CREATINE KINASE ISOENZYME MB A Critical Review by Wendy ...

CREATINE KINASE ISOENZYME MB
A Critical Review by Wendy Ward, M.D.
September 30, 1988
Creatine kinase isoenzyme MB (CK MB) is a more sensitive and
specific serum marker of acute myocardial injury (AMI) than the
older, alternative laboratory assays; total CK, LD, and AST.
Total CK, LD, and AST are notoriously nonspecific enzymes. Even
minor trauma releases CK from skeletal muscle. LD, present in all
organs, is elevated in serum after any organ injury, in
carcinomatosis, and in various anemias. AST, too, is elevated in
a spectrum of clinical situations other than AMI, including liver
disease, biliary obstruction, muscle disease, and trauma.
Likewise, electrocardiography, lacks specificity and sensitivity
in the diagnosis of acute MI. Authentic infarctions often show
only nonspecific EKG changes, which cannot be distinguished from
those seen in angina. Q-waves, although specific, are not always
present in acute MI, so lack sensitivity.
Assay of CK MB, on the other hand, is an extremely specific test
for the diagnosis of AMI.^'J'4'a CK MB is highly.concentrated in
myocardial tissue in comparison to other tissues. During
myocardial injury, CK MB is released from ischemic myocardial
cells, and is significantly increased in the serum four to six
hours after the onset of symptoms. Serum levels of CK MB reach a
peak within 12 to 24 hours after infarction. The elevation
persists from 48 to 72 hours. 'J Only when patients are admitted
later than 72 hours after the onset of cardiac symptoms, is LDH-1
a more appropriate assay. LDH 1 peaks at 48 to 72 hours-after the
infarct and remains elevated for one to two weeks. '' The
majority of patients, however, present with ongoing chest pain of
recent onset. CK MB is currently the laboratory assay of choice
for diagnosing or ruling out acute MI in these patients.
Several CK MB assay methods are currently available, and new
options are being developed rapidly. Electrophoretic separation
>f CK isoenzymes was the original method and is still the gold
tandardin many labs. If total CK activity is assayed in
< onjunction with electrophoresis, the percentage of CK MB measured
1 / scanning the CK MB band with a densitometer can be used to
c ilculate approximate activity due to the MB isoenzyme.
R grettably, CK isoenzyme electrophoresis^ has numerous
d. sadvantages. It is a cumbersome, time-consuming laboratory test.
Ir many hospitals, it is not available in evenings or on weekends.
Me mwhile, patients must be held in expensive cardiac care unit
ho pital beds, awaiting results from the next routine laboratory

f s h i f t .
Furthermore, electrophoresis is semi-quantitative, at best, and
lacks sensitivity in low ranges of CK MB. A CK MB band is only
visualized when serum CK MB fraction reaches 3% to 4% of total CK.
The ability to visualize a CK MB band is generally interpreted as
evidence of MI. Using this cutoff range, a large number of false
positives are found among healthy subjects. '4 Most laboratories
decide upon a lower limit of total CK, below which they refuse to
fractionate isoenzymes in attempt to reduce false positive
reporting. Commonly, the upper limit of the 95% confidence
interval for the total CK reference range is chosen as the lower
limit for indicating CK isoenzyme electrophoresis.
A more recent method of CK MB assay, which is now offerred by
Kodak Ektachem, uses anti-human CK-M antibody to inhibit CK-M
subunit_activity. The remaining CK-B activity, in most cases, is
proportional to CK-MB activity. Activity is measured using
creatine phosphate and ADP as substrates to generate creatine and
ATP. In a coupled reaction sequence, hydrogen peroxide oxidizes a
dye precursor. The rate of chromophore production, measured by
reflectance spectrophotometry at 670 nm, is proportional to CK MB
activity.
creatine kinase
c r e a t i n e p h o s p h a t e + A D P > c r e a t i n e + A T P
N-acetylcysteine
glycerokinase
A T P + g y l c e r o l > L - a l p h a - g l y c e r o p h o s p h a t e + A D P
L-alpha-glycerophosphate oxidase
L - a l p h a - g l y c e r o p h o s p h a t e + 0 2 > d i h y d r o x y a c e t o n e
phosphate + H,0,
peroxidase 2 2
H 2 ° 2 + d y e P r e c u r s o r > d y e + 2 H 2 0 2
Although this method is rapid, sensitive and inexpensive, it is
subject to several interferences which lead to false positives.
The false positive rate has been reported to be 5.3%, the majority
of which are caused by excess CK BB. A small number of false
positives are caused by macro CK. Rarely, mitochondrial CK is
responsible for a false positvive. Nevertheless, sensitivity
approaches 100%. Many laboratories believe that the Ektachem CK
M immunoinhibition assay is an excellent screening tool for the
specific use of rapidly excluding the diagnosis of acute MI, and
allowing rapid dismissal of patients from the cardiac care unit
during late hours and weekends. Most laboratories, however, feel
that it is necessary to confirm positive Ektachem results with a
more specific CK MB assay, such as CK isoenzyme electrophoresis,
or another immunoassay.
Recently, immunodiagnostic methods to assay CK MB in mass units,
rather than units of enzyme activity have been developed and are
offered as highly specific tests for the diagnosis of acute MI, or

