skeletal muscle injury.  Any ratio between 5.5-20% is consistent with acute myocardial infarction (10.14.)

These values are suggested guidelines.  It is recommended that each laboratory establish the normal range for the area in which it is located.

6.2     LIMITATIONS OF PROCEDURE

If the total CK activity of the sample is greater than 2000 U/L at 37°C, use physiological saline to dilute the sample before proceeding with the CK-MB assay.

This method will also measure CK-BB activity.  If a significant CK-BB activity is present, the CK-MB activity will be overestimated (10.11.)

A macro form of BB (immunoglobulin complexed) has been observed which will be measured as B in this assay.  If the measured CK-B activity is greater than 20% of the total CK, the presence of macro BB should be suspected (10.12, 10.13.)

 

7.0     QUALITY CONTROL

Standard practice for quality control should be applied to this system.  Commercially available lyophilized controls can be used to monitor the daily acceptable variations.  Normal and abnormal controls should be assayed at the beginning of each run of patient samples, whenever a new reagent or a different lot number is being used, and following any system maintenance.

A satisfactory level of performance is achieved when the analyte values obtained are within the "acceptable range" established by the laboratory.

8.0     CALIBRATION PROCEDURES

No routine reagent calibration is necessary as the CK-MB activity is calculated by use of the molar absorptivity of NADH, which is taken as 6.22 at 340 nm.

 

9.0     PERFORMANCE CHARACTERISTICS

9.1     PRECISION

The estimates of precision shown below were obtained from assays of human control serum.

Within-Run

          N         Mean(U/L)  SD (U/L)         CV (%)

          20      19.1                   ± 0.46      2.4

          20      42.4                   ± 0.55      1.3

          20        116.4          ± 2.25             1.9

Between-Run

          N         Mean(U/L)  SD (U/L)         CV (%)

          20      18.8                   ± 0.78      4.1

          20      42.8                   ± 0.98      2.3

          20        116.9          ± 2.27             1.9

9.2     CORRELATION

A correlation study was done comparing this method (y) and a comparative CK-MB method (x₁).  A manual procedure was used.  The samples ranged from 9 to 264 U/L.  A second study was done comparing this method (y) and a comparative manual CK-MB method (x₂).  An automated procedure was used. The samples ranged from 6 to 756 U/L.  The study yielded:

                      Regression Equation           Correlation

          N         y=Biotron , x=Comparative               Coefficient

          44        y = 0.99 x₁ - 1.3                 .997

          91        y = 1.07 x₂ - 4.4                 .999

9.3     SPECIFICITY

The ability of the polyclonal antibody to inhibit the M-subunit of human CK was determined by testing the activity of CK-MM at a level over 2000 U/L at 37°C.  The CK-MM isoenzyme was inhibited >99.5%.

9.4     SENSITIVITY

An absorbance change of 0.001 corresponds to approximately 6.8 U/L CK-MB activity.

 

10.0   REFERENCES

10.1   Dawson, D.M., et al, Biochem Biophys Res Comm 21:346, 1965.

10.2   Orth, H.D., Creatine Kinase Isoenzymes.  Springer Varlag, New York, pp 10-18, 1981.

10.3   Lang, H., Wuerzburg, U., Clin. Chem., 1439, 1982.

10.4   Wagner, G.S., et al, Circulation 47:263, 1973.

10.5   Roberts, R., Henry, P.D., Witteeveen, S.A., Sobal, B.E., Am. J. Cardiol. 33,650, 1974.

10.6   Mercer, D.W. Clin. Chem 20, 36, 1974.

10.7   Wuerzburg, U., Heinrich, N., Lang, H., Prellwitz, W., Neumeter, D., Knedel. M., Klin. Worchenschz., 54, 357, 1976.

10.8   Gerhardt, W., Ljungdahl, L. Borjesson, J., Hofvendahl, S., Hedenas, B., Clin. Chem. Acta 78, 29, 1977.

10.9   Pesce, A.J. and Kaplan, L.A., Methods in Clinical Chemistry. Editors, The C.V. Mosby Company, St. Louis, 1987.

10.10 Young, D.S., Effect of Drugs on Clinical Laboratory Tests. Third Edition. AACC press, WAshington, D.C. 1990.

10.11 Jockers-Wretou, E., Pfleiderer G., Clin Chem Acta 58:223, 1975.

10.12 Ljungdahl, L., Gerhardt, W., Clin Chem 24:832, 1978.

10.13 Urdal P., Landaas, S., Clin Chem 25:461, 1979.

10.14 Wu, A.H.B., and Bowers, G.N. Jr., Clin. Chem. 28, 2017, 1982.