4.26: Cardiac Isoenzymes

A physician called the laboratory concerned about the results of a cardiac profile (total CK and LD) obtained on his patient. While the specimen had normal levels of lactate dehydrogenase (LD) activity, the total creatine kinase (CK) levels were significantly elevated: 159 U/L (upper limit of normal for a Caucasian male, 100 U/L). Since the laboratory’s policy is to perform isoenzyme analysis only for cardiac profiles with an elevated total CK result, CK isoenzyme analysis had already been performed. The result, obtained by an immuno-inhibition assay, was 9 ng/mL (upper limit of normal, 6 ng/mL). The sample was non-icteric and non-hemolyzed.

Although previously there had not been any concern about the patient’s cardiac status, the physician is now worried about a possible myocardial infarct (MI), and is asking the laboratory to perform isoenzyme analysis on a number of serum samples drawn subsequently to the one processed for a cardiac profile.

QUESTION

What advice should the laboratory give to the physician regarding the need for CK isoenzyme analysis?

Questions to Consider

1. What is the principle of the "sandwich" immunoassay for CK-MB? What are the major sources of interference of the assay?
2. What alternatives to the sandwich immunoassay are there for CK-MB analysis? What are the relative advantages and disadvantages of each?
3. What is the distribution of the 3 CK isoenzymes in prostate gland tissue?

Answer

Upon further discussion with the laboratory’s clinical chemist and supervisor, the physician reveals that the initial sample, for which a cardiac profile was ordered, was drawn immediately after a surgical procedure for resection of a prostate gland. Postoperatively, the patient experienced no EKG changes, chest pains, or arrhythmias.

The laboratory should advise the physician that further CK isoenzyme analyses are not needed and that the patient most likely did not have an MI. Since the prostate gland is rich in CK-BB, prostatic surgery will result in relatively large amounts of CK-BB being released into blood. Because this laboratory’s immunoinhibition method does not blank out CK-BB activity, CK-BB causes a positive interference. The degree of interference is actually twice as large as the actual level of CK-BB present because the procedure calculates the final CK-MB activity by doubling the B activity to estimate the entire MB activity. Thus, CK-BB activity from both B chains is multiplied by 2.

The extent of such interference can be seen in the original data: total CK, 159 U/L, CK-MB activity, 66 U/L: that is 42% MB. Usually the % CK-MB in most known MIs is between 5-15%. The 42% MB with low total CK activity is a result of the presence of unblanked CK-BB activity.

Answers to Questions to Consider

1. The immunoinhibition technique was once in wide use, but it rarely employed today (See Methods of Creatine Kinase Isoenzymes on the CD-ROM). This assay worked by adding to the sample an antibody directed towards the "M" subunit of CK. The binding of this antibody would inhibit the enzyme activity of the "M" subunits, from CK-MM amd any CK-MB, but would not inhibit the "B" unit activity in CK-MB. After the addition of the antibody, the remaining CK activity of the solution would be measured. Since it was assumed that there is no CK-BB activity present, the measured "B" activity would be multiplied by a factor of two to give the full CK-MB activity.
   
   The primary interference with the assay would be the presence of CK-BB, which can come from a number of tissue sources. The presence of HAMA antibodies may cause falsely high results by binding to the anti-B antibody and preventing inhibition.
2. Immunometric CK-MB measurements work by adding to the sample a primary antibody (Ab) preparation that is directed to either the M or B subunit of CK. These antibodies, linked to some solid phase, can be monoclonal (MAb) or polycloncal (See see chapter 11 and 12 and Methods of Creatine Kinase Isoenzymes on the CD-ROM). After binding of any CK to the 'capture' antibody, there is a wash step of the solid phase to remove any interfering substances. A second, labeled antibody is added to the solid phase. The second antibody can be labeled with radioactive, fluorescent, cehmiluminescent, or enzyme tags (see chapter 12 and 13). The secondary antibody is also either a monoclononal or polyclonal antibody, but is always the opposite of the first; that is, if the primary antibody was directed toward epitopes on the B subunit, than the secondary antibody is directed towards sites on the M subunit. This gives specificty to only the MB isoenzyme form of CK. In addiiton, most current assays use a monoclonal antibody for one of the steps (primary or secondary) and a polyclonal antibody preparation for the other step. The use of a monoclonal antibody increases the specificity of the assay.
   
   The are very few interferences with the assay itself. The most frequent is the presence of HAMA antibodies, which can cause either falsely elevated or falsely low results, depending on the assay configuration.
   
   Measurement of CK isoenzymes by separation techniques can also be performed by agarose gel electrophoresis (See Creatine Kinase Isoenzymes on CD-ROM.). With this method one can detect other CK isoenzymes as well as rare, atypical CK forms. This method is relatively inexpensive, difficult to automate, rather slow, and infrequently used today.
3. The tissue distribution of CK isoenzymes is shown in Table 55-1 of the 4th Edition. Of interest is the very high percentage of CK-BB in prostate gland tissue.