f a s c o n fi r m a t o r y t e s t s . T h e c l e a r a d v a n t a g e o f t h e s e a s s a y s o v e r
electrophoresis is ease and speed of performance. Mass
measurement of CK MB is inherently more accurate and specific than
assays of enzyme activity. The potential for interference by
adenylate kinase, which necessitates inhibitory steps in enzymatic
assays, is totally eliminated by mass measurement. Furthermore,
mass concentration assays, by RIA, EIA, or chemiluminescence,
utilize anti-CK M and anti-CK B to sandwich CK MB, or they utilize
new monoclonal anti-CK MB antibodies. '3 Interference by CK BB.
excess CK MM, macro CK or other forms is virtually eliminated. '5
False negatives could theoretically result, if CK BB is present in
concentrations high enough to saturate all the anti CK B provided
in the kit. This would be an uncommon clinical situation. CK BB
is present in appreciable quantities only in the brain, and is
prevented from entering circulating blood by the blood brain
barrier, even in cases of head trauma and CVA. Occasionally, CK BB
is elevated in association with certain carcinomas, in which case
the clinician should anticipate the possibility of interference.
Ciba Corning has recently placed an immunochemiluminometric assay
of CK MB on the market, which provides a representative model of
the many new mass concentration immunoassays. This system is
selected for discussion here because of the apparent time
advantage it offers over two site sandwich capture assays
^ ( c u r r e n t l y o f f e r e d b y H y b r i t e c h , a n d B e h r i n g ) . T h e
(; immunochemiluminometric method utilizes a magnetic bead,
covalently bound to anti-CK B, which provides a solid support.
When added to serum, all CK BB and CK MB is bound by the magnetic
particles. The magnetic particle-CK MB and CK BB complexes, are
magnetically held to the sides of the test tube while free CK MM
is washed off. A monoclonal antibody to CK MB, labelled with
acridinium ester is also utilized. It binds only to CK MB, which
is bound and immobilized by the magnetic beads. Excess labelled
anti-CK MB is removed through vortexing and rinses. Quantitation
of CK MB is effected through addition of H-0. to activate
chemiluminescent photon emission from the acridinium label, and
measurement with a luminometer. Photon emission is proportional
to the amount of CK MB present in the specimen (Figure 1).
The immunochemiluminometric assay correlates well with
electrophoresis, r = .938 and with immunoenzymatic assays, r =
.942. The assay is extremely sensitive, able to detect 1 ug/L CK
MB, and therefore minimizes false negatives. Sensitvity and
specificity are reported to be 94% by Ciba Corning Diagnostic
Corp.
In summary, CK MB assays, of crucial importance to CCU's, have
undergone extensive evolution and improvement in the last several
years. Laboratories and hospitals should be aware and receptive
of new immunoenzymatic and mass concentration immunoassay options.
^ W h e n
STAT
a p p l i
screening
e d i n a
with
t h o u
Ektachem
g h t f u l s c
CK
r e e n
MB
i n g /
slides
c o n fi r m
and
a t i o
confirmation
n s t r a t e g y
with
( e . g .
Ciba Corning immunochemiluminometric methods), these new assays
can expediate results, save unnecessary CCU days, and contain

laboratory costs, with improved sensitivity and specificity.
/!ifcSy
/^^\
Figure 1
CK Isoenzymes C K - M M M ^ M
CK-B8 \£}^
C K - M B ~ H B
-< E-0
e L r T ) / ^ N
yW
-^ + &0 +ny
^< &e )Mq
Lite Reagent. Antigen Solid Phase
AE-LaDeiea in Patient Monocionai Antmoav
M o n o c l o n a l S a m o i e ! o C K - 8 B B o u n a t o
A n t i D o a y t o C K - M B P a r a m a g n e t i c P a r t i c i e s
.**» I -A1 r
l^K£)))=Q
->r*i lSa
0®^[-K->©
Separate
& Measure

BIBLIOGRAPHY
Allbright, A., editor. Clinical Laboratory Manual, San
Francisco General Hospital Medical Center. 28th edition.
1988.
2 R. Roberts. "Enzymatic Diagnosis of Acute Myocardial Infarction."
Chest. Vol. 93. 1988. 3s-6s.
3 J. Lott, J.Stang. "Serum Enzymes and Isoenzymes in the Diagnosis
and Differential Diagnosis of Myocardial Ischemia and
Necrosis." Clinical Chemistry. Vol. 26:9. 1980. 1241-1250.
T. Koch, U. Mehtz, H. Nipper. "Clinical and Analytical
Evaluation of Kits For Measurement of Creatine Kinase
Isoenzyme MB." Clinical Chemistry. Vol 32:1. 1986.
186-191.
5 .
Piran, Kohn, Uretsky, Bernier, Barlow, Niswander, stastny.
"Immunochemiluminometric Assay of CK MB with a Monoclonal
Antibody to the MB Isoenzyme." Clinical Chemistry. Vol.
33:9. 1987. 1517-1520.
g J. Sanderson. Recent Advances in the Measurement of CK-MB By
Immuno-inhibition. (Seradyn, Inc: Indianapolils, IN).
1983.
7 C. Greiner, C. Leiendecker-Foster, M. Basara, K. Willard.
"An Efficient Testing Strategy Using the Ektachem CK-MB."
Clinical Chemistry. Vol 32:6. 1988. Abstract #631.
T. Green. "Evaluation of Seradyn CK MB reagent on the Monarch
761." Veterans Administration Medical Center. Portland,
Oregon. 1988.
9 Henry, J., editor. Todd. Sanford. Davidsohn; Clinical Diagnosis
and Management by Laboratory Methods. (W.B. Saunders Co.:
Philadelphia). 1979. pp. 372-374.